COMPOSITE PARTICLES FOR CONTROLLING ARTHROPOD INFESTATION

Abstract

Composite particles comprising baculovirus particles in a coating of wax that is degradable in the gut of a larva of an arthropod species, optionally in conjunction with an insecticide, methods of manufacture, uses thereof, and methods of controlling arthropod infestations.

Claims

1. A crop plant coated with composite particles, wherein each composite particle comprises: i) at least one baculovirus particle; and ii) an enveloping coating of wax for the baculovirus particle of i) made up of at least one wax that is degradable and/or soluble in the gut of a larva of an arthropod species.

2. The crop plant according to claim 1, wherein each composite particle further comprises an insecticide such as a pyrethroid and/or an organophosphate.

3. The crop plant according to claim 1, wherein the baculovirus particle is selected from an alphabaculovirus particle and a betabaculovirus particle.

4. The crop plant according to claim 1, wherein the baculovirus particle is in the form of a baculovirus occlusion body.

5. The crop plant according to claim 1, wherein the baculovirus particle is selected from Heliothis zea NPV, Helicoverpa armigera NPV, Spodoptera exigua NPV, Spodoptera littoralis NPV, Spodoptera exempta NPV, Anticarsia gemmatalis NPV, Lymantria dispar MNPV, Neodiprion abietis NPV, Orygia pseudotsugata NPV, Neodiprion lecontei NPV, Trichoplusia ni NPV, Autographa californica NPV, Spodoptera albula NPV, Spodoptera litura NPV, Cydia pomonella GV, Plutella xylostella GV, Cryptophlebia leucotreta GV, Phthorimaea operculella GV, Adoxphyes orana GV, Homona maganima GV, Plodia interpunctella GV, and Adoxophyes honmai GV.

6. The crop plant according to claim 1, wherein the baculovirus particle is selected from Heliothis zea NPV, Helicoverpa armigera NPV, Spodoptera exigua NPV, Spodoptera littoralis NPV, Spodoptera exempta NPV, Anticarsia gemmatalis NPV, Cydia pomonella GV and Plodia interpunctella GV.

7. The crop plant according to claim 1, wherein the baculovirus particle is completely enveloped by the coating.

8. The crop plant according to claim 1, wherein the wax is selected from waxes that are ingestible by a larva.

9. The crop plant according to claim 1, wherein the wax is a natural wax that is degradable and/or soluble in the gut of a larva.

10. The crop plant according to claim 1, wherein the wax is a mixture of waxes selected from mineral waxes and synthetic waxes and at least one natural wax wherein at least the natural wax is degradable and/or soluble in the gut of a larva.

11. The crop plant according to claim 1, wherein the natural wax is selected from carnauba wax, rice bran wax and candelilla wax and a mixture of two or more thereof.

12. The crop plant according to claim 1, wherein the arthropod species is a species from the order Lepidoptera and wherein the gut of the larvae of the arthropod species has a pH from 8 to 12.

13. A method of producing particles for coating the crop plant according to claim 1 by i) melting a wax; ii) adding a baculovirus particle to the molten wax of i) and admixing therewith for a time period sufficient to at least partially coat the biological agent; iii) rapidly cooling the product of ii) to a solid; and iv) kibbling and comminuting the product of iii) to a particle size for ingestion by a larva.

14. A method according to claim 13, wherein the admixing step ii) is performed in a period of less than or equal to 5 minutes.

15. A method according to claim 13, wherein the cooling step is performed in a time period of less than or equal to 12 hours.

16. A method according to claim 13, wherein the particles have a volume mean diameter of less than 30 m, preferably in the range 8 m to 15 m, and most preferably in the range 10 m to 15 m.

17. A method of controlling larval infestation on crop plants in the field or in harvested and/or processed crop produce, preferably in the reduction of viable larvae on crop plants or in harvested and/or processed crop produce by coating a crop plant with composite particles to produce the crop plant according to claim 1.

18. The method according to claim 17 that is conducted on crop plants in the field selected from fruit trees, such as apple, pear and peach trees, crucifer vegetables such as cabbage, broccoli, brussel sprouts and kale, cereals such as corn (maize), wheat, rye, barley and sorghum, nuts trees such as almond, pecan, hazelnut, brazil, and cashew, grape vines, tobacco, evergreen trees such as Douglas fir, Loblolly pine, cotton, beans such as velvet bean, solanaceous species such as potato, tomato, eggplant, capsicum, tobacco and petunia.

19. The method according to claim 17 that is conducted on harvested and/or processed crop produce selected from dried commodities including whole grains such as wheat, rice, barley and corn (maize), pulses, beans, and lentils and products derived from or made with dried commodities such as processed foods including pasta, grain flours, couscous, breakfast cereals, dried herbs, domestic livestock feed, for example for pigs, cows, sheep and horses, semolina, breads, nuts (ground, flaked and/or whole nuts), snacking food, such as sweet and savoury items including biscuits, potato crisps, vegetable crisps, pretzels, cheese biscuits, and dried wafers.

20. A composition for application to crop plants to produce the crop plant coated with cornposite particles as defined in claim 1.

Description

EXAMPLES SECTION

Introduction

[0072] This work describes the method required to formulate virus in wax particle formulations.

Key:

[0073] BV: Baculovirus Occlusion Bodies raw material [0074] NPV: Nucleopolyhedrovirus, a genera of the baculoviridae family of viruses included under the BV umbrella [0075] GV: Granulovirus, a genera of the baculoviridae family of viruses included under the [0076] BV umbrella [0077] EBV: Baculovirus occlusion bodies formulated into Entostat particles through the methods described below [0078] EBV+: Baculovirus occlusion bodies formulated into Entostat particles through the methods described below, which also contain other additives of the types described previously in this document such as phagostimulants and aggregation pheromones.

1. Materials:

[0079] Active ingredient (BV of the chosen speciesSpliNPV targeting Spodoptera littoralis, or the Egyptian cotton leaf worm) sourced from NRI Candelilla wax and Rice bran wax.

2. Equipment:

[0080] Hotplate that heats up to 150 C. (Stuart scientific Ltd)

[0081] Two decimal place balance (Ohaus)

[0082] Freezer that cools down to at least 24 C. (any make/model will do)

[0083] High shear mixer/homogenizer (IKA T18 digital)

[0084] Kibbler mill (KT handling limited model 04)

[0085] Comminuting mill (Apex LTD type 314s)

[0086] Air jet mill (any make/model will do)

[0087] Suitable size sample pots

3. Detailed Procedure

[0088] 3.1. Using a calibrated balance weigh out the required quantity of carrier waxes125,0 g of rice bran wax and 125,0 g of candelilla wax.

[0089] Place a 50:50 mixture candelilla wax: rice bran wax (% w/w) into a pan and place onto a hotplate set to a temperature of 120 C. The wax is heated until completely melted and a clear liquid with no solids is observed.

[0090] Using a calibrated balance a quantity of the active BV at 1% w/w is weighed out.

[0091] 3.1.1. The BV is added to the molten wax quickly over a total time period of 60 s and dispersed within the molten wax by high shear mixing (using a high shear mixer IKA T18) to ensure even distribution within the wax.

[0092] 3.2. The wax is then transferred to a foil lined shallow tray and transferred to a freezer set at 24 C. to rapidly cool and solidify within approximately 1-2 h.

[0093] 3.3. Once the formulated material is frozen into a completely solid block or slab it is broken into large chunks and sent for milling. The chunks are ground in a kibbler mill (KT Handling Limited, Model 04) to particles of approximately 2 mm average diameter

[0094] 3.4. The kibbled material is then comminuted into smaller particles in a comminuting mill (model 314s, from Apex Ltd) to particles of 150 m average diameter.

[0095] 3.5. The comminuted material is further micronized in a jet mill (Hosokawa Alpine Jet AFG 100 fluidised bed jet mill) to achieve granulation of mean average particle size 10 um.

[0096] 3.6. The micronized material containing baculovirus particles (EBV) is stored in a suitable sample container under refrigeration conditions at 4 C., until use.

Bioassay Methods

[0097] The efficacy of the different EBV formulations is tested using two methodsthe droplet method, using young neonate larvae, and the diet-plug method, using older (L3) larvae. All experiments will use larvae of the Egyptian cotton leafworm (Spodoptera littoralis).

[0098] Droplet method: EBV formulations are added to 10% sucrose solution containing food dye and a wetting agent (e.g. 1% Lankem AEP 66). A dilution series provides six EBV concentrations to provide reliable comparisons of LC50 values across different formulations (i.e. the concentration of EBV required to achieve 50% mortality). As positive and negative controls, the EBV concentrations are compared against the LC50 dose of the non-formulated SpliNPV virus, and the solution without any virus (i.e. sucrose solution, dye and wetting agent alone). For the bioassay, starved neonate larvae are placed in the centre of a Petri dish containing concentric circles of droplets of EBV solution. After 30 min feeding, larvae with blue guts (indicating that they have ingested the solution) are removed and placed singly into diet pots in an incubator at 27 C. Twice-daily monitoring of the larvae provides data on timing of death (or pupation) and its causelarvae dying of viral infection are easily diagnosed visually, but is confirmed by microscopy. Bioassays use 50 larvae per treatment at each virus concentration, and the LC50 values of each EBV are calculated from five replicate assays using logistic regression.

[0099] Diet plug method: Starved 3rd-instar larvae are presented individually with a small cube (2 mm.sup.3) of semi-artificial diet laced with 2 ml of the requisite EBV solution (or control solution) and left in an incubator overnight and next morning, larvae that have not eaten all of the diet plug are discarded. The remainder of the larvae are moved into individual diet pots and again monitored twice-daily for mortality/pupation. Sample sizes and analytical methods are the same as for the droplet bioassay.

Testing Effects of UV on EBV and EBV.SUP.+ Formulations

[0100] Trial EBV and EBV.sup.+ formulations are tested initially in a sunlight simulator to quantify the effects of UV on the viability of the different EBV formulations compared to non-formulated virus and current commercial formulations (e.g. Littovir). The EBV is exposed as dried suspensions of virus on mylothene laminated sheetsa system that has been developed at University of Greenwich to mimic plant leaf exposures. They are then exposed for 24 h to a Nereus CPS laboratory sunlight simulator which produces a UV spectrum comparable to sunlight. The test samples are cooled underneath by circulating temperature-controlled water. Exposed virus is then washed using a standard recovery protocol (sonication for 3 min then by wash for 1 h in 0.2% sodium dodecyl sulphate) to extract the exposed virus for neonate bioassay at LU. Promising candidate EBV.sup.+ are identified, longer-term (0-30 day) evaluations are carried out in UV weathering equipment, simulating both tropical and temperate cropping conditions with respect to UV, temperature and humidity levels. All treatments are applied using a droplet sprayer to mimic field application. The UV stability of the most promising EBV.sup.+ is compared to both non-formulated virus and commercially-formulated viruses. Targets are exposed to sunlight day-night cycles, and EBV.sup.+ samples are harvested at 0, 1, 2, 4, 8, 16 & 32 days for virus recovery. Exposed EBV.sup.+ is then neonate bioassayed at LU to determine biological activity and EBV.sup.+ half-life. Physical characteristics of exposed EBV.sup.+ are also determined by SEM.

Quantifying Persistence of EBV and EBV.SUP.+ Formulations on the Crop

[0101] The persistence of the candidate EBV and EBV.sup.+ formulations is determined on two representative crop species: tomato and cabbage, grown in a glasshouse. Mature plants (30 per treatment) are sprayed with one of 2 rates of EBV.sup.+ solution (based upon commercial field application rates, validated via pilot studies), or with the EBV.sup.+ carrier alone (i.e. water+wetting agent; negative control) or the non-formulated SpliNPV virus solution (+wetting agent; positive control) or a commercial SpliNPV biopesticide (e.g. Littovir) Immediately post-spraying, and then after 1, 2, 5, 10 or 20 days, a subset of plants is harvested for bioassays. Small disks (1 cm diameter) of leaf from sprayed plants are fed to starved third-instar larvae overnight in an incubator (27 C.). Larvae that have eaten all of the leaf the next morning are then transferred to a diet pot and their mortality/pupation monitored twice daily (30 larvae per sampling point per treatment group). In addition, and building on initial assessments made during EBV.sup.+ selection, at each sampling point the condition of the host plants used in the different treatments is compared to establish whether there are any short- or long-term negative effects of the UV blockers. Traits to be quantified include plant height, plant dry mass (above- and below-ground), leaf area and leaf colour.

[0102] The persistence of baculovirus particles of the invention is shown to be superior when compared to that of conventional formulations containing baculovirus.