1. 2 nd TSB project meeting Bio-control of grain storage insect pests Bryony Taylor, Dave Moore, Emma Thompson, Steve Edgington

2. Aims of work Create a formulation that: is compatible with existing spray equipment produces a droplet range appropriate for a contact biopesticide has an even coverage. The undiluted formulation will: need to be stored without affecting the viability of the conidia have minimal separation or sedimentation

3. Aims of work If the formulation is to be used with a knapsack sprayer: it will need to suspend readily in a water based tank mix produce no clogging of the nozzle. If a ULV sprayer is considered the formulation will need to be: oil based compatible with ULV sprayer technology.

4. Considerations-Current application technology used in grain stores 61% of farms use fabric treatments only to treat their grain stores and a further 34% use both fabric and grain treatments 56% of fabric treatments were applied using spray technology which included knapsack and hand held sprayers Other methodologies include fogging, dusting (Pirimiphos methyl and silica), mist and smoke

5. Formulation issues Conidia are hydrophobic therefore need co-formulants to disperse in water Bb conidia lose viability quickly when stored in a water based formulation Emulsifiers need to be added to break the surface tension Some have been shown to affect conidial viability Careful screening needs to be carried out

6. Co-formulants Co-formulants to be tested were carefully considered and chosen on the basis of: suitability previous research listing in the adjuvant section of the UK pesticide guide 2010 availability (some have been withdrawn)

7. Q: Are these co-formulants toxic to Bb? Dispersents:

8. Q: Are these co-formulants toxic to Bb?

10. Concentration ● For contact biopesticides it is recommended that 50-70 drops per cm 2 ● Normal amount of spray to apply to a grainstore is 5l/100m 2 which is also the recommended amount on the Actellic 900 label ● The FAO recommend this may be increased on more porous surfaces to 10-20l/100m 2 or reduced when applied to metalwork. ● We aimed to test formulations at these concentrations AIM: to deliver a dose of both 5 x 10 9 conidia per m 2 and 2.5 x 10 10 conidia per m 2

11. Concentration If the target application rate is 10L per 100m 2 for a porous surface, then we will need to apply: 1L for 10m 2 100ml for 1m 2. AIM: 5 x 10 9 and 2.5 x 10 10 per m 2 Need to suspend these amounts of conidia in co-formulant and then make up to 100ml using H 2 O

12. Concentration Spores per gram were calculated from a previously mass produced lot of IMI 389521 (received July 2008). The average spores per gram were calculated to be 6.7 x 10 10 Recent trials have shown that 75% entostat:25% conidia has been effective in trials using the dust formulation, thus we will test at these levels initially.

13. Experiment 1 Codacide Addit Cropoil Output Cropspray 11E Silwet L77

14. Results All adjuvants mixed well after 1 minute on the whirlimixer

15. Results 1 ml paste was mixed with 99ml water to see if a stable emulsion could be formed

16. Results Codacide suspended easily forming a consistent emulsion Addit was slightly more difficult to suspend and had ~10ml foam Codacide Addit

17. Results Cropoil suspended well, but residue left on glass Output adhered to the original tube but with vigorous shaking suspended. It dispersed well, but had ~8ml of foam Output Cropoil

18. Results Cropspray 11E did not suspend in water Silwet L77 suspended easily. Roughly 10ml foaming Silwet L77 Cropspray 11E

19. After 16h Codacide: Yellow scum on top, large particles settled at the bottom of the tube

20. After 16h Addit: Foam reduced however layer of white scum at top and settling powder at the bottom

21. After 16h Cropoil: Little sedimentation at bottom, however layer at top observed

22. After 16h Output: Settling observed, little floating matter

23. After 16h Silwet L77: Foam subsided, settling of powder at the bottom

24. Experiment 2: Viability study 0.1 g conidia and 0.3g entostat mixed with 8 co-formulants Conidia only and conidia+entostat controls Kept at 5˚C and 25 ˚C Viability regularly checked

25. Viability Tests 5˚C

26. Viability Tests 25˚C

27. Conclusion ● Viability lower than previous batches (problem in transport?) ● Output co-formulant can be discounted ● Variability may be due to large amount on entostat/particles on plates ● Continue and replicate studies

28. Observations During viability study, at both 5˚C and 25˚C ; all liquid formulations re-suspended easily, even after 28 days of storage All formulations showed settling of a fine powder at the bottom of tube; apart from Silwet L77 However, there was a gelatinous ‘blob’ in the middle of the 5˚C tube Settling

29. Experiment 3 ● Codacide ● Addit ● Cropoil ● Silwet L77 ● *Break-thru S 240* (new co-formulant) ● 0.373g conidia + 1.193g of entostat (1:3) ● 1ml of adjuvant added initially, then a further 1ml ● Water added to tube and inverted 10,20,30 times and 10sec, 20sec and 1min on the whirlimixer ● Those emulsifying were added to 98ml water 2.5 x 10 10 formulations

30. Results 1 ml of liquid was not enough to form a paste 2ml enough to form paste All paste were very viscous

31. Codacide: Was not able to mix into water Waxy floating particles present

32. Addit: Was difficult to form an emulsion after inversions/using whirlimixer Eventual emulsion formed, however large particles present In 98ml water, 10-20ml foam

33. Cropoil Was not able to mix into water A waxy mat formed on top of the water

34. Silwet L77: Formed emulsion easily, although some paste stuck to side of tube Dispersed well in water with 20- 30ml foam

35. Break thru S 240: Mixed easily with water, with a little sticking to sides of tube In 98ml water, after 10 inversions 30-40ml foam

36. Future experiments ● Reduce settling with addition of varying levels of clay ● Repeat viability experiments and include Breakthru ● Look at particle sizing of promising formulations ● Investigate how well formulations spray ● AOB: ● Project student started this week ● Molecular characterisation of isolate underway

37. Thank You