Bio-control of grainstore pests TSB quarterly meeting Bryony Taylor, Marine Guerret, Rajpreet Grewal, Belinda Luke, Dave Moore Analysis and compilation by Bryony Taylor www.cabi.org KNOWLEDGE FOR LIFE
Overview of CABI’s Viability of spores objectives ● Study effects of co-formulants on: ● Ability to suspend conidia ● Viability of conidia ● Stability and virulence of wet and dry formulations to be tested under refrigerated and ambient conditions (Jan 2011-Oct 2012)
Overview of CABI’s Application objectives Assess the use of conventional equipment to ● apply wet and dry formulations with special reference to using those as used to apply chemical insecticides as part of an IPM programme Assess adherence and coverage on different ● surfaces (metal, wood, concrete, grain bags etc) (Jan-December 2011) What level of Bb is transferred to grain and is ● this above normal background levels? (assess types of storage facilities and look at likelihood of direct transfer from treated surfaces) (27-30 months) Rate chemical application equipment for use ● with Bb formulations
Overview of CABI’s Persistence and uptake from objectives surfaces Examine the persistence of Bb in realistic ● conditions in a range of places i.e. at Grainstore humidity ● On grainstore type surfaces ● In artificial crevices ●
Overview Work so far to be presented today ● Effects of co-formulants on: ● Ability to suspend conidia ● Viability of conidia ● Assess the use of conventional equipment to apply wet formulations ● Assess adherence on different surfaces (metal, wood, concrete) ● What level of Bb is transferred to grain and is this above normal background levels?
Formulation 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
How to approach formulations… ● A) Store spores in oil based adjuvant i.e. Addit or Codacide then mix with water ● B) Store as powder and mix with wetter prior to spraying ● …each has advantages and disadvantages
Co-formulants
Co-formulants
Ability to suspend conidia ● Cropoil, cropspray 11E and Output did not suspend very well from a paste ● Break thru and Silwet L77 suspended easily ● Addit and Codacide suspended but needed more mixing
Ability to suspend conidia ● For Addit and Codacide, the minimum amount of oil needed to suspend 5 x 10 9 conidia is 0.3ml ● For 1 sqm to apply 5 x 10 9 conidia you will need 30ml water= 300L/ha (assuming 1% oil) ● For 2% oil = 150L/ha
Further testing ● As concentration of conidia increases there is at tendency for clumping using Codacide- this needs to be investigated further i.e. does this occur with higher concentrations of Codacide? ● (shown: 0.5g conidia; 1.1ml codacide; 23ml water)
Silwet and Break thru ● Powders can be mixed on site to using wetters such as Break thru or Silwet L77 ● Trials using 0.5g conidia and 1% and 0.1% Silwet and Break thru were set up using a water control
● 0.1% Silwet L77 and Break thru were able to suspend conidia, however a lot of mixing was required to suspend the powder ● Look at increasing wettability of powders using various substances ● Ensure viability is not affected
Experiment 1: Viability study of conidia in pastes • 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
Formulations kept at 5°C
Formulations kept at 25°C
Formulation Effects of co-formulants on viability of conidia ● 1 st batch of conidia (129/10) did not maintain viability at 25 ° C Conidia at 5 ° C retained viability ● ● Silwet L77 and Output were not suitable for long term storage in paste Break-thru retained promising viability at 5 ° C ● ● Second batch of conidia tested with Addit and Codacide (131/11) (other formulants discounted for other reasons)
Codacide and Addit viability study
Formulation Effects of co-formulants on viability of conidia ● Germination appeared to decrease after 5 months of storage at 25 ° C However, spores did not look dead when ● germination counts were done at 24h Therefore 46h counts carried out ● Conidia germinating slowly after 24h at 25 ° C. They had been formulated in Addit for 1 month
Codacide and Addit viability study (46h germination count)
24h and 46h viability after storage for 5 months (tested on standard agar)
Do spores germinate more readily in presence of insect nutrients?
Do spores germinate more readily in presence of insect nutrients?
Formulations Anti-compaction of conidia in pastes Compaction of conidia in pastes is currently ● being investigated By adding clays such as Bentone, ● compaction of conidia may be avoided Two types of Bentone have been tested for ● Addit, Codacide and Mineral Oil Construction of cracks and crevices
Formulations Anti-compaction of conidia in pastes Construction of cracks and crevices
Formulations Anti-compaction of conidia in pastes
Formulations Anti-compaction of conidia in pastes
Field trials Viability of spores in cracks and crevices Longevity of conidia in grainstores including ● crevices need to be investigated Work package 3:2 states that “CABI will ● examine the persistence of Bb in realistic conditions (grainstore humidity, surfaces and artificial crevices) in the laboratory to see how long the conidia survive and remain infective Artificial crevices were prepared to ● investigate conidial longevity in the Construction of cracks and grainstore in artificial crevices compared to crevices open surfaces
Viability of spores in cracks and Field trials crevices Concrete ready mix was prepared and ● poured into petri dishes (55mm) and a metal ‘spacer’ was added. This was removed leaving a concrete ● ‘crevice’ in which spore viability could be assessed Metal crevices also prepared ● Conidia were applied to filter paper sections ● using a paint brush Filter paper was applied to crevices and a ● control paper left in the open in the grainstore (in petri dish) Construction of cracks and Viability assessed on Day 1, 14 and 28 in ● crevices line with field trial data collection points
Viability of spores in cracks and Field trials crevices (results so far) Construction of cracks and crevices
Application Application equipment currently used in UK grainstores (Garthwaite et al., 2008)
Application Viability post spraying Formulations were prepared and sprayed ● onto microscope slides Viability was assessed after 1, 3, 7 and 10 ● days Formulations were Codacide and Shellsol T ● as a control (shown not to affect viability of conidia of IMI 389521) Results to be repeated- spray delivery was ● not optimal for day 1 (Codacide and Shellsol T) and day 10 (Shellsol T)
Application Viability post spraying *too few to count
Application Future work Need to work on a tank mix for application ● with knapsack/lance sprayer Further work on ● Viability of conidia (post spraying) ● Adherence and coverage of surfaces ● Do ULV formulations have a place? ●
Adherence and transfer Do spores transfer onto grain post of spores spraying? Experiment set up assessing different ● formulations: Addit, Shellsol T and conidia powder Surfaces (metal, concrete and wood) were ● sprayed /conidia applied These were placed face down on sterilised ● grain Grain was washed with sterilised Tween and ● CFU counts prepared This was done after day 1 and day 7 ● Day 1, CFU counts were too high to count; ● therefore on day 7 a -2 dilution was made
Adherence and transfer Do spores transfer onto grain post of spores spraying?
Adherence and transfer Do spores transfer onto grain post of spores spraying? Preliminary results showed: ● Unformulated conidia have highest ● transference Shellsol T has the next highest transfer ● (although only significant on concrete in this experiment) Addit had lowest transfer ● Further analysis to be carried out on this data ● to allow for spray volumes etc
Adherence and transfer If spores transfer onto grain will they of spores grow? Beauveria bassiana will grow over sterile ● grain that has been soaked in water; however it will not grow over non-sterile grain treated the same way It will not grow on dry grain ● Implications are that it is unlikely to compete ● with other opportunistic microbes if it contaminates the grain; thus will not grow/ produce more conidia unless on the insect hosts
Temperature Growth of Beauveria bassiana at assays grain store temperatures
Temperature Growth of Beauveria bassiana at high assays temperatures
Temperature Radial growth of Beauveria bassiana assays at high temperatures
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