Bio-control of grainstore pests TSB quarterly meeting Bryony Taylor, - - PowerPoint PPT Presentation

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KNOWLEDGE FOR LIFE

Bio-control of grainstore pests

TSB quarterly meeting

Bryony Taylor, Marine Guerret, Rajpreet Grewal, Belinda Luke, Dave Moore Analysis and compilation by Bryony Taylor

Overview of CABI’s

• bjectives

Viability of spores

• 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

• bjectives
• 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

Application

Overview of CABI’s

• bjectives
• 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

Persistence and uptake from surfaces

Overview

• 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?

Work so far to be presented today

Formulation

• 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

Formulation issues

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 109 conidia is 0.3ml

• For 1 sqm to apply 5 x 109 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

• r 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

• 1st 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)

Effects of co-formulants on viability of conidia

Codacide and Addit viability study

Conidia germinating slowly after 24h at 25°C. They had been formulated in Addit for 1 month

Formulation

• 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

Effects of co-formulants on viability of conidia

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?

Construction of cracks and crevices

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

Formulations

Anti-compaction of conidia in pastes

Formulations

Anti-compaction of conidia in pastes

Construction of cracks and crevices

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 grainstore in artificial crevices compared to

• pen surfaces

Construction of cracks and crevices

Field trials

• 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)

• Viability assessed on Day 1, 14 and 28 in

line with field trial data collection points

Viability of spores in cracks and crevices

Construction of cracks and crevices

Field trials

Viability of spores in cracks and crevices (results so far)

Application

Application equipment currently used in UK grainstores (Garthwaite et al., 2008)

Application

• Formulations were prepared and sprayed
• nto 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)

Viability post spraying

Application

Viability post spraying

*too few to count

Application

• 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?

Future work

Adherence and transfer

• f spores
• 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

Do spores transfer onto grain post spraying?

Adherence and transfer

• f spores

Do spores transfer onto grain post spraying?

Adherence and transfer

• f spores
• 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

Do spores transfer onto grain post spraying?

Adherence and transfer

• f spores
• 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

If spores transfer onto grain will they grow?

Temperature assays

Growth of Beauveria bassiana at grain store temperatures

Temperature assays

Growth of Beauveria bassiana at high temperatures

Temperature assays

Radial growth of Beauveria bassiana at high temperatures

And finally…

• IMI 389521 has been sequenced (ITS

region)

• Fingerprinting work to commence in next few

weeks

• Isolate to be compared to commercial

Beauveria bassiana

• Thank you to our students Rajpreet Grewal

and Marine Guerret for their work on this project

Other work

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KNOWLEDGE FOR LIFE

Thank You