Fungal Bio-Control agents in
Agriculture
Dr Diana Leemon
Agri-Science Qld
Fungi are unique
ANIMALS
FUNGI
PROTISTS – unicellular
eukaryotes
MONERA – unicellular
prokaryotes
PLANTS
Fungi are unique
 Multicellular heterotrophs with cell
walls that digest food outside their
bodies
Plasticity
Morphological
Reproductive
Functional
Morphological Plasticity
Pseudo hyphae
Tubular growth – hyphae
(mycelium)
Single cells – yeast
like growth
Reproductive plasticity
• Asexual reproduction
(anamorphic/mitosporic
state)
• Sexual reproduction
(teleomorphic/meiosporic
state)
Functional plasticity
PATHOGENS
= help maintain a dynamic
equilibrium through population
control
SAPROTROPHS
(decomposers)
MUTUALISTS
(Mycorrhizal fungi, endophytes,
lichens)
Plant Pathogens
Fungal Pathogens
Animal Pathogens
Disturbed Ecosystems
Dynamic equilibrium disturbed = rise in pest species of
plants, animals & fungi
Introduction of foreign species to natural
ecosystems can create invasive species
Agricultural systems are simple unbalanced
ecosystems
Pest Problems? – NO Worries!
Cattle tick infestation
The Cost of the Chemical Century
•
•
•
•
•
Resistance
Residues
Beneficial organisms wiped out
Environmental and OHS concerns
Consumer pressure
Look back to natural ecosystems
for a Solution
Fungal Bio-control (giving a pathogen a makeover)
• Inoculative (Classical) – pathogen ex native ecosystem of
target problem introduced
– Best funded by public bodies
• Competitive Inhibition – benign strain of pathogen
selected, mass produced and applied to the environment of
the target problem
• Inundative – pathogen, selected (often local), mass
produced, formulated & applied to target problem
– Myco-insecticdes, Biopesticides, Myco-herbicides
– Potential for commercial gain
Invertebrate Pest Bio-control
• Mostly Inoculative
• Number of products registered overseas
• Most commonly used: Metarhzium & Beauveria
• Main success story in Australia: Green Guard for
locust control
Entomopathogenic Fungi
Entomophothorales (Zygomycetes)
– Cause effective epizootics
– Obligate pathogens
– Difficult to produce
Entomopathogenic Fungi
• Teleomorphic/sexual states of Hypocreales (Ascomycota)
•
Fantastic looking but not suitable for biocontrol
Entomopathogenic Fungi
Anamorphic/asexual state of Hypocreales: Beauveria bassiana
(white muscardine disease)
Entomopathogenic Fungi
Anamorphic state of Hypocreales: Metarhizium anisopliae
(green muscardine disease)
Characteristics for Innundative Biocontrol agent
• Comparatively narrow host range
• Stable
• Compatible growth characteristics for target
environment (temperature, moisture)
• Easy to produce
• Easy to Store
• Easy to formulate and apply
Mode of Action
• Spores germinate on insect
cuticle
• Invade an growth through the
insect haemocoel
• Insect death from physical
damage and toxin production
(sometimes)
• Insect body can be totally
consumed by fungus
Spores germinating on tick surface
Constraints on Success
Successful development on Fungal Bio-control
agents multidisciplinary
• Mycology
• Pathology
• Ecology
• Genetics
• Host Biology & Physiology
• Mass production
• Formulation
• Application strategies
• Ecology not so important for the development of
traditional chemical control agents
•
Ecology of Bio-control
FUNGAL PATHOGEN
• Life stage/Age
• Nutrition
• Population Density
• Genetics
TARGET HOST
• Stability
• Virulence
• Viability
• Specificity
TIME
•Temperature
• Humidity
• Solar radiation
• Substrate
ENVIRONMENT
Tick control- promising idea but field
conditions for fungus too narrow to
be commercial –
Sheep lice control – potential due to
ecology of microclimate potential
But…. road to registration hampered
long, expensive and expertise limited
House fly (Musca domestica) control in cattle
feedlots
THE PROBLEM – Effective economic
control
• Chemical Control
– Limited efficacy
– Residues – issue for export market
meat
– Resistance
• Integrated Pest Management preferred
– Manure Management
– Additional tools required
Myco-insecticide for house fly control
Flies highly susceptible to Metarhizium infection
Two Research Phases:
Phase I: 2004 - 2008 (proof of concept)
Phase II: 2013 - 2016 (develop & test
commercial formulation
Phase I
 Isolate selection
• Temperature
• Spore production stability
• Virulence
 Dose studies
 Understand pathogenesis
Phase I
Observation of fly behaviour
• Flies rest in the shade of vegetation for
much of the day & all night in “territorial”
areas
• Spray formulation in the late afternoon
onto vegetation where flies rest
– Protection from heat and UV
– Sugar and molasses added to
formulation to increase uptake when
flies return to the vegetation
Phase I Trial Method
Targeted spraying of vegetation in feedlots
over two seasons
– Season 1, Brisbane Valley
• Treated ~ 3,000 head
• Control ~ 1,000 head
– Season 2, Brisbane Valley and
Warwick Shire
• Treated ~ 3,000 head & 1,000 head
• Control ~1,000 head & 2,000 head
Phase I Trial Method
• Flies netted in control feedlots and
before and after spraying in treated
feedlots
• Flies transported back to lab for
incubation
• Fly mortality assessed after 7 days
• Isolations from dead flies to confirm
Metarhzium infection
• Alsynite Traps used to monitor fly
numbers
Number of flies trapped in the
treated (A) and Untreated (C)
feedlots over four seasons,
treatment only occurred
during the last two seasons
Average percent mortality in netted flies and percent
flies infected with Metarhizium in Brisbane Valley
feedlots during 2006-2007 and 2007-2008
Values with different superscripts are significantly different (P<0.05
Phase I confirmed the potential for a Metarhizium
based myco-insecticide for fly control
But formulation needed improvement to be
commercial (spore concentration, application,
formulation volume)
A better measure of efficacy against flies also
needed
Phase II
Focus on development and evaluation of
economic myco-insecticide:
• Optimise spore production
• Formulation with decreased spore
concentration and volume
• Large scale application method
• Better fly monitoring methods
• Track fungal deposition, viability and effect
on flies
Phase II
• Lab studies to develop formulations
– Very Low Volume (VLV) oil/water emulsions
• 5 – 50 L/Ha
– Ultra Low Volume (ULV) oil
• ≤ 5 L/Ha
– Bait (grain based +/- additives)
Phase II
• Formulation testing
– Cage trials – semi controlled
field
– Pilot field trial
– Full field trial across 4
commercial feedlots
Full Field Trial
ULV formulation
• Spores suspended in canola oil:ShellsolT
• 55 g spores/L forumulation
• Applied with vehicle mounted Micronair AU8000
ULV motorised sprayer
• ~1 L/Ha (2.2 × 1012 spores/Ha)
Monitoring of fly populations
Visual assessments
Alsynite traps
Sticky sheets
Spot cards
Fungal Investigations
 Flies netted and incubated
• Fly mortality and Metarhizium
infection assessed
 Vegetation sampled
• Metarhizium colonies assessed to
estimate spore deposition and
spore viability over time
Overall fly index (combination of 4 monitoring methods)
showed significant decrease in flies in treated feedlots with
effect increasing with season
Season 1
Feedlot A
Feedlot B
Feedlot C
Feedlot D
Season 2
Feedlot A
Feedlot B
Feedlot C
Feedlot D
Season 3
Feedlot A
Feedlot B
Feedlot C
Feedlot D
All seasons
Feedlot A
Feedlot B
Feedlot C
Feedlot D
Fly Index
Visual scores
log10 (spot)
log10 (sticky)
log10 (alsynite)
0.84ab
0.82a
0.94bc
1.02c
0.81a
0.78a
1.29b
1.60c
0.16a
0.19a
0.22a
0.37b
0.13a
0.14a
0.34b
0.28b
2.38b
2.14ab
1.90a
1.88a
1.12ab
1.24b
0.97a
1.13ab
1.58ab
1.99b
1.38a
1.98b
0.52ab
0.62b
0.45a
0.58b
0.29ab
0.26a
0.38b
0.31ab
1.84b
1.88b
1.57a
1.47a
1.19a
1.36b
1.50c
1.58c
1.82a
2.20ab
2.38bc
2.52c
0.54a
0.65ab
0.69b
0.87c
0.26a
0.28a
0.49b
0.41b
1.06a
1.14ab
1.16b
1.25c
1.38a
1.59b
1.68b
2.00c
0.42a
0.49b
0.47ab
0.61c
0.23a
0.24a
0.40c
0.34b
Spore
Deposition
(Log10
spores)
Total
Infection
(%)
Infection in
Dead Flies
(%)
Infection in
Live Flies
(%)
Fly Mortality
(%)
<0.001
<0.001
<0.001
<0.001
<0.001
Control
1.3a ± 0.12
0.7a ± 0.15
1.9a ± 0.48
0.0a ± 0.00
16.0a ± 1.00
Post Spray
4.6c
41.9b ±
1.00
73.4b ± 1.33
3.5b ± 0.56
43.7b ± 2.17
1 Week
Post Spray
3.4c ± 0.23
1.1a ± 0.43
4.8a ± 2.01
0.0a ± 0.02
21.0a ± 4.49
2 Weeks
Post Spray
2.2b ± 0.16
0.3a ± 0.15
1.00a ± 0.54
0.0a ± 0.00
17.2a ± 1.74
3 Weeks
Post Spray
2.0ab ± 0.39
0.9a ± 0.63
3.6a ± 2.77
0.0a ± 0.00
16.4a ± 4.52
Treatment
Means
F
probability
± 0.15
N.B. Means within columns with the same superscript are not significantly different at the P=0.05 level.
Fungal Bio-control in Agriculture the future
• Use of fungal pathogens - simple principle,
but the practical application is often difficult
• Some good success stories but potential for
more
• Imperative for safe & sustainable pest control
will increase
• Requires political and legislative support
• Requires committed research with a range of
expertise
Acknowledgments:
• Meat and Livestock Australia
• Steven Rice, David Mayer
• Peter James, Dalton Baker, Rosie
Godwin
• Rudolf Urech, Jerry Hogsette,
Peter Green
• Gary Everingham, Jacinta
McMahon