Transcript An experimental and mathematical study of M. oryzae spore

An experimental and mathematical
study of M. oryzae spore germination
and dispersal in the presence of host
and non-host volatiles
Kyle Stern
Amanda Romag, Dr. Harsh Bias, Dr. Nicole Donofrio, Dr. John Pelesko
Magnaporthe oryzae
• Fungus is also known as “rice blast” disease
• Thought to be a potential bio-terrorism weapon
during the mid-twentieth century
• Kills enough rice per year to feed over 60 million
people worldwide
• Also infects barley and wheat crops
The destructive process
• Spore lands on a leaf via dispersal through the air
• Spore sticks to the leaf with sticky substance on
surface of its body
• Germination begins:
• Moisture
• Hard surface
• Dark
• Room temperature
The destructive process
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Spore begins to pump fluids from its body into the end of
the germ tube
Causes a swelling at the end of the germ tube
Appressorium develops
Pressure causes appressorium to swell
Penetration peg infiltrates the plant leaf
Fungus invades the plant
Noticeable brownish-yellow lesions in the plant leaves
Plant dies
Normal barley leaf
After the infection
Volatile Compounds
• Emitted from a plant in gas form
• Farnesyl acetate (C17H28O2 ), a volatile of broad
bean, inhibits spread of bean rust fungus
• Limonene (C10H16) – volatile of rice
• Other volatiles?
– Gas chromatography/ mass spectrometry
– None found yet
Limonene:
The Two Assays
• Germ tube assay
– Do volatile compounds assist in M. oryzae germ tube
growth?
– Do germ tubes grow in specific directions?
• Spore dispersal/sedimentation assay
– Are spores actively or passively released from their
stalks?
– Do volatile compounds assist in M. oryzae spore
dispersal?
– At what velocity and acceleration are spores
released?
– Is there a particular force causing the release?
The Germ Tube Assay
• Volatile incorporated into water agar
• Spore suspension created using
sporulating colony
• Spore suspension dropped on empty plate
of plain water agar
• Strip of volatile in water agar cut out and
placed in plate containing spore
suspension
The Germ Tube Assay
• Plate sealed and placed in dark drawer for
24 hours
• Viewed at 6.3x magnification under
dissecting microscope
The Germ Tube Assay
The Germ Tube Assay
Concentration Gradient
• Volatiles must diffuse into the agar where the spores
are germinating.
• The concentration gradient of a compound in water
agar, C(x,t), is found via the following partial
differential equation:
Spores
Volatile
Solution:
The Dispersal & Sedimentation Assay
• Empty Petri dish prepared with two sterile glass
slides
• V8 agar cut in half through the diameter and
placed directly on top of glass slides
• Side of V8 agar perpendicular to bottom of dish
swabbed with sporulating M. oryzae
• Volatile placed in non-control plates
The Dispersal & Sedimentation Assay
• Plate left unsealed and placed in fungal
growth chamber for eight to ten days
• Viewed under dissecting microscope
M. oryzae
The Dispersal & Sedimentation Assay
The Dispersal & Sedimentation Assay
Germ Tube Results
• Initial results show that germ tube growth
direction is random
Germ Tube Results
Rose Plot
Random
N = 100
N = 27
M. oryzae
M. oryzae
Farnesyl
Acetate
Limonene
N = 45
Germ Tube Results
Rose Plot
N = 1000
N = 100000
Dispersal & Sedimentation Results
The Volume of an M. oryzae Spore
- 30 spores measured using ocular micrometer
Mean length: 26.2 μm
Standard deviation: 3.585 μm
Mean width: 11.233 μm
Standard deviation: 1.612 μm
Dispersal & Sedimentation Results
The Volume of an M. oryzae Spore
- Is a spore ellipsoidal or something else?
Dispersal & Sedimentation Results
The Volume of an M. oryzae Spore
Dispersal & Sedimentation Results
The Volume of an M. oryzae Spore
Let w = h
V = (πlwh)/6 = 1730.98 μm3
Dispersal & Sedimentation Results
The Mass of an M. oryzae Spore
m = ρV
Let ρ = 1000 kg/m3, the density of water
m = 1000 * 1.731 x 10-15 kg
m = 1.731 x 10-12 kg
Dispersal & Sedimentation Results
The mechanics of spore dispersal
a = radius of the spore,
μ = absolute viscosity of air at room temperature,
K = shape factor of the ellipsoid given by:
Solution:
Dispersal & Sedimentation Results
The mechanics of spore dispersal
Velocity of a spore in freefall:
Time it takes a free-falling spore to reach the ground:
between 70 and 110 seconds.
Terminal vertical velocity:
between 56.96μm/s and 90.86μm/s downward
Dispersal & Sedimentation Results
Distribution of Dispersing Spores
Dispersal & Sedimentation Results
Distribution of Dispersing Spores
Control
N = 1340
Mean: 510.8527
Std. Dev.: 334.2456
F. Acetate
N = 68
Mean: 556.6809
Std. Dev.: 398.3656
Limonene
N = 289
Mean: 823.1248
Std. Dev.: 397.2171
Dispersal & Sedimentation Results
Random Walk of a Spore
• A spore that does not avoid the block of agar will hit it
and either
– stick to it
– bounce off of it
Dispersal & Sedimentation Results
Random Walk of a Spore
• The distributions are almost identical.
Bounce, N=10000
Frequency
Frequency
Stick, N=10000
Simulated Distance
Simulated Distance
Conclusions
• Spores are actively released.
• Some force is pushing them from their
stalks.
• The presence of limonene is assisting in
the dispersal process.
• Germ tubes grow in random directions
regardless of any volatiles present in the
assay.
Future Work
• GC-MS testing on rice, lima bean, and
barley plants
• Determine the diffusion coefficients of the
volatiles
• Determine the underlying force causing
spores to disperse
Future Work
• Direct extraction of volatiles
The Dispersal & Sedimentation Assay
• Optimize
spore dispersal assay so that healthy leaves can be
placed in the dish with the fungus
References
• 1 Trail, F., Gaffoor, I., Vogel, S. 2005. “Ejection mechanics and
trajectory of the ascospores of Gibberella zeae”. Fungal 42, 528-533.
• 2 Clarkson University. “Drag Force and Drag Coefficient”.
<http://people.clarkson.edu/~rayb/aerosol/hydrodynamic/hydro4.htm>
.
• 3 Mendgen, K., Wirsel, S., Jux, A., Hoffmann, J., Boland, W. 2006.
“Volatiles modulate the development of plant pathogenic rust fungi”.
Planta 224, 1353-1361.
Acknowledgments
Thanks:
Howard Hughes Medical Institute
University of Delaware Undergraduate Research Program
University of Delaware Department of Mathematical Sciences
University of Delaware Department of Plant and Soil Sciences
Dr. Harsh Bais
Dr. Nicole Donofrio
Dr. John Pelesko
And…
Acknowledgments
My awesome lab partner, Mandy, who had to put up with me.