An experimental and mathematical study of
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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

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An experimental and mathematical study of M. oryzae spore germinationand 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
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
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 process1
The destructive process

  • 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




Volatile compounds
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
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
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 assay1
The Germ Tube Assay

  • Plate sealed and placed in dark drawer for 24 hours

  • Viewed at 6.3x magnification under dissecting microscope




Concentration gradient
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
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 assay1
The Dispersal & Sedimentation Assay

  • Plate left unsealed and placed in fungal growth chamber for eight to ten days

  • Viewed under dissecting microscope

M. oryzae




Germ tube results
Germ Tube Results

  • Initial results show that germ tube growth direction is random


Germ tube results rose plot
Germ Tube ResultsRose Plot

Random

N = 100

M. oryzae

M. oryzae

Farnesyl Acetate

N = 27

Limonene

N = 45


Germ tube results rose plot1
Germ Tube ResultsRose Plot

N = 1000

N = 100000


Dispersal sedimentation results
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 results1
Dispersal & Sedimentation Results

The Volume of an M. oryzae Spore

- Is a spore ellipsoidal or something else?


Dispersal sedimentation results2
Dispersal & Sedimentation Results

The Volume of an M. oryzae Spore


Dispersal sedimentation results3
Dispersal & Sedimentation Results

The Volume of an M. oryzae Spore

Let w = h

V = (πlwh)/6 = 1730.98 μm3


Dispersal sedimentation results4
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
Dispersal & Sedimentation ResultsThe 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 dispersal1
Dispersal & Sedimentation ResultsThe 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 ResultsDistribution of Dispersing Spores


Dispersal sedimentation results distribution of dispersing spores1
Dispersal & Sedimentation ResultsDistribution of Dispersing Spores

Control

N = 1340

Mean: 510.8527

Std. Dev.: 334.2456

F. Acetate

N = 68

Mean: 556.6809

Std. Dev.: 398.3656

LimoneneN = 289

Mean: 823.1248

Std. Dev.: 397.2171


Dispersal sedimentation results random walk of a spore
Dispersal & Sedimentation ResultsRandom 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 spore1
Dispersal & Sedimentation ResultsRandom Walk of a Spore

  • The distributions are almost identical.

Stick, N=10000

Bounce, N=10000

Frequency

Frequency

Simulated Distance

Simulated Distance


Conclusions
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
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 work1
Future Work

  • Direct extraction of volatiles


The dispersal sedimentation assay4
The Dispersal & Sedimentation Assay

  • Optimize spore dispersal assay so that healthy leaves can be placed in the dish with the fungus


References
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
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…


Acknowledgments1
Acknowledgments

My awesome lab partner, Mandy, who had to put up with me.


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