Kyle SternKyle SternAmanda Romag, Dr. Harsh Bias, Dr. Nicole Donofrio, Dr. John PeleskoAmanda Romag, Dr. Harsh Bias, Dr. Nicole Donofrio, Dr. John Pelesko

An experimental and mathematical An experimental and mathematical study of study of M. oryzae M. oryzae spore germinationspore germinationand dispersal in the presence of host and dispersal in the presence of host

and non-host volatilesand non-host volatiles

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• 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:

Solution:

SporesVolatile

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

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

• Viewed under dissecting microscope

The Dispersal & Sedimentation Assay

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 ResultsRose Plot

N = 100

N = 45N = 27 Farnesyl Acetate Limonene

Random

M. oryzae M. oryzae

Germ Tube ResultsRose Plot

N = 100000N = 1000

Dispersal & Sedimentation Results

The Volume of an M. oryzae Spore

- 30 spores measured using ocular micrometer

Mean length: 26.2 μmStandard deviation: 3.585 μm

Mean width: 11.233 μmStandard deviation: 1.612 μm

Dispersal & Sedimentation Results

The Volume of an M. oryzae Spore

- Is a spore ellipsoidal or something else?

Spore.mw

Dispersal & Sedimentation Results

The Volume of an M. oryzae Spore

Dispersal & Sedimentation ResultsThe Volume of an M. oryzae Spore

Let w = hV = (πlwh)/6 = 1730.98 μm3

Dispersal & Sedimentation ResultsThe Mass of an M. oryzae Spore

m = ρVm = ρV

Let ρ = 1000 kg/mLet ρ = 1000 kg/m33, the density of water, the density of waterm = 1000 * 1.731 x 10m = 1000 * 1.731 x 10-15-15 kg kg

m = 1.731 x 10m = 1.731 x 10-12-12 kg kg

Dispersal & Sedimentation ResultsThe mechanics of spore dispersal

Solution:

a = radius of the spore,μ = absolute viscosity of air at room temperature,K = shape factor of the ellipsoid given by:

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

Velocity of a spore in freefall:

Dispersal & Sedimentation ResultsThe mechanics of spore dispersal

Dispersal & Sedimentation ResultsDistribution of Dispersing Spores

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 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 ResultsRandom Walk of a Spore

• The distributions are almost identical.

Stick, N=10000 Bounce, N=10000

Simulated Distance Simulated Distance

Freq

uenc

y

Freq

uenc

y

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.

AcknowledgmentsThanks:

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…

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