An experimental and mathematical study of M oryzae

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 • • 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 (C 17 H 28 O 2 ), a volatile of broad bean, inhibits spread of bean rust fungus • Limonene (C 10 H 16) – 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. 3 x 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 • V 8 agar cut in half through the diameter and placed directly on top of glass slides • Side of V 8 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 Farnesyl Acetate Limonene N = 45

Germ Tube Results Rose Plot 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 μm 3

Dispersal & Sedimentation Results The Mass of an M. oryzae Spore m = ρV Let ρ = 1000 kg/m 3, 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 Stick, N=10000 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/hydro 4. 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.