An experimental and mathematical

choppedspleenMechanics

Feb 22, 2014 (3 years and 8 months ago)

101 views

Kyle Stern


Amanda Romag, Dr. Harsh Bias, Dr. Nicole Donofrio, Dr. John Pelesko

An experimental and mathematical
study of
M. oryzae
spore germination

and dispersal in the presence of host
and 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 (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 6.3x 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:

Spores

Volatile

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 Results

Rose Plot

N = 100

N = 45

N = 27

Farnesyl
Acetate

Limonene

Random

M. oryzae

M. oryzae

Germ Tube Results

Rose Plot

N = 100000

N = 1000

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

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 Results

The mechanics of spore dispersal

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.

Stick, N=10000

Bounce, N=10000

Simulated Distance

Simulated Distance

Frequency

Frequency

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.