Presentation on theme: "Amanda Romag, Dr. Harsh Bias, Dr. Nicole Donofrio, Dr. John Pelesko"— Presentation transcript:
1Amanda 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 volatilesKyle SternAmanda Romag, Dr. Harsh Bias, Dr. Nicole Donofrio, Dr. John Pelesko
2Magnaporthe oryzae Fungus is also known as “rice blast” disease Thought to be a potential bio-terrorism weapon during the mid-twentieth centuryKills enough rice per year to feed over 60 million people worldwideAlso infects barley and wheat crops
3The destructive process Spore lands on a leaf via dispersal through the airSpore sticks to the leaf with sticky substance on surface of its bodyGermination begins:MoistureHard surfaceDarkRoom temperature
4The destructive process Spore begins to pump fluids from its body into the end of the germ tubeCauses a swelling at the end of the germ tubeAppressorium developsPressure causes appressorium to swellPenetration peg infiltrates the plant leafFungus invades the plantNoticeable brownish-yellow lesions in the plant leavesPlant dies
7Volatile Compounds Emitted from a plant in gas form Farnesyl acetate (C17H28O2 ), a volatile of broad bean, inhibits spread of bean rust fungusLimonene (C10H16) – volatile of riceOther volatiles?Gas chromatography/ mass spectrometryNone found yetLimonene:
8The 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 assayAre 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?
9The Germ Tube Assay Volatile incorporated into water agar Spore suspension created using sporulating colonySpore suspension dropped on empty plate of plain water agarStrip of volatile in water agar cut out and placed in plate containing spore suspension
10The Germ Tube AssayPlate sealed and placed in dark drawer for 24 hoursViewed at 6.3x magnification under dissecting microscope
13Concentration 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:SporesVolatileSolution:
14The Dispersal & Sedimentation Assay Empty Petri dish prepared with two sterile glass slidesV8 agar cut in half through the diameter and placed directly on top of glass slidesSide of V8 agar perpendicular to bottom of dish swabbed with sporulating M. oryzaeVolatile placed in non-control plates
15The Dispersal & Sedimentation Assay Plate left unsealed and placed in fungal growth chamber for eight to ten daysViewed under dissecting microscopeM. oryzae
21Dispersal & Sedimentation Results The Volume of an M. oryzae Spore- 30 spores measured using ocular micrometerMean length: μmStandard deviation: μmMean width: μmStandard deviation: μm
22Dispersal & Sedimentation Results The Volume of an M. oryzae Spore- Is a spore ellipsoidal or something else?
23Dispersal & Sedimentation Results The Volume of an M. oryzae Spore
24Dispersal & Sedimentation Results The Volume of an M. oryzae SporeLet w = hV = (πlwh)/6 = μm3
25Dispersal & Sedimentation Results The Mass of an M. oryzae Sporem = ρVLet ρ = 1000 kg/m3, the density of waterm = 1000 * x kgm = x kg
26Dispersal & 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:
27Dispersal & 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
28Dispersal & Sedimentation Results Distribution of Dispersing Spores
29Dispersal & Sedimentation Results Distribution of Dispersing Spores ControlN = 1340Mean:Std. Dev.:F. AcetateN = 68Mean:Std. Dev.:Limonene N = 289Mean:Std. Dev.:
30Dispersal & Sedimentation Results Random Walk of a Spore A spore that does not avoid the block of agar will hit it and eitherstick to itbounce off of it
31Dispersal & Sedimentation Results Random Walk of a Spore The distributions are almost identical.Stick, N=10000Bounce, N=10000FrequencyFrequencySimulated DistanceSimulated Distance
32Conclusions 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.
33Future Work GC-MS testing on rice, lima bean, and barley plants Determine the diffusion coefficients of the volatilesDetermine the underlying force causing spores to disperse
35The Dispersal & Sedimentation Assay Optimize spore dispersal assay so that healthy leaves can be placed in the dish with the fungus
36References1 Trail, F., Gaffoor, I., Vogel, S “Ejection mechanics and trajectory of the ascospores of Gibberella zeae”. Fungal 42,2 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 “Volatiles modulate the development of plant pathogenic rust fungi”. Planta 224,
37Acknowledgments Dr. Harsh Bais Dr. Nicole Donofrio Dr. John Pelesko Thanks:Howard Hughes Medical InstituteUniversity of Delaware Undergraduate Research ProgramUniversity of Delaware Department of Mathematical SciencesUniversity of Delaware Department of Plant and Soil SciencesDr. Harsh BaisDr. Nicole DonofrioDr. John PeleskoAnd…
38AcknowledgmentsMy awesome lab partner, Mandy, who had to put up with me.