Initiation of Cercospera sojina Sexual Reproduction (1)
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Transcript of Initiation of Cercospera sojina Sexual Reproduction (1)
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Shantal PaiWallace Carver InternUSDA ARSMay 31- August 19, 2016
Initiation of Cercospera sojina Sexual Reproduction
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•Frogeye Leaf Spot Disease▫Losses of up to 60%
•Resistance and Virulence Genes▫Strobulin resistance is a growing
problem for control of Frogeye Leaf Spot Disease
•Spread of Fungicide Resistance▫Understanding sexual mating makes
it possible to predict how fungicide resistance will spread
Importance of Cercospera sojina
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•Sexual vs. Asexual Reproduction▫Sexual: two parents come together to produce genetically unique offspring▫Asexual: one parent produces identical offspring (except for random
mutations)•Frogeye Leaf Spot sexual stage has never been observed•University of Arkansas study found sexual reproduction likely exists▫Mating Type Ratio
Goal: Initiate Sexual Reproduction
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Media Type
• Stem and Pod media
• V8
Growth Environment
• Stress (?)• Ethylene
Parent Isolate
• MAT 1 x MAT 2• Fungicide
Resistant x Susceptible
Time
• 4 weeks• 6 weeks• 8 weeks• More?
Manipulatable Variables
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Method: Mimic Field Conditions
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• Isolate mating type was identified using genetic analysis• Isolates of opposite mating type were crossed▫Stem and Pod Media▫V8 Media ▫Ethylene Treated
•Crosses were grown to overlap, then overlaid with water agar and placed at 16 ? Under dark light (?) in a cooler, darker incubator▫“Make the fungi think they’re the last two on earth and the world is ending,
they must mate to stand a chance” – Shantal▫Actually: Make them “think” it is winter
Method
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Sexual Cross
• Isolates of MAT 1 were crossed with MAT 2 on media and placed in an environment to stimulate growth
Overlay
• Sexual crosses were overlain with 2% water agar to create a stressed environment
Fall Simulation
• Overlain sexual crosses were placed in an incubator under black light at 16 C to mimic conditions at the end of a growing season
Method
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•Variable type: Media (Host) •Purpose: To mimic field conditions by using a media that emulates in-
field host. •Method:
Stem and Pod
MAT 1 MAT 2TN 88 TN 20TN 160 TN 22TN 161 TN 65TN 183
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•Variable type: Media Concentration (Host, Environment)•Purpose: To determine what concentration of V8 media is best for C.
sojina.•Method:
V8 Media Concentration
MAT 1 MAT 2TN 161 TN 22TN 183 TN 65
Media Concentration5% V810% V815% V820% V8
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•Variable type: Pathogen•Purpose: To determine which pairs of isolates generate sexual bodies
more effectively. •Method:
Isolate Effect
MAT 1 MAT 2TN 124 TN 35*
TN 51 TN 63*TN 39 TN 170*TN 89 TN 32TN 160 TN 84TN 410 TN 210 TN 213Isolates marked with a * were also crossed with TN 216 (MAT 2)
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•Variable type: Pathogen•Purpose: •Method:
Fungicide Resistant x Susceptible
Mating Type Resistant SusceptibleTN 39 (MAT 1) TN 399 TN 183TN 210 (MAT 2) TN 204 TN 208 TN 42
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•Variable type: Environment•Purpose: To mimic senescence with phytochemical environment•Method:
Ethylene Effect
MAT1 MAT 2161 22183 65
Treatments: 0, 1, 2, or 3 Fruits
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Results: Stem and Pod
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Results: V8 Concentration161 183
2 2
6 5
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Results: Isolate TestMAT 2x MAT 2
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Results: Fungicide Resistant x Susceptible
The remaining samples are currently in the incubator and will be viewed this Wednesday.
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0 Plantains 1 Plantain 2 Plantains 3 Plantains
Contaminated 3 7 6 9
Uncontaminated 13 9 10 7
Percent Contamination
18% 43% 40% 43%
Results: Ethylene
Rate of Contamination when Plantains Used to Introduce Ethylene gas
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•Once sexual mating is confirmed, we can use the results of these studies to determine the ideal conditions for sexual mating
• If Ethylene gas successfully introduces sexual mating, it will be necessary to repeat the procedure with compressed gas
What next?
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Questions?