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Shantal PaiWallace Carver InternUSDA ARSMay 31- August 19, 2016
Initiation of Cercospera sojina Sexual Reproduction
•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
•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
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
Method: Mimic Field Conditions
• 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
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
•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
•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
•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)
•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
•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
Results: Stem and Pod
Results: V8 Concentration161 183
2 2
6 5
Results: Isolate TestMAT 2x MAT 2
Results: Fungicide Resistant x Susceptible
The remaining samples are currently in the incubator and will be viewed this Wednesday.
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
•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?
Questions?