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?