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United States Department of Agriculture

Agricultural Research Service

Research Project: Characterization of Frog Eye Leaf Spot Isolates Using Molecular and Host Pathotype

Location: Crop Genetics Research Unit

2013 Annual Report


1a.Objectives (from AD-416):
The objectives of this research project are to (1) characterize the pathogenicity of more than 100 C. sojina isolates. (2) develop SSR molecular markers specifically for C. sojina, (3) employ the SSR markers to molecularly differentiate C. sojina isolates. .
4)identify race-specific sources of resistance to C. sojina for use in the breeding program. Objective 4 is a long-term objective, whereas the objectives of pathologically and molecularly characterizing C. sojina isolates are a critical first steps in developing race specific resistance.


1b.Approach (from AD-416):
Isolates from Northern and Southern US will be collected and used to inoculate a set of twelve soybean differentials to detect the potential variation within C. sojina. These differentials will include soybean varieties possessing the Rcs1, Rcs2, and Rcs3 resistance genes and soybean differentials cited in other studies as well as those listed in the “Compendium of Soybean Disease”. These differentials are Davis, Peking, Kent, CNS, Palmetto, Tracy, Hood, Lincoln, Lee, S-100, Blackhawk and Richland. Plants will be grown in a growth chamber, and inoculation will be performed when plants are 10 days old. Pure cultures will be derived from spores generated from a single spore. A spore concentration of 1 x 105 ml-1 will be sprayed over the entire plant until runoff occurs. The plants will be incubated in a dew chamber for 12 hr and transferred to a growth chamber. The temperature will be maintained at 28 'C with a 14 hr photoperiod. Ten days post inoculation, data on the severity of symptoms produced will be taken based on a standardized rating system of 0-9 scale, where 0= immune, and 9= very susceptible. Culture media will include lima bean and soybean pod and stem media to measure fungal morphology and sporulation to better develop and compare culture characteristics. C. sojina cultures will be grown in a liquid media for approximately three weeks. Cultures will then be freeze dried in a Model 2400 freeze dryer (The Freeze Dry Company, Nisswa, MN 56468). The freeze dried tissue will then be ground to a fine powder using a tissue pulverizer (Garcia Manufacturing, Visalia, CA 93292). DNA will be isolated from the pulverized tissue using a Maxwell 16™ automated DNA isolation machine (Promega, Madison, WI 53711) following the manufacturer’s protocols. SSR-enriched libraries will be generated following the detailed procedures we have described in detail elsewhere (Arias et al., 2011). Libraries will be sequenced in the MidSouth Genomics Center (Stoneville, MS) and specific SSR’s identified. SSR primers will be designed and synthesized by Integrated DNA Technologies (Coralville, IA 52241) with either a hexachlorofluorescein (HEX) or 6-carboxyfluorescein (FAM) 5’-fluorescent label. These SSR markers will then be applied to each C. sojina isolate DNA to determine molecular classification. Depending on the degree of polymorphisms identified with the SSR developed, we expect to apply 24 unique SSR markers on each of the approximately 100 C. sojina isolates.


3.Progress Report:

Frogeye leaf spot isolates were collected, cultured and DNA isolated from over 80 isolates from 11 states and three countries. The pathogenicity of these isolates on a set of soybean differentials was determined, and results indicated there were differences among isolates. A set of molecular markers were applied to the isolates, and the markers were able to both genetically identify individual isolates and cluster genetically related isolates. Additionally, it appears that some markers may be associated with pathogenicity groups. Lastly, parents with different responses to Cercospora sojina were crossed, F1 seed produced and that seed sent to Puerto Rico for seed increase. These populations are being evaluated in 2013. Overall, these efforts provide a solid foundation for continued research and breeding efforts as well as provide a basis for scientific publications.


Last Modified: 11/23/2014
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