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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

2012 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:

Pathogenicity assays on 50 isolates has been conducted. The results indicate the existence of variation even within just nine isolates. Among the nine isolates, two had a score of 6 or above on 11 of the 12 differentials indicating that there are aggressive isolates that potentially attack many cultivars. Preliminary molecular marker analysis was performed on selected isolates collected from eleven different states and two other countries (Brazil and China). Although, more than 100 polymorphic loci were identified among these isolates, a select group of 33 loci were used to conduct a preliminary evaluation of genetic relatedness among the isolates. The results indicate that the molecular markers were capable of distinguishing among isolates. Several soybean accessions were evaluated and accession specific to resistance haplotype of cultivar Davis. In addition, several other soybean accessions predicted not to have the resistance allele and with no frog eye leaf symptoms in field trials have also been identified. Based on the results of the pathogenicity tests, parental lines have been identified that contain different genetic responses. It is anticipated that these lines will be crossed this summer (2012) to begin the process of creating segregating populations to be used in elucidating the genetic control.


Last Modified: 4/23/2014
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