Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) This research will continue with screening soybean accessions and germplasm for resistance to rust, monitoring gene expression in soybean lines exhibiting rust resistance, and in developing disease management and control strategies for U.S. growers. Approach (from AD-416) Adult plant resistance will be evaluated on accessions that were identified in seedling screens as having some resistance. Commercial cultivars identified as resistant in areas where the rust occurs evaluated in comparison to accessions from the germplasm bank. Populations with the mot promising sources of resistance will be developed. U.S. soybean cultivars evaluated for tolerance to soybean rust in areas where the pathogen occurs and compared to rust severity to determine the relationship of partial resistance and tolerance. Profile gene expression in soybean lines exhibiting resistance to soybean rust. Identify regulatory/coordinated interactions between resistant and susceptible reactions. Develop a network of state extension specialists to evaluate application methods that fit current U.S. agronomic and economic conditions. Use this information to develop recommendations for fungicide selection and usage for U.S. soybean growers. Source areas for over-wintering of the soybean rust pathogen will be further defined. The determination and availability of alternate host plants will be determined for source areas. Significant Activities that Support Special Target Populations This final report serves to document research conducted under a reimbursable agreement between ARS and the United Soybean Board, under 1920-22000-035-03R (formerly 1920-22000-027-19R and 1920-22000-033-04R). Additional details of research can be found in the report for the parent 1920-22000-035-00D. Soybean rust, caused by Phakopsora pachyrhizi, is a serious foliar disease of soybeans in most soybean producing countries throughout the world, and the disease was discovered in the continental U. S. for the first time in November 2004. The 805 accessions selected from the previous preliminary germplasm screen were assembled into a replicated seedling evaluation using P. pachyrhizi isolate Louisiana 04-1 collected in 2004 for intermediate screens in the USDA-ARS BioSafety Level 3 Containment Facility at Ft. Detrick, MD. From this experiment, approximately 200 accessions were selected that had reduced disease severity and fewer sporulating pustules. These 200 accessions will be evaluated further using additional isolates of P. pachyrhizi that vary in virulence to determine if resistance is broad spectrum or limited to specific isolates. The soybean cultivar Komata (soybean accession PI200492), which contains the single rust resistance gene Rpp1, confers a resistant reaction (immune response) to the rust isolates India 73-1, Australia 79-1, Hawaii 94-1 and Hawaii 98-1. RNA was extracted from rust infected Komata plants from both susceptible and resistant rust interactions at 0, 6, 12, 24, and 48 hours post inoculation. The messenger RNA from susceptible and resistant interactions from each of the five time points was labeled a fluorescent dye, hybridized to the Affymetrix Soybean GeneChip microarray, and scanned as part of a collaboration with the National Cancer Institute's Laboratory of Molecular Technology (LMT) at Ft. Detrick, MD. A total of 1112 genes were identified as being expressed significantly different in resistant and susceptible samples. The induced and suppressed genes were identified by comparison with sequences deposited in the GenBank and UniProt public databases. Over 300 of those significantly different genes have been identified as �defense related� and many are genes that have been previously identified in other plant-microbe interactions as being involved in defense and stress responses. This project was monitored by meeting with the USB representatives at national scientific meetings, numerous e-mails, telephone calls, and quarterly research reports to USB.
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Progress 06/01/04 to 12/31/06
Outputs
Progress Report Objectives (from AD-416) This research will continue with screening soybean accessions and germplasm for resistance to rust, monitoring gene expression in soybean lines exhibiting rust resistance, and in developing disease management and control strategies for U.S. growers. Approach (from AD-416) Adult plant resistance will be evaluated on accessions that were identified in seedling screens as having some resistance. Commercial cultivars identified as resistant in areas where the rust occurs evaluated in comparison to accessions from the germplasm bank. Populations with the mot promising sources of resistance will be developed. U.S. soybean cultivars evaluated for tolerance to soybean rust in areas where the pathogen occurs and compared to rust severity to determine the relationship of partial resistance and tolerance. Profile gene expression in soybean lines exhibiting resistance to soybean rust. Identify regulatory/coordinated interactions between resistant and susceptible reactions. Develop a network of state extension specialists to evaluate application methods that fit current U.S. agronomic and economic conditions. Use this information to develop recommendations for fungicide selection and usage for U.S. soybean growers. Source areas for over-wintering of the soybean rust pathogen will be further defined. The determination and availability of alternate host plants will be determined for source areas. Significant Activities that Support Special Target Populations The goals of this research were to identify soybean line with resistance to soybean rust and to monitor changes gene expression in resistant and susceptible lines containing the Rpp1 resistance gene using the Affymetrix Soybean GeneChip microarray. A total of 805 soybean accessions selected from our previous germplasm screen were assembled into a replicated seedling evaluation using P. pachyrhizi isolate Louisiana 04-1 collected in 2004 in the USDA-ARS BioSafety Level 3 Containment Facility at Ft. Detrick, MD. From this experiment approximately 200 accessions were selected that had reduced disease severity and fewer sporulating pustules. These 200 accessions will be evaluated further using additional isolates of P. pachyrhizi that vary in virulence to determine if resistance is broad spectrum or limited to specific isolates. The soybean cultivar Komata (soybean accession PI200492) contains the single rust resistance gene Rpp1 and confers a resistant reaction (immune response) to the rust isolates India 73-1, Australia 79-1, Hawaii 94-1 and Hawaii 98-1. RNA was extracted from rust infected Komata plants from both susceptible and resistant rust interactions at 0, 6, 12, 24, and 48 hours post inoculation. The messenger RNA from susceptible and resistant interactions from each of the five time points was labeled a fluorescent dye, hybridized to the Affymetrix Soybean GeneChip microarray, and scanned as part of a collaboration with the National Cancer Institute's Laboratory of Molecular Technology (LMT) at Ft. Detrick, MD. A total of 1112 genes were identified as being expressed significantly different in resistant and susceptible samples. The induced and suppressed genes were identified by comparison with sequences deposited in the GenBank and UniProt public databases. Over 300 of those significantly different genes have been identified as �defense related� and many are genes that have been previously identified in other plant-microbe interactions as being involved in defense and stress responses. This information will be valuable to soybean breeders and to government, academic and private researchers for developing soybean lines with resistance to rust. This project concluded at the end of December 2006 and no additional research was conducted in FY 2008.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a reimbursable agreement between ARS and the United Soybean Board. Additional details of research can be found in the report for the parent project 1920-22000-027- 00D, Identification and Characterization of Emerging Foreign Plant Pathogenic Fungi, under NP 303. Germplasm screening continued at the USDA- ARS BioSafety Level 3 Containment Facility at Ft. Detrick, MD on approximately 800 soybean accessions from the USDA Germplasm Bank at Urbana, IL identified in previously preliminary screens. Plants were inoculated with the U.S. isolate Louisiana 04-1, and the lesion type (Tan or RB), disease severity and the number of sporulating pustules/cm2 were measured at 8, 10, 13, 15, and 17 days after inoculation. This data was used to generate the Area Under the Disease Progress Curve for each accession. A second replication of the inoculation with Louisiana 04-1 is planned for later in
2006. Multiple accessions of commercial non- soybean legume crops including varieties of lima bean, clovers, cowpeas, green beans, and green peas; in addition to accessions of various winter legumes from the southern U.S., including kudzu, lupines, beggarweed, coffee senna, hemp sesbania, partridge pea, sweet hyacinth bean, and showy crotaleria, were inoculated with urediniospore suspensions of P. pachyrhizi in the Containment Facility at Ft. Detrick, MD. Some accessions of green beans and green peas were moderately susceptible, and produced urediniospores. Green peas, although susceptible, dropped their infected leaves very soon after initial infection. Clovers, in general, were not susceptible. Other than soybean, kudzu was the most susceptible host, producing large amounts of urediniospores. Accessions of kudzu, however, varied considerably in degrees of susceptibility and sporulation. Photographs of infected plants from this study were placed on the USDA Soybean Rust Web
Page: http://www.usda.gov/soybeanrust/identification. shtml The soybean cultivar Komata (Accession PI200492) contains the single soybean rust resistance gene, Rpp1, that confers a resistant reaction (immune response) to the rust isolates India 73-1, Australia 79-1, Hawaii 94-1 and Hawaii 98-1. Nucleic acids were extracted from rust infected Komata plants from 3 different biological experiments from both susceptible and resistant rust interactions. The nucleic acid from susceptible and resistant interactions were analyzed using a soybean gene microarray, as part of a research collaboration with the National Cancer Institute's Laboratory of Molecular Technology (LMT) at Ft. Detrick, MD. The data indicated possible changes in gene expression between resistant and susceptible interactions.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a reimbursable agreement between ARS and the United Soybean Board. Additional details of research can be found in the report for the parent CRIS 1920-22000-027- 00D, Identification and Characterization of Emerging Foreign Plant Pathogenic Fungi, under NP 303. Germplasm screening continued at the USDA-ARS BioSafety Level 3 Containment Facility at Ft. Detrick, MD on the soybean accessions from the USDA Germplasm Bank at Urbana, IL using the seedling inoculation method developed in the previous year. A mixture of four rust isolates, Brazil 01-1, Paraguay 01-2, Zimbabwe 01-1, and Thailand 01-1, were used in the initial screening process, and approximately 25,000 accessions were evaluated. From these seedling screens approximately 800 accessions have been identified for further testing and evaluation, and list of these accessions has been released to public and university breeders and
pathologists in 23 states by USDA-ARS, Urbana, IL to evaluate for rust resistance under field conditions. The accessions consist of 13 maturity groups ranging from 000 to X. The 800 lines that were identified in preliminary screens were evaluated further by inoculating separately with the four rust isolates Thailand 01-1, Zimbabwe 01-1, Brazil 01-1, and Paraguay 01-2. The lesion type (Tan or RB), disease severity and the number of sporulating pustules/cm2 were measured at 8, 10, 13, 15, and 17 days after inoculation. This data was used to generate the Area Under the Disease Progress Curve for each accession. Histological examination of fixed tissue from the day 17 time point was used to count the number and measure the size of uredinia per lesion from these intermediate inoculations in an attempt to quantifying resistance. In addition to the inoculations in BSL-3 Containment Facility at Ft. Detrick, MD, soybean accessions were evaluated for a second year at three international
locations: South Africa, Thailand, and China by our foreign collaborators. At each location lines that were identified as resistant the previous year were re-evaluated in greenhouse or field trials using local isolates of soybean rust. The soybean cultivar Komata (Accession PI200492) was previously described as containing a single rust resistance gene, Rpp1, that confers a resistant reaction (immune response) to the rust isolates India 73-1, Australia 79- 1, Hawaii 94-1 and Hawaii 98-1. Approximately 1000 clones from a subtractive suppressive hybridization (SSH) library derived from the soybean cultivar Komata following inoculation with the rust isolate Hawaii 94-1 were sequenced at the ARS Nucleic Acid Facility in Wyndmoor, PA, edited and compared to the non-redundant protein and Expressed Sequence Tag (EST) public databases using the computer algorithm BLAST. Of particular interest, 11.8% of the sequences are involved in cell rescue/defense/cell death/ageing, 7.12% are involved in
signaling, 14% of the sequences have putative functions related to regulation of transcription and protein synthesis/destination. DNA was extracted from each of the 1056 SSH clones and spotted onto glass slides for gene expression analysis using microarrays in collaboration with ARS, Beltsville, MD. RNA was extracted from rust infected tissue from 3 different biological experiments encompassing 8 time points, labeled with two fluorescent dyes, and hybridized to the SSH microarray slides. The microarray slides were washed, scanned, and data was extracted and partially analyzed. Of the 1056 forward suppressive subtractive hybridization (SSH) clones, 133 putative genes were up-regulated in all three biological replicates from the resistant Rpp1 interaction. ARS, Urbana, IL planned and coordinated fungicide field trials in as part of a collaboration with the Centro Regional de Investigacion Agricola in Capitan Miranda, Paraguay and the Commercial Oilseed Producers Association of
Zimbabwe in Harare, Zimbabwe. The results of the fungicide field trials that were conducted in Paraguay and Zimbabwe have been posted at the following Web page: http://www.ipmcenters. org/NewsAlerts/soybeanrust/efficacy.cfm
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