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United States Department of Agriculture

Agricultural Research Service

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Location: Soybean/maize Germplasm, Pathology, and Genetics Research

2009 Annual Report

1a. Objectives (from AD-416)
1: Develop and use quantitative methodologies to detect, identify, and characterize soybean pathogens and monitor the spread of disease epidemics. 2: Identify viral and host determinants of horizontal and vertical transmission of soybean viruses. 3: Identify and improve germplasm with soybean disease resistance using phenotypic and marker-assisted selection. 4: Develop and improve integrated strategies for sustainable disease management in soybean. Sub-objectives: a. Determine the efficacy of fungicides and optimize cultural practices for the management of soybean rust. b. Develop and validate biological control protocols for bacterial agents against soybean cyst nematode.

1b. Approach (from AD-416)
1) Pathogens will be collected from diseased plants, air, soil, and water and phenotypically and molecularly characterized to identify nucleotide sequences that will be used to quantitatively identify pathogenic organisms and to determine genetic variability of pathogens. 2) To identify viral determinants of transmission, transmission phenotypes of chimeric viruses constructed from viruses differing in transmission properties will be evaluated; regions of the soybean genome involved in seed transmission will be identified using molecular markers and populations of soybean plants differing in transmission of virus through seed. 3) Potential sources of soybean resistance genes and populations segregating for resistance will be evaluated in nurseries and other sites appropriate for resistance screening tests. 4) Soybean germplasm accessions with resistance will be crossed with selected cultivars and lines to create populations used for inheritance and mapping studies. Fungicide trials will evaluate fungicide efficacy, sprayer/delivery technology, timing and frequency of applications, possible interactions between fungicides and soybean genotypes, row spacing, irrigation, and plant architecture on soybean rust infection. Molecular techniques will be developed to identify and quantify obligately parasitic, biological control bacteria in soybean cyst nematode populations and in field soil and culture of bacteria will be attempted using proprietary technology.

3. Progress Report
Objective 1: The aggressiveness of Fusarium virguliforme isolates that cause sudden death syndrome were evaluated and the fungus was quantified in soybean roots by colony-forming unit assays and real-time quantitative polymerase chain reaction (QRT-PCR). Alternative hosts of Phakopsora pachyrhizi, the fungus that causes soybean rust, were identified in field evaluations in Florida. Artificial diets for rearing soybean aphids, Aphis glycines, were investigated and two species of symbiotic bacteria were found to be present in soybean aphids. Soybean mosaic virus (SMV) was detected for the first time in commercial soybean fields in North Dakota. Sensitive fluorescence assays were developed and validated for a newly discovered soybean-infecting virus, Soybean yellow mottle mosaic virus. The virulence of 32 soybean cyst nematode populations obtained from fields with a history of planting resistant cultivars in east central Illinois was determined. Objective 2: To assess the roles of virus-encoded proteins involved in transmission of SMV by aphids in transmission of SMV through seed, a series of site-specific mutations were introduced into the genes that produce two of the proteins primarily responsible for aphid transmission. Soybean plants were inoculated with the mutant and unaltered viruses using recombinant DNA copies of the viral genome. Inoculated plants became infected and are producing seeds. Analysis of molecular markers in populations of soybean plants segregating genes for transmission of SMV through seed and seed-coat mottling when infected by SMV were completed and are being analyzed. The variability of genomic regions that encode aphid transmission proteins of 24 Midwestern Soybean dwarf virus isolates was characterized. Objective 3: A new allele at Rpp1, which conditions resistance to soybean aphids, was mapped and confirmed from soybean in PI 504538A. Methods were developed to evaluate soybean resistance to sudden death syndrome. Glyphosate-resistant soybean cultivars for resistance to bacterial pustule were evaluated. The inheritance of resistance to soybean aphids in soybean plant introduction (PI) 200538 was reported. Quantitative trait loci (QTL) were identified for resistance to Sclerotinia Stem Rot in PI 194639. Ninety-six soybean germplasm accessions were re-evaluated for resistance to soybean rust at seven locations between Louisiana and South Carolina, and at least 45 had moderate to high resistance in two or more locations. Lesion color, density and sporulation on rust resistant germplasm accessions with variable reactions to a Florida isolate of soybean rust were studied in the greenhouse. Objective 4: The effect of fungicide treatment and timing of application on soybean rust severity and yield was determined. Interactions between fungicide application rates and moderate resistance to rust were studied in the field. In field plots either protected or unprotected by fungicide, tolerance to soybean rust was evaluated in 12 breeding lines developed by breeding programs at southeastern universities.

4. Accomplishments
1. Differential responses of resistant soybean genotypes to isolates of Phakopsora pachyrhizi. Soybean rust, caused by the fungus Phakopsora pachyrhizi, is an important disease in many soybean-producing countries. Several new sources of resistance to soybean rust have been identified in soybean. However, there is limited information about how well the resistance will protect soybean plants from infection by other U.S. and international soybean rust isolates. ARS researchers in the Soybean/Maize Germplasm, Pathology & Genetics Research Unit in Urbana, IL compared disease responses of 20 soybean lines after inoculation with 10 P. pachyrhizi isolates. Soybean entries included 2 susceptible cultivars, 4 sources of soybean rust resistance genes (Rpp1–4), and 4 and 10 resistant entries selected from field trials in Paraguay and Vietnam, respectively. Plants with previously described Rpp1–4 sources of resistance showed consistent resistance responses to most P. pachyrhizi isolates. The resistant entries selected from Paraguay and Vietnam varied from susceptible to resistant in their responses to the 10 P. pachyrhizi isolates. The reaction patterns on the resistant entries to the P. pachyrhizi isolates were different compared with the four soybean accessions with the Rpp genes, indicating that they may contain novel sources of rust resistance. Phakopsora pachyrhizi isolates from Taiwan and India produced the most susceptible and resistant reactions, respectively, on the soybean entries. This research is important to the soybean research community and identified soybean lines that may be sources of resistance to soybean rust that could be incorporated into commercial cultivars.

2. Effects of virus-derived small RNAs on host gene expression examined. Posttranscriptional gene silencing (PTGS) serves as a potent antiviral defense that degrades RNAs of infecting viruses into small pieces. These small pieces of viral RNAs have the potential to direct the degradation of host messenger RNAs (mRNAs) and alter normal cellular processes. In a collaborative project involving ARS researchers at Urbana, IL and scientists at the Universities of Illinois and Kentucky, soybean messenger RNAs (mRNAs) were identified with regions of complementarity to small RNAs from Bean pod mottle virus (BPMV), an economically important pathogen of soybean. Accumulation of a subset of the corresponding mRNAs was significantly reduced and some were cleaved proximal to regions of BPMV complementarity in BPMV-infected plants. These results show that small viral RNAs produced by PTGS can alter expression of host genes. These findings will be of interest to researchers investigating the interactions of viruses and hosts that lead to disease symptoms.

3. Resistance of soybean germplasm accessions to soybean rust confirmed. To identify potential sources of resistance to soybean rust, ARS personnel from Urbana, IL and Ft. Detrick, MD had collaborated in 2004 and 2005 to screen 16,595 accessions from the USDA Soybean Germplasm Collection. After two rounds of screening seedlings inoculated with a mixture of four foreign isolates of P. pachyrhizi, 805 PIs were selected for further evaluation on the basis of low disease severity and/or development of reddish-brown lesions indicating resistance. In collaboration with researchers from Louisiana State, Auburn, Clemson, Georgia and Florida between 2006 and 2008, most of the 805 PIs were evaluated for rust resistance in the southeastern U.S. Forty-five PIs showed relatively high levels of resistance at most locations in 2008, and another 18 accessions had at least moderate resistance at two or more locations. The evaluation data indicated that many of the PIs are resistant in either the western locations (Louisiana) or the eastern locations (such as Georgia and Florida), but few are highly resistant in both regions. Soybean breeders are using this information to make decisions about which crosses to make and which existing breeding populations to continue working with. Development of cultivars with broad and durable resistance to soybean rust in North America therefore will require combinations of genes from different sources, and continued re-testing of those sources is important to ensure that the genes continue to provide sufficient resistance to reduce or eliminate the need for fungicide applications. A subset of these resistant PIs was crossed with elite soybean lines to combine potentially novel rust resistance genes with genes for other desirable traits, including high yields.

4. New technique developed for isolation of Pasteuria DNA from soil. Pasteuria nishizawae is a soil-borne bacterium that has been used successfully in field trials to control soybean cyst nematode. Initial success in quantifying P. nishizawae was the development of a protocol for QRT-PCR amplification of the 16S ribosomal RNA gene of P. nishizawae. The use of this protocol has allowed detection of minute amounts (less than 1,000 copies) of the target from crude DNA extracts obtained from suspensions of Pasteuria endospores. However, the application of the assay to detect P. nishizawae from soil known to contain the bacterium has been a more daunting task than expected. The use of commercial kits for soil DNA isolation was not successful. It was hypothesized that the inability of the assay to detect P. nishizawae from soil DNA extracts likely stemmed from the “drowning” of the rare DNA molecules of P. nishizawae in a heterogeneous background of DNA from other microbial sources in the soil. This hypothesis has now been verified using the magnetic bead hybridization capture (MBHC) technique. The technique uses a magnetic bead-streptavidin-linked biotinylated P. nishizawae probe to capture the rare target molecules from the soil DNA extracts. The MBHC technique has allowed consistent detection of P. nishizawae from DNA extracts from soil, including extracts from which the bacterium could not be detected in the past. This research, a cooperative endeavor between ARS and University of Illinois scientists, will allow researchers to better monitor population levels of P. nishizawae in field trials to evaluate its effectiveness as a biological control agent.

5. Movement of Phakopsora pachyrhizi spores by non-conventional means. Phakopsora pachyrhizi, which causes soybean rust, is the most important foliar pathogen infecting soybean. Historically, the disease was important only in the Eastern Hemisphere, but since 1994 it has been reported in many countries in Africa and the Americas. In the U.S.A., soybean rust has been perceived as a threat to soybean production and monitoring of the disease occurs throughout the country where soybean is grown. ARS scientists in the Soybean/Maize Germplasm, Pathology & Genetics Research Unit in Urbana, IL conducted a study to show conclusive evidence that soybean rust spores can be transported by means other than through air currents, such as clothing. Findings from this research are relevant to scientists involved in monitoring the spread of soybean rust and may affect precautions taken by researchers as they move equipment and personnel from areas where soybean rust is present to regions where soybean rust has not yet been detected.

6. Pathogenic variation of Phakopsora pachyrhizi. Soybean rust, caused by the fungus Phakopsora pachyrhizi, is an important disease in many soybean-producing countries. It was first reported in the continental United States in 2004. Widespread epidemics have occurred since, but the intensity and time of infection has been late in the season and has not caused significant yield losses. In this study, researchers from the University of Ibadan in Nigeria and ARS scientists in the Soybean/Maize Germplasm, Pathology & Genetics Research Unit in Urbana, IL examined the distribution and genetic variation of soybean rust isolates in Nigeria, a country where rust has caused soybean yield losses. To determine the geographical distribution of soybean rust in Nigeria, soybean fields were surveyed in three agroecological zones. Disease incidence was significantly different between the zones. During the survey, 116 purified isolates were established in culture on detached soybean leaves. Based on rates of reproduction of the fungi on selected soybean lines, the isolates were separated into seven groups. This study was the first to report grouping of soybean rust isolates based on their abilities to reproduce on different soybean lines. The results will be useful to soybean researchers who are interested in studying the occurrence and distribution of rust based on disease surveys and for those working on variability of the pathogen as it relates to host resistance.

7. Additional sources of resistance to North American populations of soybean rust identified. Soybean rust is a foliar disease of soybean that can cause significant yield losses even when adult plants become infected. Adult-plant resistance to soybean rust was evaluated on 134 germplasm accessions. The accessions were identified in the first round of screening of seedlings of 16,595 soybean accessions from the USDA Soybean Germplasm Collection for resistance by ARS personnel from Urbana, IL and Ft. Detrick, MD. Three accessions from maturity group (MG) IX were resistant at Fairhope, AL and Quincy, FL, and another accession from MG X was resistant in Quincy. Twenty MG VII and VIII accessions tested in Attapulgus, GA also were resistant to rust. These results suggested that some of the accessions that were rust resistant after the first, but not the second round of selection in the Ft. Detrick seedling assays are resistant as adult plants to North American isolates of the rust fungus. Identification of additional sources of resistance genes will allow development of cultivars with greater genetic diversity and will lessen selection pressure on the rust pathogen.

8. Timing of fungicide application on control of rust and its effects on yield. Soybean rust is a devastating foliar disease of soybean and yield losses of up to 80% have been reported in experimental trials in Asia. Currently there are no commercial cultivars in the U.S. that are resistant to soybean rust making it feasible that millions of soybean hectares may be sprayed with fungicides if soybean rust becomes widespread. In a collaboration among scientists from USDA-ARS in the Soybean/Maize Germplasm, Pathology & Genetics Research Unit in Urbana, IL, CRIA in Itapua Paraguay, University of Florida, University of Georgia, and the Commercial Farmers Union of Zimbabwe in Harare, Zimbabwe, field trials were completed in Paraguay (four locations), United States (two locations), and Zimbabwe (one location) to determine the effect of fungicide timing on soybean rust severity and soybean yield. Mean yields from plants in fungicide plots ranged from 16 to 114% greater than yields in non-fungicide control plots in four locations in Paraguay, 31% greater in Zimbabwe, and 12 to 55% greater in two locations in the U.S. In all locations, rust severity was negatively correlated with yield. The effectiveness of fungicide treatments were often dependent on when rust was first detected and the intensity of its development. This information will help soybean producers, pathologists and others interested in management of soybean rust.

9. A new soybean gene for resistance to the soybean aphid. Since the first discovery of soybean aphids in North America in 2000, the insect has become a major soybean pest. Sources of resistance to soybean aphids have been identified, and one of these sources with a high level of resistance is plant introduction (PI) 200538. University of Illinois and USDA-ARS researchers in the Soybean/Maize Germplasm, Pathology & Genetics Research Unit in Urbana, IL determined the inheritance of aphid resistance and mapped the gene controlling resistance in PI 200538. Segregation of resistance in multiple populations indicated that a single dominant gene controlled soybean aphid resistance. The resistance gene in PI 200538 mapped to the same location and is most likely the same gene as Rag2, a previously mapped aphid resistance gene. This information is important to the soybean industry and others who are developing soybean cultivars that have resistance to the soybean aphid.

Review Publications
Lynch, T.N., Steinlage, T.A., Miles, M.R., Marois, J.J., Wright, D.L., Hartman, G.L. 2008. New Legume Hosts of Phakopsora pachyrhizi Identified from Field Studies in Florida. Plant Disease. 92:767-771.

Scaboo, A.M., Pantalone, V.R., Walker, D.R., West, D.R., Walker, F.R., Sams, C.E., Boerma, H. 2009. Confirmation of Molecular Markers and Agronomic Traits Associated with Seed Phytate Content in Two Soybean RIL Populations. Crop Science. 49:426-432.

Wille, B., Hartman, G.L. 2008. Evaluation of Artificial Diets for Rearing Aphis Glycines (Hemiptera: Aphididae). Journal of Economic Entomology. 101:1228-1232.

De. Fraias Neto, A.L., Schmidt, M., Hartman, G.L., Li, S., Diers, B.W. 2008. Greenhouse Inoculation Methods for Evaluating Resistance of Soybean to Sudden Death Syndrome. Brazilian Journal of Agricultural Research. 43:1475-1482.

Pham, T.A., Miles, M.R., Frederick, R.D., Hill, C.B., Hartman, G.L. 2009. Differential Responses of Resistant Soybean Genotypes to Ten Isolates of Phakopsora Pachyrhizi. Plant Disease. 93:224-228.

Twizeyimana, M., Ojiambo, P.S., Sonder, K., Ikotun, T., Hartman, G.L., Bandyopadhyay, R. 2009. Pathogenic Variation of Phakopsora pachyrhizi Infecting Soybean in Nigeria. Phytopathology. 99:353-361.

Chakroaborty, N., Curley, J., Frederick, R.D., Hyten, D.L., Nelson, R.L. Hartman, G.L., Diers, B.W. 2009. Mapping and Confirmation of a New Allele at Rpp1 from Soybean PI 504538A Conferring RB Lesion Type Resistance to Soybean Rust. Crop Science. 49:783-790.

Hartman, G.L., Haudenshield, S. 2009. Movement of Phakopsora pachyrhizi (Soybean Rust) Spores by Non-Conventional Means. European Journal of Plant Pathology. 123:225-228.

Li, S., Hartman, G.L., Domier, L.L., Boykin, D.L. 2008. Quantification of Fusarium solani f. sp. glycines Isolates in Soybean Roots by Colony-forming Unit Assays and Real-time Quantitative PCR. Journal of Theoretical and Applied Genetics. 117:343-352.

Mueller, T.A., Morel, W., Marois, J.J., Wright, D.L., Kemerait, R.C., Miles, M.R., Levy, C., Hartman, G.L. 2009. Effect of Fungicide and Time of Application on Soybean Rust Severity and Yield. Plant Disease. 93(3):243-248.

Sugimoto, T., Watanabe, K., Furiki, M., Walker, D.R., Yoshida, S., Aino, M., Kanto, T., Irie, K. 2009. The Effect of Potassium Nitrate on the Reduction of Phytophthora Stem Rot Disease of Soybeans, the Growth Rate and Zoospore Release of Phytophthora sojae. Journal of Phytopathology. 157:379-389.

Voung, T., Diers, B., Hartman, G.L. 2008. Identification of QTL for Resistance to Sclerotinia Stem Rot (Sclerotinia sclerotiorum) in Soybean Plant Introduction 194639. Crop Science. 48:2209-2214.

Last Modified: 2/23/2016
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