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: Image processing methods were used to quantify soybean rust from multispectral images on infected leaves. Exogenous controls were developed to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi. Carbon utilization profiles were developed for 18 Fusarium virguliforme isolates. Phakopsora pachyrhizi was cultured on detached leaves, and uredinio spore survival was studied in different temperature and humidity conditions. Models for soybean rust epidemics were developed based on observations of sequentially planted soybean cultivars. Multiplexed quantitative PCR assays were developed and verified for viruses infecting soybean in Illinois based on metagenomic analysis of virus populations present in soybean plants collected from production fields. Objective 2: Allele-specific assays were developed to detect variation in the genes, DCL3 and RDR6, putatively associated with seed transmission of Soybean mosaic virus (SMV) and used in preliminary association genetics studies. In those experiments, RDR6 haplotypes were the same as the resistant parent in five soybean plant introductions (PIs) resistant to seed transmission and DCL3 haplotypes of five PIs susceptible to SMV seed transmission were the same as the susceptible parent. Construction of a set of 14 chimeric clones between SMV isolates that differ in their seed transmission rates in soybean was completed. Multiple replications of soybean plants were inoculated with the chimeric viruses and seed have been collected for grow-out assays. Objective 3: Resistance in soybean to Phakopsora pachyrhizi was characterized by defining the traits that describe complete and partial resistance. Differentially expressed genes were identified in leaves of Glycine tomentella during infection by Phakopsora pachyrhizi. Plant introductions, breeding lines and mapping populations were evaluated in Attapulgus, GA, Baton Rouge, LA, Bossier City, LA, Fairhope, AL, Quincy, FL, Stoneville, MS, and/or Urbana, IL for a variety of traits including, resistance to premature dehiscence (pod shattering) and resistance to Cercospora blight, frogeye leaf spot, soybean rust, and virus infection. Experiments were initiated to evaluate and map genes conditioning resistance to Phomopsis seed decay and Phytophthora root rot, and to measure the growth of Phakopsora pachyrhizi in resistant soybean germplasm accessions that develop distinctly different types of lesions when challenged by the same population or isolate of the fungus. Objective 4: Field studies to test the ability of low levels of soybean rust resistance to reduce yield losses from soybean rust when fungicide applications are sub-optimal were planted in Quincy, FL in 2010 to obtain a final year of data. However, severe drought conditions and high temperatures prevented the local soybean rust epidemic from developing in time to provide useful data. Because of drought conditions again early in the 2011 growing season, only an experiment to evaluate tolerance of soybean cultivars to soybean rust was planted in June, 2011.
1. Quantified soybean rust on soybean leaves using photographic images. Soybean rust can cause yield losses in soybean. In this study, different digital imaging techniques were compared for automated quantification of soybean rust severity. The threshold-setting method differentiated degrees of soybean rust severity based on color saturation and a rust severity index, but required user input. In contrast, a method that identified the center and area of each lesion was deemed the more practical for automated disease severity determination. These results provide several approaches for automated quantification of rust severity using digital imaging. This information is important to soybean pathologists, plant diagnosticians, and others interested in measuring severity of soybean rust.
2. Demonstrated that saccharin application can reduce soybean rust severity and incidence. Soybean rust can cause losses in profitability to soybean producers by reducing yields and necessitating the application of fungicides for disease control. In a collaborative research project with the University of Florida, it was found that a solution of the artificial sweetener saccharin, particularly when applied as a root drench, provided protection against rust infection for at least 15 days after application. These results indicate that it is possible to reduce soybean rust incidence and disease severity through the application of a nontoxic substance, which induces plant defenses as an alternative to the application of fungicides. These results are of interest to soybean agronomists and pathologists who are interested in alternative methods for reducing losses caused by soybean rust and other fungal diseases of soybean.
3. Identified regions of the Soybean mosaic virus (SMV) genome involved in transmission by aphids. Soybean mosaic virus is a seed and aphid transmitted virus that infects soybean and is capable of significantly decreasing yields and grain quality. Reducing transmission of SMV through seed and by viruses could reduce losses caused by the virus. By analyzing the rates of transmission of recombinant viruses, two regions in the SMV genome were identified that are important for transmission of SMV by soybean aphids. These two regions of the SMV genome have also been implicated in spread of SMV within plants and transmission of SMV through seed. This information is of interest to other scientists who are interested in the interactions of viral proteins in seed and aphid transmission.
4. Developed indicators for the presence of inhibitors in molecular assays for detecting soybean rust spores. Soybean rust is a destructive foliar disease of soybean. Rapid and accurate detection of soybean rust spores is important for the management of soybean rust through timely application of fungicides. However, inhibitors of assays to detect soybean rust DNA are sometimes present in samples collected from the field that can cause the fungus to be missed in samples that actually contain rust spores. To alert diagnosticians to this problem, ARS scientists at Urbana, Illinois designed, synthesized and validated three synthetic DNA molecules that, when included in assays for soybean rust DNA, provide an indication of the success or failure of the detection assay. The methods outlined give diagnosticians more confidence that samples that are negative for soybean rust DNA do not contain soybean rust spores. This information will be useful to molecular biologists, plant pathologists, and other scientists interested in using internal controls with their molecular detection assays.
5. Identification of soybean lines with partial resistance to soybean rust. Soybean rust is one of the most important disease constraints to soybean production worldwide. Five independently inherited single genes that confer nearly complete resistance to soybean rust have been identified. However, some soybean rust strains overcome this resistance. Partial resistance, which is produced by the interaction of multiple genes, slows the rate of disease development over time, and may be less likely to be overcome than resistance conditioned by single-genes. ARS scientists at Urbana, Illinois identified characteristics related to partial resistance to soybean rust in 34 soybean germplasm lines. Two of the most resistant germplasm lines had responses to inoculation with soybean rust that resembled those produced by single genes for resistance. Most of the other accessions were susceptible to soybean rust, but some were partially resistant. These experiments identified lines with partial resistance that may be incorporated into high yielding soybean cultivars, and is important to soybean researchers that are interested in developing resistance to soybean rust.
6. Identification of differentially expressed genes for resistance to soybean rust in Glycine tomentella, a wild relative of cultivated soybean. Soybean rust is a destructive foliar disease of soybean. With the goal of identifying genes that are associated with resistance to soybean rust, ARS scientists at Urbana, Illinois compared responses of rust resistant and rust susceptible lines of Glycine tomentella to infection with soybean rust. The experiments identified 1342 genes in 12 functional categories that were expressed differently in uninfected and soybean-rust-infected leaves. These results provide insights into mechanisms underlying resistance and general activation of plant defense pathways in response to rust infection. This information is important to soybean pathologists, plant molecular biologists and others that are interested in basic studies on host-pathogen interactions.Ma, A., Hill, C., Hartman, G.L. 2010. Production of Macrophomina phaseolina conidia by multiple soybean isolates in culture. Plant Disease. 94:1088-1092.