1a. Objectives (from AD-416):
Objective 1: Use pathogen/pest virulence and genomics data to develop quantitative detection methodologies that will be used to monitor the spread, diversity, and impact of these biotic agents. Sub-objective 1.A. Characterize soybean pathogens and pests in terms of their aggressiveness, virulence, and molecular and genetic diversity. Sub-objective 1.B. Evaluate the impact of soybean cyst nematode and F. virguliforme on yield when using host resistance genes and plant defense elicitors. Sub-objective 1.C. Develop and utilize molecular markers to monitor soybean pathogens and pests. Objective 2: Identify and genetically characterize resistance in cultivated soybean and related annual and perennial species to pathogens/pests of soybean. Sub-objective 2.A. Identify new sources of pathogen/pest resistance in annual and perennial accessions from the USDA Soybean Germplasm Collection. Sub-objective 2.B. Genetically and phenotypically characterize identified resistance to soybean pathogens and pests. Sub-objective 2.C. Develop improved soybean germplasm and breeding lines that carry disease and pest resistance genes.
1b. Approach (from AD-416):
The distribution and diversity of soybean pests and pathogens will be monitored using phenotypic evaluations and molecular diagnostic assays developed from pathogen genome sequence data. The impacts on soybean yields of selected pathogens and pests with and without the application of chemical inducers of disease resistance will be characterized in replicated field trials over multiple years using soybean lines that differ in their levels of resistance. Resistance to pathogen and pests will be identified and characterized in cultivated soybean and related annual and perennial accessions from the USDA Soybean Germplasm Collection through field and greenhouse evaluations. Molecular markers for regions of soybean chromosomes associated with pathogen/pest resistance and regions of pathogen chromosomes associated with virulence will be identified by molecular mapping techniques. The information gained during the characterization of soybean germplasm accessions will be used to produce breeding lines with enhanced resistance to pathogens and pests by a combination of breeding and marker assisted selection techniques.
3. Progress Report:
For Objective 1, high-throughput sequencing of mRNA from soybean leaf samples collected from North Dakota revealed the presence of a novel soybean-associated virus that has a genome structure similar to a family of viruses called the Dicistroviridae that have RNA genomes and infect invertebrates. The genome of the new virus contains two large protein-coding regions. The first protein-coding region is predicted to express proteins that show similarity to structural proteins of invertebrate viruses like Aphid lethal paralysis virus and Cricket paralysis virus. The second protein-coding region is predicted to encode proteins that function in RNA replication and show similarity to replication-related proteins of animal viruses (e.g., Hepatitis A virus), invertebrate viruses (e.g., Solenopsis invicta virus 1), and plant viruses (e.g., Maize chlorotic dwarf virus). The unique genome organization and modes of gene expression suggests that the new virus belongs to a new taxonomic group. Experiments are in progress to determine the host(s) of the new virus. Plant pathogenic fungi like Fusarium virguliforme, the causal agent of soybean sudden death syndrome, secrete cell-wall degrading enzymes in the process of colonizing soybean roots. Soybean plants respond to the secreted enzymes by enhancing cell walls and increasing the expression of defense-related compounds. To better understand the complex interactions between F. virguliforme and soybean, the abundance of transcripts from fungal genes encoding secreted cell-wall degrading enzymes was evaluated from 5-day and 20-day old cultures. This analysis identified 161 F. virguliforme genes that showed increased transcript abundance in 20-day old cultures compared to only 14 that showed reduced transcript abundance. Structural modeling and protein-protein docking analysis of one of the F. virguliforme enzymes suggested that it is susceptible to plant-expressed inhibitors of cell-wall degrading enzymes, which further suggests the possibility of developing transgenic soybean plants with exotic inhibitory proteins to reduce the severity of F. virguliforme infections. Like all crops, soybeans are under constant threat of disease from invading pathogens. One potential management strategy is to treat plants with chemicals called elicitors that induce protective resistance. To determine if chemical elicitors that induced resistance to pathogen infection in other crops would reduce disease severity in soybean, two soybean genotypes producing high and low amounts of reactive oxygen species (ROS) after elicitation were evaluated in field experiments. In two consecutive growing seasons, five different elicitors and a water control were sprayed onto soybean plants in the field. Foliage and stems were evaluated for disease incidence and severity. Two elicitors reduced disease severity in the soybean genotype with high ROS, but not in the genotype with low ROS. Selected elicitors will be further evaluated in greenhouse studies to determine if applications under controlled pathogen inoculations reduce disease severity. To characterize the proteins from soybean and Soybean mosaic virus (SMV) that are involved in transmission of SMV through seed, experiments were started to evaluate soybean seedlings from a population of 200 SMV-infected soybean lines for their incidence of SMV infections in the greenhouse. Previously, we cloned soybean and SMV genes hypothesized to express proteins that interact in SMV seed transmission into expression plasmids for bimolecular fluorescence complementation studies. The tagged proteins were expressed in tobacco leaves and examined by confocal fluorescence microscopy to detect possible genotype-specific interactions between the soybean and SMV proteins. However, no interactions were detected between the cloned soybean and SMV proteins. Interactions were observed between two SMV proteins previously shown to interact in plants, which confirmed the ability of the methods to detect protein-protein interactions. The results may indicate that the interactions of the selected soybean and SMV proteins are more complex than hypothesized or that the virus and host proteins do not directly interact in seed transmission of SMV. For Objective 2, in collaboration with the University of Florida and the University of Georgia, 349 soybean germplasm accessions were screened for resistance to soybean rust in the field in late 2013. A subset of 108 accessions not previously reported to be resistant was also evaluated in greenhouse assays, and 31 accessions were confirmed to be resistant. A set of 349 breeding lines from this project and 96 lines from another Urbana ARS breeding program were also screened for rust resistance in Florida and resistant lines were found. A workshop on soybean rust research was organized in Quincy, FL in November 2013. Identification of new sources of resistance genes and of which soybean lines in the ARS program have resistance is critical for the development of resistant soybean cultivars. More than 1,800 lines derived from crosses to plant introductions with resistance to soybean rust, Phomopsis seed decay, or Phytophthora root and stem rot were evaluated for agronomic and resistance traits in Urbana. Soybean like many cultivated crops, has a relatively narrow genetic base and lacks diversity for some economically important traits, including resistance to Sclerotinia stem rot (SSR), which is caused by Sclerotinia sclerotiorum. Unlike soybean, Glycine latifolia, a perennial wild relative of soybean in the subgenus Glycine, shows high levels of resistance to SSR. In G. latifolia, populations of 186 F2 individuals and 90 F5 lines that segregated resistance to SSR were analyzed for segregation of molecular markers and sensitivity to oxalic acid (a pathogenicity determinant for S. sclerotiorum). The analysis identified a region on G. latifolia linkage group 19 for resistance to oxalic acid that contained 69 genes and corresponded to a locus mapped in soybean for resistance to SSR. Additional studies are in progress to refine the position of the genes for insensitivity of oxalic acid in G. latifolia. Soybean vein necrosis virus (SVNV) has become the most prevalent and widely distributed virus pathogen of soybean in North America. During 2013, a population of 364 soybean lines was evaluated for sensitivity to SVNV in replicated field trials planted late to maximize exposure of young sensitive soybean plants to SVNV and the thrips that transmit the virus. Each entry was evaluated for the numbers and severity of symptomatic plants. Symptom phenotypes were highly reproducible between replications. The numbers of symptomatic leaves per entry formed a near normal distribution with the tails of the distribution having either few plants showing very mild or very severe symptoms. The results suggest that the soybean lines differed significantly in their responses to SVNV infection in the field. The evaluations are being repeated in 2014.
1. Identified and named a new fungal species that causes soybean anthracnose. Anthracnose of soybean was first reported in Korea in 1917, and now is known to occur wherever the crop is grown. The disease has been reported to cause significant yield losses in soybean in southern areas of the USA. The most common pathogen that causes soybean anthracnose is Colletotrichum truncatum. In 2009, an ARS scientist at Urbana, Illinois in cooperation with a University of Illinois scientist surveyed the distribution of Colletotrichum species in soybean fields in Illinois and characterized the collected species using traditional morphological observations and molecular characteristics of multiple gene sequences. Multiple-gene sequence analyses identified one isolate that was genetically distinct from other established Colletotrichum species. The isolate was further examined closely for its morphology, growth characteristics in culture, and pathogenicity on soybean. Based on the molecular phylogenetic, morphological and pathogenicity analyses, a new species was proposed named Colletotrichum incanum. This information is important for the development of soybean cultivars that are resistant to anthracnose, which will reduce the use of fungicides to control this disease thereby reducing costs of production for soybean growers and enhancing environmental quality.
2. Confirmed that the 2013 population of the soybean rust fungus in north-central Florida was less virulent than the 2012 population. Soybean rust, a potentially devastating foliar disease, is currently managed using fungicide applications, but previous studies discovered soybean genotypes with resistance to the disease. ARS scientists from Urbana, IL collaborated with researchers from the University of Florida and the University of Georgia to evaluate the reactions of historically resistant soybean germplasm accessions in 2013. These accessions developed much less disease in the field in Quincy, FL in 2013 than most of them had in 2012, while known susceptible lines had severe rust symptoms in both years. This dramatic decrease in virulence was unexpected, and is an important discovery because it indicates that loss of rust resistance in one growing season does not necessarily mean that the resistance genes will fail to provide protection in the following season. It also demonstrates that the virulence of the rust population at a location does not always increase from one season to the next. These results suggest that rust-resistant soybean cultivars maybe effective longer than previously thought for reducing yield losses from the disease and the economic and environmental costs of fungicide applications to manage soybean rust.
3. Compared the genome organizations of soybean and one of its wild perennial relatives. Soybean, like many cultivated crops, lacks genetic diversity for some economically important traits, while soybean’s wild perennial relatives are more genetically diverse and show high levels of resistance to multiple soybean pathogens and pests. However, it has been extremely difficult to cross soybean with its wild perennial relatives to capture genes for these valuable traits. ARS scientists at Urbana, Illinois, in collaboration with researchers from the University of Illinois, investigated differences in chromosome structure between soybean and one of its perennial relatives, Glycine latifolia, and showed that 12 of 20 soybean and G. latifolia chromosomes were very similar. The remaining eight chromosomes appeared to contain multiple interchromosomal rearrangements, which could reduce the fertility of offspring between the two species. The results from these experiments will aid in the development of methods to transfer genes from soybean’s wild perennial relatives to improve soybean yields and resistance to pathogens and pests thereby reducing both costs of production and applications of chemicals applied to the crop that will in turn enhance rural economies and environmental quality.
4. Aggressiveness of the fungus that causes soybean rust from Nigeria and the United States assessed using quantitative traits. Soybean rust is one of the most important foliar diseases of soybean worldwide. An ARS scientist at Urbana, Illinois cooperated with University of Illinois scientists to compare isolates of soybean rust from Nigeria and the U.S. for six quantitative traits to assess aggressiveness on two soybean genotypes. Five of the six quantitative measures were significantly different among the isolates within each soybean genotype within each country. By defining and understanding the traits used to measure aggressiveness, comparisons among fungal isolates can be used to determine if geographic or environment differences are associated with quantitative aspects of aggressiveness. This research will allow scientists to develop soybean cultivars to better manage pathogenically diverse populations of the soybean rust pathogen, which will reduce the need for costly fungicide applications and in turn enhance rural economies and environmental quality.
5. Identified additional soybean germplasm accessions with resistance to soybean rust. Populations of the rust fungus in different locations in the southern USA have shown considerable variation in terms of their ability to cause disease on plants with genes for resistance for soybean rust. To develop soybean cultivars with broad and durable resistance to soybean rust, it is important for breeders to have different resistance genes available. ARS scientists at Urbana, Illinois collaborated with researchers from the University of Georgia and the University of Florida in 2013 to identify 47 rust-resistant soybean plant introductions previously unreported to be resistant in the United States. The resistance of 31 of these was subsequently confirmed in greenhouse assays, and experiments have been started to determine which of these have unique resistance genes that differ from those reported in other plant introductions. The potential to reduce fungicide use by planting resistant crop cultivars developed from these and other sources has economic benefits for growers in addition to environmental benefits.
6. Identified regions of the soybean mosaic virus (SMV) genome that are required for transmission of SMV through seed. SMV is a seed and aphid-transmitted virus that can cause significant yield reductions and reduce seed quality in soybean. In North America, seed transmission serves as the primary source of inoculum for SMV. ARS scientists at Urbana, Illinois, in collaboration with researchers from the University of Illinois, investigated the roles of SMV encoded proteins in seed and aphid transmission. The results showed that some mutations that reduced transmission of SMV through seed also reduced transmission of SMV by aphids, which suggested that specific interactions between SMV-encoded proteins are important for multiple functions in the virus life cycle. The results of this study will help scientists develop soybean cultivars that reduce economic losses caused by SMV and thereby enhance rural economies.
7. Characterized the infection process of the fungus that causes soybean rust in different soybean genotypes. Soybean rust is an economically important disease of soybean with potential to cause severe epidemics resulting in significant yield losses. Host resistance is one of the management tools to control this disease. ARS scientist at Urbana, Illinois, cooperated with University of Illinois scientists to study in detail the infection process of the fungus in soybean using microscopic observations and quantitative measures of changes in the amounts of fungal DNA in soybean plants over time. Differences in infection among soybean genotypes were evident once the fungus invaded the leaves. Soybean genotypes completely or partially resistant to soybean rust had significantly lower quantities of fungal sporulation and fungal DNA than soybean genotypes with lower levels of soybean rust resistance. These results demonstrated that resistance in soybean results from restricted fungal development because plants kill infected cells to reduce disease spread. This information is important for the development of soybean cultivars that are resistant to soybean rust, which will reduce the use of fungicides to control this disease thereby reducing costs of production for soybean growers and enhancing environmental quality.
8. Developed a novel assay method to screen soybean germplasm for resistance to Phomopsis seed decay (PSD). PSD is a fungal disease that causes soybean seeds to become moldy when harvest is delayed by rain, thereby decreasing seed weight and oil and protein contents, which can result in substantial economic losses to producers. Because PSD is a seed disease, assessment of plant resistance is more difficult than it is for root or foliar diseases that can be seen prior to maturity. Furthermore, maintaining adult plants under conditions that promote the disease is difficult in the field and expensive in the greenhouse due to space requirements. ARS scientists from Urbana, Illinois developed a novel greenhouse assay in which soybean plants were grown to maturity at high densities in sand under a misting system that enhanced PSD infection. This method kept the plants diminutive but healthy, and allowed 20-30 plants to be grown to maturity in the same area that a single mature plant grown in standard potting mix would have required. Use of greenhouse space and overhead sprinklers was more efficient with the smaller plants, and plants could be induced to flower early by imposing a shortened photoperiod. Seed harvested from plants known to be susceptible to PSD exhibited higher than 90% disease incidence. These methods will facilitate the development of PSD-resistant soybean cultivars, which will maintain the quality of soybean seed through harvest, and in turn support soybean profitability and the health of rural economies.
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Yang, H., Haudenshield, J.S., Hartman, G.L. 2014. Colletotrichum incanum sp. nov., a curved-conidial species causing soybean anthracnose in USA. Mycologia. 106(1):32-42. DOI: 10.3852/13-013.
Vittal, R., Paul, C., Hill, C.B., Hartman, G.L. 2014. Characterization and quantification of fungal colonization of Phakopsora pachyrhizi in soybean genotypes. Phytopathology. 104:86-94.
Paul, C., Hartman, G.L., Marois, J., Wright, D., Walker, D.R. 2013. First report of Phakopsora pachyrhizi adapting to soybean genotypes with Rpp1 or Rpp6 rust resistance genes in field plots in the United States. Plant Disease. 97:1379.
Yang, H., Stewart, J.M., Hartman, G.L. 2013. First report of Colletotrichum chlorophyti infecting soybean seed in Arkansas, United States. Plant Disease. 97:1510. Available: http://dx.doi.org/10.1094/PDIS-04-13-0441-PDN.
Radwan, O., Rouhana, L., Hartman, G.L., Korban, S.L. 2013. Genetic mechanisms of host-pathogen interactions for charcoal rot in soybean. Plant Molecular Biology Reporter. 32:617-629. DOI 10.1007/s11105-013-0686-9.
Xiang, Y., Herman, T., Hartman, G.L. 2014. Utilizing soybean milk to culture soybean pathogens. Advances in Microbiology. 4:126-132.
Diers, B.W., Kim, K., Frederick, R.D., Hartman, G.L., Unfried, J.R., Schultz, S.J., Cary, T.R. 2013. Registration of eight soybean germplasm lines resistant to soybean rust. Journal of Plant Registrations. 8:96-101.
Bonin, C.M., Kim, K., Cregan, P.B., Hill, C.B., Hartman, G.L., Diers, B.W. 2013. Inheritance of soybean aphid resistance in 21 soybean plant introductions. Theoretical and Applied Genetics. 127(1):43-50.
Hill, C.B., Bowen, C.R., Hartman, G.L. 2013. Effect of fungicide application and cultivar on soybean green stem disorder. Plant Disease. 97:1212-1220.
Lygin, A., Zernova, O., Hill, C., Kholina, N., Widholm, J., Hartman, G.L., Lozovaya, V. 2013. Glyceollin is an important component of soybean plant defense against Phytophthora sojae and Macrophomina phaseolina. Phytopathology. 103:984-994.
Marvelli, R.A., Hobbs, H.A., Li, S., McCoppin, N.K., Domier, L.L., Hartman, G.L., Eastburn, D.M. 2014. Identification of novel double-stranded RNA mycoviruses of Fusarium virguliforme and evidence of their effects on virulence. Archives of Virology. 159(2):349-352.
Kim, K., Chirumamilla, A., Hill, C.B., Hartman, G.L., Diers, B.W. 2014. Identification and molecular mapping of two soybean aphid resistance genes in soybean PI 587732. Theoretical and Applied Genetics. 127:1251-1259.
Twizeyimama, M., Ojiambo, P., Bandyopadhyay, R., Hartman, G.L. 2014. Use of quantitative traits to assess aggressiveness of Phakopsora pachyrhizi isolates from Nigeria and the United States. Plant Disease. 98:1261-1266. Available: http://dx.DOI.org/10.1094/PDIS-12-13-1247-RE.
Jossey, S., Hobbs, H.A., Domier, L.L. 2013. Role of Soybean mosaic virus-encoded proteins in seed and aphid transmission in soybean. Phytopathology. 103(9):941-948.
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Chang, S., Thurber, C.S., Brown, P.J., Hartman, G.L., Lambert, K.N., Domier, L.L. 2014. Comparative mapping of the wild perennial Glycine latifolia and soybean (G. max) reveals extensive chromosome rearrangements in the genus Glycine. PLoS One. 9(6):e99427.