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 Archaeophytopathology of P. pachyrhizi was investigated by determining which species of rust occurred in herbarium specimens collected from 1887 to 2006. One specimen was positive for P. pachyrhizi as far back as 1912 based on quantitative PCR primers specific to P. pachyrhizi. These results demonstrated the feasibility of DNA genotyping in archaeophytopathological investigations and found that P. pachyrhizi was of Australasian origin and was not found in the archival specimens from the Western Hemisphere. The soybean aphid, Aphis glycines, is one of the most destructive insect pests on soybeans in the United States. One method for managing this pest is through host plant resistance, although in recent years biotypes of the aphid have been found including variants within biotypes. This year we reported on a soybean aphid isolate from Moline, Illinois as a variant of aphid biotype 3, which is the first report of a variant within a soybean aphid biotype. Little is known about the microbial composition of the soybean phyllosphere, which could contain microorganisms that are either directly or indirectly antagonistic to economically important fungal plant pathogens. This year, we compared the metatranscriptomes from soybean leaves collected from soybean fields at multiple locations in Illinois in 2008, 2009, 2010, 2013 and 2014. Each year, total RNA was extracted from the leaf samples, pooled, depleted of ribosomal RNAs and analyzed by high throughput sequencing. Based on the normalized reads assigned to kingdoms, bacteria represented from 1% to 4% of the total aligned reads. An average of 2.8% of the reads aligned to virus sequences. The remaining reads were mostly eukaryotic in origin. Among the bacterial reads, 48% on average belonged to Cyanobacteria, followed by 39% to Proteobacteria, 9% to Firmicutes and 1.1% to Actinobacteria. Among the eukaryotic reads, 95% were assigned to plants (soybean), 1.1% to Ascomycota, followed by 0.3% to Arthopoda, specifically aphids, followed by less than 0.01% each to Basidiomycota and Oomycetes, and less than 0.01% to Nematoda. Using the genetic system developed for Sclerotinia sclerotiorum hypovirus 2L (SsHV2L), the effects of virus infection on fungal gene expression and small RNA accumulation were compared using S. sclerotiorum isolate DK3 either transfected with SsHV2L or not transfected. Significant changes in the accumulation of mRNAs encoding genes involved in sucrose sequestration and membrane remodeling were observed in cultures transfected with SsHV2L compared to the non-transfected culture. SsHV2L infection was also associated with changes in the accumulation of classes of small RNAs expressed from the S. sclerotiorum genome. For Objective 2 the resistance in soybeans to soybean aphid biotypes was tested under different temperatures to determine the stability of the resistance genes across different temperatures; no gene-by-temperature interaction was found indicating that the resistance genes discovered so far are not temperature sensitive, which is not the case for all resistance genes found in soybean. Resistance to anthracnose and charcoal rot was identified and provides for stronger resistance than previously reported. In late 2014, 187 plant introductions (PIs) from the USDA Soybean Germplasm Collection were planted in soybean rust (SBR) screening nurseries through collaboration of an ARS scientist from Urbana, IL with researchers from the University of Florida and the University of Georgia. The main field test included 144 PIs that had either shown resistance to U.S. populations of Phakopsora pachyrhizi (the SBR fungus) in previous years or were known to carry specific Rpp resistance genes. Forty-three other PIs that had not previously been screened for SBR resistance in the field were planted in another field test. Unfortunately, early frosts at the two nursery locations (north-central Florida and southeastern Georgia) damaged the leaves of the plants before disease ratings could be made. In collaboration with the University of Georgia greenhouse assays were conducted to test the reactions of subsets of the field test entries for SBR resistance. Of 24 PIs challenged with a 2014 Florida isolate of P. pachyrhizi in an APHIS-approved greenhouse in Urbana, IL, three Japanese accessions and two Indonesian accessions had no visible SBR lesions when inspected two weeks after inoculation. Eight other PIs developed reddish-brown lesions with low sporulation, indicating a high level of resistance (but not immunity). More than 2,000 lines derived from crosses to plant introductions with resistance to soybean rust, Phomopsis seed decay, or Phytophthora root and stem rot were planted in Urbana or Quincy, FL for agronomic evaluation (with many in replicated plots). Individual F1 plants and F2 populations obtained from crosses to PIs with unexploited resistance to P. sojae were planted. Crosses were made in July and August to 21 other PIs with putative resistance to soil pathogens. Through two Material Transfer Agreements (MTA), 810 breeding lines derived from crosses with rust-resistant PIs were shared with the soybean breeder at Clemson University. U.S. populations of Phakopsora pachyrhizi, the soybean rust fungus, have been found to have considerable pathogenic diversity, both within and across growing seasons. In FY 2015, fungal isolates were collected from three counties in Florida and were obtained from several locations in Louisiana, where they were collected by colleagues from Louisiana State University. Experiments were conducted in growth chambers and greenhouse to assess pathogenic diversity across years in the same location in Florida, and in several different locations in the 2014 growing season. Four pathogenically distinct domestic isolates from previous years were provided to Syngenta through an MTA. To characterize amino acid sequence variability in soybean proteins putatively associated with transmission of Soybean mosaic virus (SMV) through seed, experiments were completed to evaluate a population of 200 soybean PIs for their incidence of SMV seed transmission. As an alternative to bimolecular fluorescence complementation studies, which have been plagued with technical difficulties, the genome of a highly seed transmitted isolate of SMV was modified to express tags for tandem-affinity purification. The modified viruses will be used to identify proteins that interact with SMV proteins in a genotype-specific manner.
1. Discovered new sources of resistance to soybean anthracnose and charcoal rot. Soybean anthracnose and charcoal rot are diseases that occur throughout the major soybean production areas of the world and are capable of producing significant yield losses. No commercial soybean varieties are available with resistance to the two diseases. Consequently, soybean growers cannot purchase resistant soybean varieties. An ARS scientist at Urbana, IL cooperated with University of Illinois researchers to discover several soybean germplasm lines with high levels of resistance to the diseases. These materials will be a valuable resource to soybean researchers working with soybean resistance to economically important fungal diseases. For example, commercial soybean breeders have contacted the ARS scientist about utilizing these sources of resistance in commercial varieties and to have their materials evaluated for resistance to anthracnose and charcoal rot.
2. Developed a culture medium for growth of soybean fungal pathogens based on soybean milk. The medium typically used for growth of soybean fungal pathogens in microbiology laboratories is expensive and does not contain soybean-specific components that could be important for understanding disease etiologies. An ARS scientist at Urbana, IL cooperated with a University of Illinois scientist to evaluate soybean milk as a medium to culture soybean pathogens. Three soybean fungal pathogens, Cercospora sojina, Colletotrichum truncatum and Fusarium virguliforme, grew faster on soybean milk dextrose agar (SDA) than commercial potato dextrose agar (PDA). In addition, Sclerotinia sclerotiorum produced significantly greater masses of survival structures on SDA than PDA. Hence, soymilk used with agar or alone as a broth is an option for replacing more expensive commercial culture media at about one-tenth of the cost. These results provide mycologists, soybean pathologists and others interested in culturing fungi that attack soybean a means to decrease their costs associated with buying commercial media.
3. Developed a genetic system to study the mechanisms by which mycoviruses reduce the virulence of fungal plant pathogens. Sclerotinia sclerotiorum is a fungal plant pathogen that causes necrotic diseases in multiple crop species that result in significant yield losses each year. However, the diseases caused by Sclerotinia have not been adequately controlled by conventional technologies. Mycoviruses have been used successfully to reduce losses caused by some fungal plant pathogens. An ARS scientist at Urbana, IL in collaboration with a researcher at the University of Illinois infected S. sclerotiorum with synthetic copies of a mycovirus RNA genome, which significantly reduced the virulence of the fungus. These results provide direct evidence that hypoviruses can significantly reduce the severity of diseases caused by S. sclerotiorum. The ability to infect the fungus with a genetically defined virus will facilitate investigations into the molecular bases of hypovirulence and aid in the development of effective and targeted biological controls for Sclerotinia diseases.
4. Mapped genes for resistance to soybean rust in soybean plant introductions. In many growing seasons, soybean rust epidemics threaten soybean production in the southern United States and increase production costs by necessitating applications of fungicides to control the disease. In addition, populations of the fungus that cause soybean rust are highly diverse and are capable of rapidly adapting to deployed resistance genes. Hence, soybean cultivars that express multiple genes for rust resistance are essential for protecting soybean yields and profitability in the region. Researchers at the University of Georgia and USDA-ARS scientists at Fort Detrick, MD, Stoneville, MS, and Urbana, IL collaborated to identify and map genes for resistance to soybean rust in more than 70 soybean plant introductions. Over 60% of the resistance genes mapped to the location of a previously identified rust resistance gene. Some of the plant introductions showed differential sensitivity to rust isolates from different geographical regions. This information will help soybean breeders decide which resistance genes to combine to achieve broader and more durable resistance to soybean rust.
5. Showed that the fungus that causes soybean rust does not require melanin accumulation to penetrate leaves. Soybean rust is one of the most important foliar diseases of soybean worldwide. The soybean rust pathogen, Phakopsora pachyrhizi, produces abundant aerial spores. To spread the disease to new plants, rust spores germinate after landing on a soybean leaf, form an appressorium, and then mechanically penetrate the cuticle and epidermis with an infection peg. In many other fungal species, the penetration process involves accumulation of high levels of melanin. Because cell penetration is an essential stage in the infection cycle, understanding the processes involved provide potential targets for control of soybean rust and related fungal diseases. An ARS scientist at Urbana, IL cooperated with University of Illinois researchers to describe the process by which P. pachyrhizi penetrates soybean leaves and showed that the rust fungus is an exception and does not require melanin accumulation to build the turgor pressure needed to penetrate the leaf. This fundamental research will aid soybean pathologists, mycologists, and other scientists in developing strategies to disrupt the penetration processes by which fungi infect plants.
Yang, H., Hartman, G.L. 2015. Methods and evaluation of soybean genotypes for resistance to Colletotrichum truncatum. Plant Disease. 99:143-148.
Pawlowski, M., Hill, C.B., Voegtlin, D., Hartman, G.L. 2015. Soybean aphid intrabiotype variability based on colonization of specific soybean genotypes. Insect Science. 22(1):1-8.
Agindotan, B.O., Domier, L.L., Bradley, C.A. 2015. Detection and characterization of the first North American mastrevirus in Switchgrass. Archives of Virology. 160(5):1313-1317. DOI 10.1007/s00705-015-2367-5.
Murithi, H., Beed, F., Madata, C.S., Haudenshield, J.S., Hartman, G.L. 2014. First report of Phakopsora pachyrhizi on soybean causing rust in Tanzania. Plant Disease. 98(11):1586. DX.DOI.ORG/10.1094/PDIS-06-14-0601-PDN.
Murithi, H., Beed, F., Soko, M., Haudenshield, J.S., Hartman, G.L. 2014. First report of Phakopsora pachyrhizi causing rust on soybean in Malawi. Plant Disease. 99(3):420. doi: 10.1094/PDIS-9-14-0924-PDN.
Pawlowski, M.L., Hill, C.B., Hartman, G.L. 2015. Resistance to charcoal rot identified in ancestral soybean germplasm. Crop Science. 55:1230-1235. DOI: 10.2135/cropsci2014.10.0687.
Xiang, Y., Scandiani, M.M., Herman, T.K., Hartman, G.L. 2015. Optimizing conditions of a cell-free toxic filtrate stem cutting assay to evaluate soybean genotype responses to Fusarium species that cause sudden death syndrome. Plant Disease. 99:502-507.
Lee Marzano, S., Hobbs, H.A., Nelson, B.D., Hartman, G.L., Eastburn, D.E., McCoppin, N.K., Domier, L.L. 2015. Transfection of Sclerotinia sclerotiorum with in vitro transcripts of a naturally occurring interspecific recombinant of Sclerotinia sclerotiorum hypovirus 2 significantly reduces virulence of the fungus. Journal of Virology. 89:5060-5071.