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: Multiple species of the fungal genus Colletotrichum cause anthracnose of soybean. A multiplex real-time PCR was developed to detect and differentiate these species. The fungus, Fusarium virguliforme, produces foliar symptoms in a disease known as sudden death syndrome. Multiple phytotoxins produced by the fungus were identified including a phytotoxic effector. Pathogenic fungi produce plant cell wall degrading enzymes. Differential expression analysis of plant cell-wall-degrading enzymes in the Fusarium virguliforme secretome and structural modeling found that the fungus produces a diverse group of enzymes that degrade plant cellulose and pectin. The complete nucleotide sequence of a new soybean-infecting member of the Nepovirus genus (provisionally named Soybean latent spherical virus [SLSV]) was identified by high-throughput sequencing of RNAs from soybean leaf samples from North Dakota, USA. The sequences of RNAs 1 and 2 were completed by rapid amplification of cDNA ends. Each contained a single long open reading frame and a 3’ nontranslated region of greater than 1,500 nt. The predicted amino acid sequences of the two ORFs were most closely related to nepoviruses in Subgroup C. Full-length cDNAs of RNAs 1 and 2 were cloned and used to inoculate soybean plants, which did not display obvious symptoms. These results suggest that SLSV represents a new species in the genus Nepovirus. For objective 2: A genome-wide association and genomic prediction study identified associated loci and predicted the sensitivity of Tobacco ringspot virus in soybean plant introductions. A field study evaluated disease and pest damage on soybean cultivars released from 1923 through 2008 under field conditions in Central Illinois and showed that, in general, cultivars released more recently had more resistance than older released cultivars. By using a semi-hydroponics system, zinc deficiency was shown to significantly enhance soybean sensitivity to bacterial pustule, Sclerotinia stem rot and soybean aphids. This is the first report on how zinc alters soybean susceptibility to pathogens and pests. In an effort to reduce the use of fungicides, application of plant elicitors may be a useful alternative to drive plant defense systems. Responses of soybean genotypes to pathogen infection after the application of elicitors varied some with a few diseases being reduced after elicitation. A draft genome was assembled for Glycine tomentella, a perennial wild relative of cultivated soybean. The assembled sequence represented approximately 80% of the Glycine tomentella genome with greater than 30-fold coverage. Soybean mosaic virus (SMV), which is transmitted by aphids and through seed, causes significant reductions in soybean yield and seed quality worldwide. In North America, seedborne infections are the primary sources of inoculum for SMV infections. Therefore, host-plant resistance to seed transmission of SMV provides a means to limit the impacts of SMV on soybean production. To identify loci associated with resistance to SMV seed transmission, genome-wide association studies were carried out by combining SMV seed transmission data of two diverse soybean populations and genotypic data derived from publically available molecular marker data for the entries in the USDA Soybean Germplasm Collection. This analysis identified a single locus significantly associated with the rate of SMV seed transmission and a second locus associated with seed yield in SMV-infected plants. The marker most significantly associated with seed transmission was located 1.3 kb upstream of gene Glyma.09g076600, which is annotated as a plant invertase/pectin methylesterase inhibitor (PMEI). Pectin methylesterases play crucial roles in plant-pathogen interactions and disease development. Other researchers showed that over expressing a PMEI gene in transgenic tobacco and Arabidopsis plants constrained the systemic movement of Tobacco mosaic virus. Hence, Glyma.09g076600 represents a good candidate for a gene involved in SMV movement into meristematic tissues. In soybean, Phomopsis seed decay is caused by a seedborne fungus, Diaporthe longicolla, and reduces yields and seed quality. An endophytic and soilborne fungus, Sarocladium kiliense, has been shown to reduce the severity of root rot diseases in tomato and vascular wilt of pistachio trees. The usefulness of S. kiliense as a biocontrol agent for Phomopsis seed decay was investigated. Even though co-inoculation of soybean seeds with both fungi failed to reduce seed rot or increase seed germination, pretreating soybean leaf pieces with S. kiliense conidia for one or three days prior to inoculation with D. longicolla eliminated the development of asexual fruiting bodies completely. The experiments highlight the potential to use S. kiliense as a protectant biocontrol for soybean fungal pathogens that should be further investigated.
1. Characterized the genes expressing plant cell wall-degrading enzymes in the genome of Fusarium virguliforme. The fungus F. virguliforme causes root rot and sudden death syndrome in soybean, both of which produce significant reductions in soybean yields each year. An ARS scientist at Urbana and collaborators showed that F. virguliforme produces a diverse set of cell wall-degrading enzymes, including several unique enzymes not reported in other plant pathogenic fungi. The enumeration of the specific types of cell wall-degrading enzymes produced by F. virguliforme will facilitate strategies to reduce the severity of root rot and sudden death syndrome by expressing proteins that inhibit specific cell wall-degrading enzymes in transgenic soybean plants.
2. Identified multiple phytotoxins produced by the fungus causing sudden death syndrome. Fusarium virguliforme, a soilborne fungal pathogen, causes soybean sudden death syndrome (SDS) by producing toxins in soybean roots that are translocated to leaves where they cause damaging foliar symptoms. ARS scientists at Urbana, IL and cooperators bioinformatically identified F. virguliforme toxin genes that when expressed in plants induced SDS foliar symptoms. The identification of F. virguliforme genes that produce phytotoxins provides important insights into the etiology of this economically important disease and molecular targets for its management.
3. Identified a locus for tolerance to Tobacco ringspot virus (TRSV) in soybean and predicted the sensitivities of soybean plant introductions to TRSV. Tobacco ringspot virus causes a problematic disease in soybean that so far has no management options. ARS scientists at Urbana, Illinois and collaborators identified a single locus on soybean chromosome 2 that was strongly associated with tolerance to TRSV infection and predicted the sensitivities of 18,955 accessions in the USDA soybean germplasm collection to TRSV based on genomic estimates. Because limited resistance is available to TRSV in the soybean gene pool, the identification of a locus for tolerance to TRSV infection provides a potentially valuable tool for management of this soybean disease.
4. Identified and molecularly characterized 49 previously undescribed viruses that infect fungal pathogens of soybean. Soybean provides essential nutrients for both humans and food animals, and is an important source of bioenergy. Each year, fungal diseases significantly reduce soybean yields and seed quality, but some fungal viruses reduce the ability of pathogenic fungi to induce disease. ARS scientists at Urbana and collaborators identified viruses that infect the fungi that cause five widely prevalent soybean diseases: anthracnose, charcoal rot, Phomopsis seed decay, Rhizoctonia root rot and Sclerotinia stem rot. Because some of the viruses reduced the virulence of their fungal hosts, these results widened the range and diversity of biological agents that can be used in the management of fungal diseases of soybean.
Helfenstein, J., Pawlowski, M.L., Hill, C., Stewart, J., Lagos-Kutz, D., Bowen, C.R., Frossard, E., Hartman, G.L. 2015. Zinc deficiency alters soybean susceptibility to pathogens and pests. Journal of Plant Nutrition and Soil Science. 178:896-903.
Kandal, Y.R., Haudenshield, J.S., Srour, A.Y., Islam, K.T., Fakhoury, A.M., Santos, P., Wang, J., Chilvers, M.I., Hartman, G.L., Malvick, D.K., Floyd, C.M., Mueller, D.S., Leandro, L. 2015. Multi-laboratory comparison of quantitative PCR assays for detection and quantification of Fusarium virguliforme from soybean roots and soil. Phytopathology. 105(12):1601-1611.
Hartman, G.L., Pawlowski, M.M., Chang, H., Hill, C.B. 2016. Successful technologies and approaches used to develop and manage resistance against crop diseases and pests. In: Macramootoo, C. editor. Emerging Technologies for Promoting Food Security. Sawston,Cambridge; Woodhead Publishing. p. 43-66.
Marzano, S.L., Domier, L.L. 2016. Novel mycoviruses discovered from metatranscriptomics survey of soybean phyllosphere phytobiomes. Virus Research. 213(2):332-342.
Bekal, S., Domier, L.L., Gonfa, B., Lakhssassi, N., Meksem, K., Lambert, K.N. 2015. A SNARE-like protein and biotin are implicated in soybean cyst nematode virulence. PLoS One. doi: 10.1371/journal.pone.0145601.
Harbach, C.J., Allen, T.W., Bowen, C.R., Davis, J.A., Hill, C.B., Leitman, M., Leonard, B.R., Mueller, D.S., Padgett, G.B., Phillips, X.A., Schneider, R.W., Sikora, E.J., Singh, A.K., Hartman, G.L. 2016. Delayed senescence in soybean: Terminology, research update, and survey results from growers. Plant Health Progress. 17:76-83.
Chang, H., Brown, P.J., Lipka, A.E., Domier, L.L., Hartman, G.L. 2016. Genome-wide association and genomic prediction identifies associated loci and predicts the sensitivity of Tobacco ringspot virus in soybean plant introduction. Biomed Central (BMC) Genomics. 17:153. doi:10.1186/s12864-12016-12487-12867.
Chang, H., Domier, L.L., Radwin, O., Yendrek, C., Hudson, M., Hartman, G.L. 2016. Identification of multiple phytotoxins produced by Fusarium virguliforme including a phytotoxic effector (FvNIS1) associated with soybean sudden death syndrome foliar symptoms. Molecular Plant-Microbe Interactions. 96:96-108.
Hartman, G.L., Bowen, C.R., Haudenshield, J.S., Fox, C.M., Cary, T.R., Diers, B.W. 2015. Evaluation of disease and pest damage on soybean cultivars released from 1923 through 2008 under field conditions in Central Illinois. Agronomy Journal. 107:2373-2380.
Hartman, G.L., Pawlowski, M.L., Herman, T.K., Eastburn, D.M. 2016. Organically grown soybean production in the USA: Constraints and management of pathogens and insect pests. Agronomy. 6(1):16. doi:10.3390/agronomy6010016.
Kelly, H.Y., Dufault, N.S., Walker, D.R., Isard, S.A., Schneider, R.W., Giesler, L.J., Wright, D.L., Marois, J.J., Hartman, G.L. 2015. From select agent to established pathogen: The response to Phakopsora pachyrhizi (soybean rust) in North America. Phytopathology. 105(7):905-916.
Pawlowski, M.L., Bowen, C.R., Hill, C.B., Hartman, G.L. 2016. Responses of soybean genotypes to pathogen infection after the application of elicitors. Crop Protection Journal. 87:78-84.
Rincker, K., Hartman, G.L., Diers, B.W. 2016. Fine mapping of resistance genes from five brown stem rot resistance sources in soybean. The Plant Genome. doi:10.3835/plantgenome2015.08.0063.
Weems, J.D., Haudenshield, J.S., Bond, J.P., Hartman, G.L., Ames, K.A., Bradely, C.A. 2015. Effect of fungicide seed treatments on Fusarium virguliforme infection of soybean and development of sudden death syndrome. Canadian Journal of Plant Pathology. 37:435-447.
Yang, H., Haudenshield, J.S., Hartman, G.L. 2015. Multiplex real-time PCR detection and differentiation of Colletotrichum species infecting soybean. Plant Disease. 99:1559-1568.
Chang, H., Yendrek, C.R., Caetano-Anolles, G., Hartman, G.L. 2016. Genomic characterization of plant cell wall degrading enzymes and in silico analysis of xylanses and polygalacturonases of Fusarium virguliforme. BMC Microbiology. 16:147. doi: 10.1186/s12866-016-0761-0.
Divilov, K., Walker, D.R. 2016. Reaction of Diaporthe longicolla to a strain of Sarocladium kiliense. Biocontrol Science and Technology. 26(7):938-950.