Research Project: Integrated Management of Soybean Pathogens and Pests

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

2019 Annual Report

Objectives
Objective 1. Relate the spatial and temporal dynamics of soybean pathogens, pests, and associated microbial communities to soybean productivity. Subobjective 1.A. Determine if novel virulent or resistance-breaking soybean pathogens/pests have emerged within the U.S. and other parts of the world. Subobjective 1.B. Determine the impact of selected biocontrol and beneficial microbes to reduce the impact of soybean pathogens and pests. Subobjective 1.C. Characterize variability and shifts in the pathogenicity of Phakopsora pachyrhizi populations in the southern U.S. to guide breeding program decisions. Objective 2: Identify, characterize, and develop improved resistance in soybean that can be used for sustainable disease management strategies that include effective host resistance and biological control. Subobjective 2.A. Identify or characterize pathogen/pest resistance using annual and perennial accessions from the USDA Soybean Germplasm Collection and selected breeding lines. Subobjective 2.B. Develop agronomically competitive soybean breeding lines with disease- or pest-resistance genes from adapted or unadapted germplasm accessions in the USDA Soybean Germplasm Collection. Subobjective 2.C. Investigate relationships between soybean yields and resistance to soybean cyst nematode and Phytophthora sojae in public breeding lines from the Northern Uniform/Preliminary Soybean Tests.

Approach
The distribution and abundance of soybean pathogens and pests will be monitored on multiple geographic scales using pathogen-specific and metagenomic assays. The impacts of beneficial and insect-borne microbes on soybean diseases and yields will be characterized in replicated trials over multiple years. Changes in pathogen virulence over time will be assessed using soybean lines expressing different pathogen resistance genes and pathogen populations collected from soybean fields each year. New sources of resistance to pathogens 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. Regions of soybean chromosomes associated with pathogen/pest resistance will be identified using phenotypic assays and molecular marker analyses of derived mapping populations. Soybean lines shown to be resistant to soybean pathogens/pests will be used to produce breeding lines with enhanced resistance using phenotypic and marker-assisted selection techniques as appropriate.

Progress Report
For objective 1 (Relate the spatial and temporal dynamics of soybean pathogens, pests, and associated microbial communities to soybean productivity): Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, was discovered in Ethiopia infecting soybean for the first time. Morphological and molecular analyses verified that the identified pathogen was S. sclerotiorum. Arbuscular mycorrhizal fungi (AMF) form associations with most terrestrial plant species and assist them by increasing their nutrient uptake, drought tolerance, and resilience against pathogens and pests in a host-genotype-specific manner. To identify genomic regions associated with mycorrhizal colonization in soybean, a diverse panel of 350 exotic soybean accessions were inoculated with AMF and microscopically evaluated for root colonization, which differed significantly among genotypes. Analysis of phenotypic and molecular marker data identified six chromosomal regions significantly associated with extent of root colonization. The results showed there was a substantial genetic component in soybean to the level of colonization by AMF. Bacterial tan spot, an important disease of beans, was first reported on soybean in Iowa in 1975 and was subsequently reported from other locations in the United States and from Brazil and Germany. However, the disease had not been reported on soybeans grown in Africa. In 2018, leaflets from plants showing symptoms of what appeared to be bacterial tan spot were collected from research plots in Zambia. Molecular, morphological and pathological analysis confirmed the presence of the bacterial species causing tan spot. We extracted DNA from the intestinal tract of 286 stinkbugs and conducted PCR amplification of conserved ribosomal regions with this DNA. After data filtering, we kept the bacterial ribosomal sequence data from 191 samples and kept the fungal ribosomal sequence data from 215 samples. These sequences were analyzed by IM-Tornado and Qiime software packages to identify putative microbes, determine some statistics associated with these sequences and predicted associated microbes. Subsequently, we isolated 42 culturable microbes from the red-banded stinkbug. DNA was extracted, and conserved ribosomal regions were PCR amplified and sequenced to provide sequence information to help identify the microbes. We recovered good PCR amplicons and usable sequence data for all but 14 of the isolates. We will repeat the DNA extractions and continue to try to obtain clean PCR products for these 14 isolates. For Objective 2 (Identify, characterize, and develop improved resistance in soybean that can be used for sustainable disease management strategies that include effective host resistance and biological control): Trichoderma are soil- and root-inhabiting fungi that can serve as plant growth promoters and biocontrol agents. The bio efficacy of two Trichoderma species from Brazil and the USA was evaluated against isolates of Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot or white mold of soybean. The two Trichoderma species suppressed reproduction of S. sclerotiorum. Soybean seeds coated with the Trichoderma species showed substantially improved seed germination and challenged with S. sclerotiorum. Even in the absence of S. sclerotiorum, the Trichoderma species increased biomass of shoots and roots, and root nodulation. These results underscore the potential of using Trichoderma species to protect soybean plants against the white mold fungus and highlights the growth promoting attributes of Trichoderma species for increasing soybean productivity. Sclerotinia stem rot or white mold is a destructive disease of soybean caused by Sclerotinia sclerotiorum. Low levels of resistance are available to white mold in cultivated soybean but some of soybean’s perennial relatives show higher levels of resistance to the disease. To identify alternative sources of resistance to white, a recombinant inbred line population produced from a cross between perennial relatives of soybean resistant or susceptible to white mold were assayed for their sensitivity to oxalic acid, a pathogenicity determinant for S. sclerotiorum, and analyzed for molecular markers. The molecular marker data and phenotypic data from oxalic acid analysis and sensitivity to the white mold fungus (analysis in progress) will be analyzed to identify chromosomal regions associated with the resistance traits in the perennial species. A soilborne fungus causes sudden death syndrome of soybean in North America, which is estimated to suppress soybean yields by over $0.5 billion annually. Resistance to the fungal pathogen in soybean is genetically complex. A genome-wide association study using 254 soybean plant introductions (PIs) and disease ratings of sudden death syndrome foliar and root rot symptoms revealed 14 chromosomal regions associated with resistance to foliar sudden death syndrome and eight with root rot resistance. The analysis identified 10 PIs with high levels of resistance and four candidate genes that may be useful in soybean breeding programs for resistance to sudden death syndrome and root rot resistance. Viruses that infect pathogens and pest of soybean are potentially useful for biological disease control and as tools to study the interactions between soybean plants and pathogens. Sugar beet cyst nematode virus 1 (SBCNV1) infects both sugar beet cyst nematodes and soybean cyst nematodes (SCN). To investigate the potential utility of SBCNV1 for use as a biological control agent for SCN, copies of the SBCNV1 genome were cloned into plasmid vectors and used to produce synthetic RNA copies of the virus genome. SCN second-stage juveniles were bombarded with gold particles coated with synthetic SBCNV1 RNA and subsequently inoculated to soybean seedlings. Accumulation of SBCNV1 RNA was detected in nematodes that had been bombarded with synthetic RNAs, but not in the negative controls. Subsequently, a sequence encoding green fluorescent protein (GFP) was inserted into the SBCNV1 genome to permit visualization of SBCNV1-infected nematodes. Following production of synthetic SBCNV1 RNA and bombardment of nematodes, a small number of SCN were observed showing GFP fluorescence. In experiments with wild-type and modified SBCNV1 RNA, the levels of virus infection have been very low, suggesting that modifications to the cloned SBCNV1 sequence and/or methods used for inoculation may be needed to achieve usable numbers of infected nematodes. Investigations of resistance of soybean to five species of the soilborne fungus Pythium were continued. Pythium fungi can kill or seriously damage soybean seed and young seedlings as they emerge from the ground in early spring. Resistance to Pythium fungi could help to reduce seedling rot when soils remain cool and damp after early planting dates, which are now popular with producers. Analysis confirmed the resistance of some germplasm accessions to specific Pythium species, but the resistance of some other soybean accessions was not confirmed. Accession by isolate/species interactions were better characterized and resistance assay protocols were improved to obtain better reproducibility. Crosses were made between high-yielding breeding lines and Pythium resistant cultivars from Canada to develop breeding lines and segregating populations that can be used to map genes associated with resistance to different Pythium species.

Accomplishments
1. Discovered trichothecene-producing fungi in the genus Fusarium associated with soybean roots in Ethiopia and Ghana. Trichothecene mycotoxins produced by Fusarium fungi can be toxic to plants and inhibit seed germination. Little research has been conducted to characterize Fusarium pathogens of soybean in Sub-Saharan Africa. ARS scientists at Urbana and Peoria, Illinois (i) identified Fusarium species isolated from diseased soybeans grown in Ethiopia and Ghana, (ii) determined whether selected isolates produced trichothecene mycotoxins in culture, and (iii) tested these isolates for pathogenicity on soybean. Molecular phylogenetic analyses revealed the trichothecene mycotoxin-producing isolates comprised three previously undescribed Fusarium species. Nearly half of the toxin-producing isolates tested from Ethiopia and Ghana completely inhibited germination of soybean seeds, and all isolates produced lesions on inoculated soybean roots. This was the first study characterizing toxin-producing Fusarium species from soybean roots in Ethiopia and Ghana. This information is important to soybean breeders and soybean producers concerned about the impact of these soybean root rotters and toxin producers on soybean production.

2. Determined the effectiveness of selected biopesticides in reducing the severity of soybean rust. Soybean rust is a fungal disease of soybean that has produced yield losses of up to 60% in the southern United States. The intensive use of synthetic fungicides for controlling soybean rust has accelerated the emergence of fungal populations with reduced sensitivity to synthetic fungicides. To provide alternatives to synthetic fungicides, ARS scientists at Urbana, Illinois, cooperated with a University of Illinois researcher to determine if application of biopesticides would reduce sporulation of the soybean rust fungus. One biopesticide made from the bacterium Bacillus subtilis caused particularly high reductions in sporulation of the soybean rust fungus. The identification of biopesticides that effectively reduce soybean rust may be valuable alternatives or compliments to synthetic fungicides that could be useful in integrated pest management programs for soybean rust control. This information is important to soybean growers, extension agents, and others interested in the use of non-synthetic fungicides to control soybean rust.

3. Mapped genes for resistance to transmission of soybean mosaic virus (SMV) through soybean seed to germinating seedlings. SMV causes significant reductions in soybean yield and seed quality. Because seedborne infections can serve as primary sources of inoculum for SMV infections, resistance to SMV seed transmission provides a means to limit the impact of SMV. An ARS scientist at Urbana, Illinois, cooperated with University of Illinois researchers to evaluate two diverse sets of soybean lines from the USDA Soybean Germplasm Collection for SMV seed transmission rate, seed coat mottling, and seed yield from SMV-infected plants. When the trait data were analyzed along with molecular marker data, genes controlling the three traits were found clustered in narrow regions on two soybean chromosomes. The results suggest that a common set of genes may control the three traits. The results of this study provide additional insight into the genetic architecture of these three important traits and suggest candidate genes for downstream functional validation. This work will be of interest to researchers working to limit the impacts of SMV infection on soybean or studying the mechanisms of transmission of plant viruses through seed.

Review Publications
Pawlowski, M., Helfenstein, J., Frossard, E., Hartman, G.L. 2019. Boron and zinc deficiencies and toxicities and their interactions with other nutrients in soybean roots, leaves, and seeds. Journal of Plant Nutrition. 41(6):634-649. https://doi.org/10.1080/01904167.2019.1567782.
Lagos-Kutz, D.M., Potter, B., DiFonzo, C., Russell, H., Hartman, G.L. 2018. Two aphid species, Phorodon cannabis and Rhopalosiphum rufiabdominale, identified as potential pests on industrial hemp, Cannabis sativa L., in the US Midwest. Crop, Forage & Turfgrass Management. 4:180032. https://doi.org/10.2134/cftm2018.04.0032.
Yuan, C., Zhang, L., Zhao, H., Wang, Y., Liu, X., Dong, Y., Hartman, G.L. 2019. RNA-seq analysis for soybean cyst nematode resistance of Glycine soja (wild soybean). Chinese Journal of Oil Crop Sciences. 4(1):32-46.
Haudenshield, J.S., Pawlowski, M.L., Miranda, C., Hartman, G.L. 2018. First report of Paramyrothecium roridum causing Myrothecium leaf spot on soybean in Africa. Plant Disease. https://doi.org/10.1094/PDIS-04-18-0624-PDN.
Muturi, E.J., Lagos-Kutz, D.M., Dunlap, C.A., Ramirez, J.L., Rooney, A.P., Hartman, G.L., Fields, C., Rendon, G., Kim, C. 2018. Mosquito microbiota cluster by host sampling location. Parasites & Vectors. 11:468. https://doi.org/10.1186/s13071-018-3036-9.
Swaminathan, S., Das, A., Assefa, T., Knight, J.M., Ferreira Da Silva, A., Hartman, G.L., Huang, X., Leandro, L.F., Cianzio, S.R., Bhattacharyya, M.K. 2019. Genome wide association study identifies novel single nucleotide polymorphic loci and candidate genes involved in the expression of sudden death syndrome resistance in soybean. PLoS One. https://doi.org/10.1371/journal.pone.0212071.
Hartman, G.L., McCormick, S.P., O'Donnell, K. 2019. Trichothecene production and pathogenicity of Fusaria isolated from symptomatic soybean roots in Ethiopia and Ghana. Plant Disease. https://doi.org/10.1094/PDIS-12-18-2286-RE.
Childs, S.P., King, Z.R., Walker, D.R., Harris, D.K., Pedley, K.F., Buck, J.W., Boerma, H.R., Li, Z. 2017. Discovery of a seventh Rpp soybean rust resistance locus in soybean accession PI 605823. Theoretical and Applied Genetics. 131:27-41. https://doi.org/10.1007/s00122-017-2983-4.
Walker, D.R. 2018. Advances in disease-resistant varieties of soybean. In: Nguyen, H.T., editor. Achieving Sustainable Cultivation of Soybeans Volume 2: Diseases, pests, food and other uses. Cambridge, UK: Burleigh Dodds Science Publishing. p. 105-143.
Rod, K.S., Walker, D.R., Bradley, C.A. 2018. Evaluation of major ancestors of North American soybean cultivars for resistance to Pythium species that cause seedling blight. Plant Disease. 102:2241-2252. https://doi.org/10.1094/PDIS-09-17-1341-RE.
Chang, H., Tan, R., Hartman, G.L., Wen, Z., Sang, H., Domier, L.L., Whitham, S.A., Wang, D., Chilvers, M.I. 2019. Characterization of soybean STAY-GREEN genes in susceptibility to foliar chlorosis of Sudden Death Syndrome. Journal of Plant Physiology. 180:711-717. https://doi.org/10.1104/pp.19.00046.
Keough, S., Danielson, J., Marshall, J.M., Lagos-Kutz, D.M., Voegtlin, D., Srinivasan, R., Nachappa, P. 2018. Factors affecting population dynamics of thrips vectors of soybean vein necrosis virus. Environmental Entomology. 47(3):734-740. https://doi.org/10.1093/ee/nvy021.
Wen, L., Chang, H., Brown, P.J., Domier, L.L., Hartman, G.L. 2019. Genome-wide association and genomic prediction identifies soybean cyst nematode resistance in common bean including a syntenic region to soybean Rhg1 locus. Horticulture Research. 6:9. https://doi.org/10.1038/s41438-018-0085-3.
Twizeyimana, M., Hartman, G.L. 2019. Effect of selected biopesticides in reducing soybean rust (Phakopsora pachyrhizi) development. Plant Disease. https://doi.org/10.1094/PDIS-02-19-0384-RE.
Pawlowski, M.L., Murithi, H., Hailemariam, M., Tesfaye, A.A., Hartman, G.L. 2019. First report of Sclerotinia sclerotiorum causing stem rot on soybeans in Ethiopia. Plant Disease. https://doi.org/10.1094/PDIS-05-19-0929-PDN.
Ju, H., Kim, I., Hu, W., Kim, B., Choi, G., Kim, J., Lim, Y., Domier, L.L., Hammond, J., Lim, H. 2019. A single nucleotide change in the overlapping MP and CP reading frames results in differences in symptoms caused by two isolates of Youcai mosaic virus. Archives of Virology. 164(6):1553-1565. https://doi.org/10.1007/s00705-019-04222-z.
Kim, B., Cho, I., Kim, I., Choi, G., Ju, H., Hu, W., Oh, J., Kim, J., Seo, E., Domier, L.L., Hammond, J., Lim, H. 2019. Length of poly(A) tail affects transcript infectivity of three ZYMV symptom variants differing at only five amino acid positions. Journal of Plant Pathology. 101:1187-1193. https://doi.org/10.1007/s42161-019-00316-4.
Kim, I., Ju, H., Gong, J., Han, J., Seo, E., Cho, S., Hu, W., Choi, S., Lim, Y., Domier, L.L., Hammond, J., Lim, H. 2019. A Turnip mosaic virus determinant of systemic necrosis in Nicotiana benthamiana, and a novel resistance-breaking determinant in Chinese cabbage identified from chimeric infectious clones. Phytopathology. 109:1638-1647. https://doi.org/10.1094/phyto-08-18-0323-r.
Marzono, S.L., Neupane, A., Domier, L.L. 2018. Transcriptional and small RNA responses of the white mold fungus Sclerotinia sclerotiorum to infection by a virulence-attenuating hypovirus. Viruses. 10:713. https://doi.org/10.3390/v10120713.
Hu, W., Seo, E., Cho, I., Kim, J., Ju, H., Kim, I., Choi, G., Kim, B., Ahn, C., Domier, L.L., Hammond, J., Lim, H. 2019. Amino acid differences in the N-terminal half of the polyprotein of Chinese turnip mosaic virus isolates affect symptom expression in Nicotiana benthamiana and radish. Archives of Virology. 164(6):1683-1689. https://doi.org/10.1007/s00705-019-04242-9.