Detection and Prevalence of Oomycete Seedling Diseases on Soybean

Martin Chilvers, Michigan State University; Austin McCoy, Michigan State University; Zachary Noel, Auburn University; Alejandro Rojas, University of Arkansas; Travis Faske, University of Arkansas; Daren Mueller, Iowa State University; Damon Smith, University of Wisconsin-Madison; Albert Tenuta, Ontario Ministry of Agriculture, Food, and Rural Affairs; and Kiersten Wise, University of Kentucky.

The oomycetes Pythium and Phytophthora are two of the main soilborne organisms causing seedling diseases of soybean in the United States and Ontario. Soybean losses due to seedling diseases have averaged over $525 million each year for the last ten years (Crop Protection Network Disease Loss, 2020). Oomycetes are more closely related to brown algae and plants than they are to fungi, and numerous oomycetes have adapted to infect plants, including corn and soybean. Wet, saturated soil favors seedling diseases caused by Pythium and Phytophthora. Cool temperatures can also favor certain Pythium species, while Phytophthora and certain Pythium species prefer warmer soil conditions. Production practices, such as reduced or no-tillage, earlier planting, and seed treatments can also influence Pythium and Phytophthora populations in the soil. Identifying the cause of the seedling disease, including accurately identifying the oomycete species, is important to optimize soybean seedling disease management, select the most appropriate varieties, and reduce losses. 

Research goals

• Identify oomycete species causing soybean seed and seedling root rot

• Determine conditions that favor various pathogenic species and disease

• Improve seedling disease management through understanding of which species are causing diseases

New tools for oomycete species identification 

Most seedling diseases that occur early in the season are difficult to differentiate in the field and even in the laboratory. Recent studies using both traditional isolation and high-throughput DNA sequencing techniques have helped to identify and determine the abundance of Pythium species that cause seedling disease (Noel et al. submitted(ab), Rojas 2017a, b, Rojas et al. 2019).

The identification of seedling disease by visual observations alone is often impossible but plant diagnostic labs across the U.S., such as the National Plant Diagnostic Network (NPDN), are available to assist farmers, agronomists, and homeowners with the identification of pests and plant diseases. Plant diagnosticians have traditionally isolated the causal organism of disease by placing affected plant parts on to special agar culture medium in which the organism can grow independently from the affected plant parts. Once the organism is isolated and in pure culture, examination of physical characteristics and/or DNA sequencing are done to confirm the causal agent’s identity (Kox et al. 2007). Unfortunately, the isolation and identification processes can be time consuming, and costly. Serological testing on plant parts is widely used to speed up time to diagnosis; however, current serological tests for Phytophthora can, and do, cross react with some Pythium species, leading to incorrect diagnosis and other methods such as DNA sequencing are necessary to confirm species identity (MacDonald et al. 1990). DNA sequencing technologies are accurate, but also take 2-3 days for results. 

New technologies are now available that can decrease the time between sample acquisition to causal organism identification from days to hours. These technologies come in two forms: laboratory-based testing (quantitative PCR - qPCR) and isothermal assays (RPA and LAMP). Laboratory-based assays like qPCR can quantify how much organism DNA is in a sample, not just presence or absence. These diagnostic assays (qPCR, RPA, and LAMP) are available for Phytophthora with some assays accurate down to the species level (Dai et al. 2012, McCoy et al. 2020, Miles et al. 2015, Miles et al. 2017, Rojas et al. 2017c, Tomlinson et al. 2010). 

Assays such as RPA and LAMP can be used in the field for identification without the need for complicated laboratory equipment. Currently, there are few assays to accurately identify Pythium species, but work is underway to design, optimize, and validate Pythium specific tests so that they will be available in the future (Miles et al. 2016).

High-throughput DNA sequencing techniques of plant and soil samples is an emerging tool for plant pathologists (Figure 1). These techniques rely on “in-depth sequencing” in which all DNA sequences present in a sample are compared to known specimen DNA sequences for identification. These data can be used to identify the presence or absence of organisms, the abundance of those organisms in the soil, as well as differences in the soil microbial communities between field locations. This work is of particular interest to farmers working toward “prescription seed treatments” or seed treatment, variety selection, or crop rotation recommendations for a single field based on its unique soil microbial community.

The research

Soybean seedlings were collected from multiple fields across the Midwest. Oomycete species were isolated from the seedlings and identified using molecular techniques in the laboratory. Eighty-four different oomycete species grew from pieces of infected soybean roots and were screened for their ability to cause disease on soybean seeds and seedlings. Of the 84 oomycete species isolated, 43 were found to cause disease on soybean seeds or seedlings (Figure 2). 

The number of individual oomycete species recovered from infected soybean roots were counted and abundance profiles for each location were created (Figure 3). Pythium species were found in every field that was sampled (Rojas et al. 2017a, b). However, the abundance of individual Pythium species varied from state to state. Phytophthora sojae was also found but was far less abundant than Pythium species. Oomycete communities were significantly different when comparing across state lines; however, neighboring states were more similar than those geographically separated (Figure 3).

This study demonstrates the abundance and diversity of oomycetes in soybean fields and that species diversity may differ from one field to another. This information indicates the need for future work in “prescription” based seed treatment recommendations for individual fields or site-specific management zones. Companies are interested in tailoring specific seed treatments to specific regions; however, soil oomycetes are complex and more information is needed before this becomes common practice.

High-throughput DNA sequencing is also being used to investigate the causal organisms of seedling disease. To date, high-throughput DNA sequencing results have demonstrated that oomycete communities are unique by geographic location which supports previous isolation data. This tool is currently being used to determine the effect of fungicide seed treatments, bio-fungicides, soybean varieties, agronomic practices, and soil properties on the oomycete communities associated with soybean roots. Knowing which species are present is helpful for management decisions, such as a seed treatment or soybean variety selection.

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References

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This research summary was based on the following manuscripts

McCoy, A. G.,  Miles, T. D., Bilodeau, G. J., Woods, P., Blomquist, C., Martin, F. N., and Chilvers, M. I. 2020. Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora species. Plants, 9(4):466. Article / Google Scholar 

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Rojas, J. A., Witte, A., Noel, Z. A., Jacobs, J. L., and Chilvers, M. I. 2019. Diversity and Characterization of Oomycetes Associated with Corn Seedlings in Michigan. Phytobiomes Journal, 3(3):224-234. Article / Google Scholar

Acknowledgments 

Authors

Martin Chilvers, Michigan State University; Austin McCoy, Michigan State University; Zachary Noel, Auburn University; Alejandro Rojas, University of Arkansas; Travis Faske, University of Arkansas; Daren Mueller, Iowa State University; Damon Smith, University of Wisconsin-Madison; Albert Tenuta, Ontario Ministry of Agriculture, Food, and Rural Affairs; and Kiersten Wise, University of Kentucky.

All photos were provided by and are the property of the authors and reviewers.

The authors wish to acknowledge the Michigan Soybean Promotion Committee, North Central Soybean Research Program, the United Soybean Board, and the United States Department of Agriculture - National Institute of Food and Agriculture (USDA-NIFA) for generous support of this research. 

This information in this publication is only a guide, and the authors assume no liability for practices implemented based on this information. Reference to products in this publication is not intended to be an endorsement to the exclusion of others that may be similar. Individuals using such products assume responsibility for their use in accordance with current directions of the manufacturer.