Source: UNIVERSITY OF DELAWARE submitted to NRP
SYSTEMS-LEVEL CHARACTERIZATION OF PYTHIUM PATHOGENESIS, AN EMERGING THREAT TO MAIZE PRODUCTION.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1029798
Grant No.
2023-67013-39165
Cumulative Award Amt.
$749,865.00
Proposal No.
2022-08799
Multistate No.
(N/A)
Project Start Date
Mar 1, 2023
Project End Date
Feb 29, 2028
Grant Year
2023
Program Code
[A1112]- Pests and Beneficial Species in Agricultural Production Systems
Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717
Performing Department
(N/A)
Non Technical Summary
In conjunction with extreme spring rains, changes in farming practices such as reduced tillage, a push for earlier planting dates, and "green bridges'' from cover crops have led to a rise in maize seedling disease incidence and subsequent death. Pathogen surveys have identified Pythium species as one source of disease and an emerging threat to maize production. Economic impacts of Pythium can include increased production costs associated with replanting and season long reductions in plant vigor that lower yield potential due to smaller ears or completely barren plants. Patterns of Pythium infection are sporadic and there are no management strategies to combat Pythium once symptoms are observed, making it difficult for growers to overcome this threat. Improved management relies on filling knowledge gaps of Pythium distribution and abundance in field soil relative to climatic variability and quantifying the impacts to plant growth and mechanics. The objectives of this project are to 1) Characterize load and distribution of Pythium spp. in field soil; 2) Quantify the impact of Pythium infection on maize root-type specific growth; 3) Assess Pythium impact on early season plant biomechanics to predict yield impacts. Through this project, we will address the increased invasiveness of Pythium and provide a foundational understanding of Pythium pathogenesis. Improved understanding of host responses will provide a basis for developing mitigation strategies for Pythium-related crop losses to increase yield, productivity, and economic potential for maize producers.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121510110250%
2161510104015%
2161510102015%
2161510202020%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 216 - Integrated Pest Management Systems;

Subject Of Investigation
1510 - Corn;

Field Of Science
1040 - Molecular biology; 2020 - Engineering; 1102 - Mycology; 1020 - Physiology;
Goals / Objectives
The goal of this project is to providea foundation to develop effectivePythium mitigation and management strategies for growers. Our central hypothesis is that load and distribution of differentPythiumspp. within the soil alters the severity of disease, which is mediated by a reduction in root growth and changes in plant mechanics that can be used to predict yield impacts. We have defined the following objectives to address the central hypothesis and answer open questions regarding howPythiumspp. are concentrated and distributed within fields, howPythiumspp. impact maize root growth, and if early season plant metrics froPythiuminfected plants can be used to predicts end of season yield impacts.Objective 1: Characterize load and distribution of Pythium spp. in field soilObjective 2: Quantify the impact of Pythium infection on maize root-type specific growthObjective 3: Assess Pythium impact on early season plant biomechanics to predict yield impacts
Project Methods
Objective 1: Characterize load and distribution of Pythium spp. in field soilMethodology:We will establish sampling gridsin grower cooperator field sites (Willin Farms, LLC) with a history of Pythium in maize.At maize growth stage vegetative (V2 to V3), paired sets consisting of a symptomatic (stunted, chlorotic, or necrotic) plant and a neighboring symptomless plant will be marked. Three sets will be destructively sampled at V2-V3 for species identification, while five sets will be marked and tracked through the season. Soil cores will be collected from the base of symptomatic and asymptomatic maize plants in each marked set using a 2.5 cm diameter soil probe. Soil cores 25 cm in depth will be collected and partitioned in 7.5 cm intervals for 3 total depth divisions (1-8.3 cm, 8.3-16.6 cm, and 16.6-25 cm). Each depth will be transferred to a separate plastic bag. We will target to collect 5-10 g of soil for each depth division bulked by symptomatic or symptomless plant type across the grid.A minimum of 2 grids will be set up within each field site (n=3). Soil samples will be collected at maize growth stage V2 to V3, vegetative tassel (VT), and dent (R5).Species quantification through plate assays.Within each grid, a subset of three symptomatic and three asymptomatic plants will be destructively sampled at the V2-V3 timing to plate out roots to confirm presence or absence of Pythium and identify species distribution. For each isolate, the ITS1-5.8S-ITS2 (ITS) (White et al. 1990) and COXII (Martin 2000) gene regions will be amplified for identification to species. Active cultures will be maintained for the duration of the project and a representative set of isolates acquired in this objective will be prepared for long term sterile water storage to be maintained for use in laboratory and greenhouse trials.Clade quantification of soil samples by diagnostic qPCR.For this experiment, DNA will be isolated from 100 mg of each soil sample using the DNeasy Powerlyzer Powersoil Kit. Quantification of Pythium clade B (arG190mod) and F (IrSy1123mod) will be completed using TaqMan probes reported in Acharya et al. 2017 and tested in our preliminary work. ITS6 and ITS7 TaqMan probes will be used to quantify general oomycete load as well. Absolute quantification of Pythium will be determined by the cycle threshold (Ct) extrapolated to the standard curve of known concentrations per 250 mg of soil using a QuantStudio3 qPCR machine.Objective 2: Quantify the impact of Pythium infection on maize root-type specific growthMethodology:Using the single hybrid maize line and two Pythium species, we will assess root type specific responses to Pythium infection using two complementary approaches.Plate-based Assays First, we will develop plate-based assays to quantify the response of the embryonic root system to Pythium infection. For plate-based assays, maize seeds will be sterilized with 35% v/v hydrogen peroxide in sterile water for 20min on a shaker followed by three washes with sterile water. Individual seeds will be plated on sterile petri dishes with 1% agar and Hoagland's nutrient solution. Maize seedlings will be inoculated with a single 6.75 mm agar plug from a 6-day old Pythium culture and control plates will be inoculated with a sterile agar plug. We will assess the primary and seminal roots of maize plants infected with Pythium for:Gross changes in morphology.After infection, roots will be imaged every-other day for up-to-3-weeks using a stereomicroscope to quantify gross changes in morphology after infection.Root growth will be tracked by marking plates daily and scanning plates at the end of the experiment to measure daily root growth. If there are gross changes in root morphology such as root hair development or lateral root formation, images will be quantified with publicly available software as appropriate. The quantitative-plant.org site collates the latest plant image analysis software tools and will be references for the appropriate analysis pipeline depending on the changes observed.Localization of Pythium within roots.We will develop confocal imaging assays to visualize the localization of Pythium within the roots. Specifically, roots will be fixed with 4% paraformaldehyde (PFA) by vacuum infiltration with a bell jar for 1 hr at room temperature and then rinsed with 1X phosphate buffered saline (PBS). Samples will be transferred to ClearSee (Kurihara et al. 2015) overnight at room temperature for tissue clearing. Samples will be stained with Calcofluor White in ClearSee to visualize cell walls of both plant and oomycete structures as described in Ursache et al. 2018. In previous work with maize leaf fungi, the hyphal structures were indistinguishable by structure, therefore we will additionally test a variety of dyes (e.g. aniline blue, safranin, ruthenium red, solophenyl flavine, propidum iodide, trypan blue with lactophenol and wheat germ agglutinin) to distinguish the Pythium structures from plant structures. Images will be obtained in whole mount confocal imaging on a Zeiss 780 or 880 microscope. As oomycete colonization occurs at the surface of the root, the penetration of the confocal should be sufficient to visualize several cell layers into the root. However, if the confocal whole mount images are unsuccessful, we will also use microtome sectioned material and/or a multi-photon microscope to localize the two Pythium species within maize root structures.Rhizobox-based Assays To complement the plate-based assays, we will assess embryonic and post-embryonic root growth in the rhizoboxes that we highlighted in our preliminary analysis. Using the same hybrid line and two Pythium spp., we will first optimize the greenhouse conditions to elicit disease symptoms.Optimization of rhizobox inoculations.We will first set up a series of experiments to assess the variation in response to Pythium load by altering the amount of cornmeal inoculum in each rhizobox. Alternatively, Pythium thrives in wet environments, so we will also experiment with different irrigation strategies to determine if the moisture conditions are what have limited symptom development in the greenhouse.Comparison of embryonic and post-embryonic roots.After optimization, we will then observe the embryonic and post-embryonic roots in Pythium infected plants. In this experiment the rhizoboxes allow us easy access to different root types within the same plant. Therefore, root growth of the primary root and 4-5 nodal roots will be monitored weekly by marking the tip of the growing root on the face of the rhizoboxes. At the end of the experiment, root tissues will be collected and plated for Pythium isolation to ensure that infection occurred and that no secondary contamination pathogens entered the system.Objective 3: Assess Pythium impact on early season plant biomechanics to predict yield impactsMethodology: The Sparks lab has developed two novel tools to non-destructively measure stalk and root biomechanics. For stalk biomechanics, a device called the Plant Pusher will be used to measure the flexural stiffness of the stalks - a measure of the stalk's ability to resist bending. For root biomechanics, a device called Sorghum and Maize Under Rotation Force (SMURF) will be used to measure the torsional stiffness of root systems - a measure of the root's ability to resist rotation. Using the same strategy of paired symptomatic and asymptomatic Pythium-infected plants as in Objective 1, we will non-destructively measure stalk and root biomechanics 2-3 times per week beginning at the V2-V3 growth stage aiming for measurements within the replant window. We also measure stalk circumference and analyze approximately 25-pairs of plants. At the end of season, we will destructively sample these plants and measure stalk circumference, root weight, kernel number, and extrapolated yield as in our preliminary results.

Progress 03/01/24 to 02/28/25

Outputs
Target Audience:Audiences that will benefit from this work will include: extension educators at state and county level growers of maize and other crops impacted by Pythium crop consultants, allied industry, and farmers reached through grower meetings, field days, publications, and social media Changes/Problems:No major problems were encountered in the second project year. We are pleased with the data that was collected and we will use this data to optimize and refine experimental approaches for year three to maximize project resources. The first M.S. student on the project will be defending in summer 2025 and a new M.S. student will join in summer 2025 to ensure project continuity. What opportunities for training and professional development has the project provided? This project has provided training for one M.S. graduate student that is developing skills in pathology and microscopy with specific thesis objectives to create a zoospore methodology that will facilitate quantification of Pythium infection on maize root-type specific growth. This student was able to participate in multiple professional development opportunities including speaking at the UD Carroll Symposium and presenting a poster at the UD CANR Research Symposium. How have the results been disseminated to communities of interest?Growers: Discussion of this project occurred on August 7, 2024 as part of the Carvel Research and Education Center summer field day in Georgetown, DE. Preliminary results were also discussed at the 2025 Delaware Ag week in Harrington, DE on January 15, 2025 and at the MD Crop Improvement Association Meeting in Annapolis, MD on January 22,2025. Academic and Federal Researchers: Two research seminars, 1 poster, and 3 extension presentations were delivered in the past year by Drs. Betts and Sparks and their graduate students to disseminate project findings to other researchers at professional meetings and universities. Academic Research Seminars: Henrickson M., Sparks E., and Betts A.K. 2024. Quantification of plant metrics and pathogen load to develop integrated pest management approaches for Pythium in corn. APS Potomac Division Meeting, Martinsburg, WV, 2nd Place. March 2024. Singh B. , Sparks E., and Betts A.K. 2024. Into the fascinating world of zoospores, the motile agents of infection. UD Carroll Symposium, Newark, DE. November 2024. Academic Posters Singh B. Sparks E.E., and Betts A.K. 2024. Method development to optimize production of Pythium graminicola zoospores. CANR Student Research Symposium, 3rd place. Nov 2024. Extension Presentations: Betts A,K, 2024. Building an Applied Research Program and Digging into Soilborne Disease, FMC New Investigator Award Reception, Newark, DE, Aug 2024 Betts A.K. 2025. Disease Management Considerations for Small Grains and Corn, Maryland Crop Improvement Associate Meeting, Annapolis, MD. Jan 2025. Betts A.K. 2025. 2024 Corn Disease Management and Research Update. Delaware Ag Week Corn Agronomy Session, Harrington, DE. Jan 2025. What do you plan to do during the next reporting period to accomplish the goals?On-farm trials will be continued to gain additional data on dynamics of Pythium recovery in qPCR soil samples and Plant Pusher stalk biomechanics. Now that we have associated Pythium symptomatic plants with reduced stalk metrics, in 2025 we will aim to begin layering potential recovery treatments to understand if a growth regulator product applied at V3/V4 could improve performance of symptomatic plants following infection. For qPCR based research we also aim to design experiments allowing comparison recovery across fields to work towards developing diagnostic approaches to evaluate risk at the field level. We will continue to expand plate based assays, microscopy screening, and rhizobox utilization to study Pythium localization in roots.

Impacts
What was accomplished under these goals? To complete project objectives plots were established in a grower collaborator field. For this season we updated the sampling style to longer rows to allow for more streamlined collection of stalk biomechanics data. This sample design allowed for increased sampling with reduced issues of moving equipment into full canopy corn stands. Symptomatic plants were marked at the V2-V3 growth stage along with a neighboring symptomless plant for tracking through the season. Three field sections were marked with 9 replicate pairs per block. For Objective 1, soil samples were collected from the base of symptomatic v. symptomless plants for qPCR analysis of pathogen abundance on May 22 (V2-V3), June 12 (V8), July 22 (R1), and Aug 22 (R5). Samples were bulked by symptom type per block for 6 samples at each collection date. As seen in preliminary data, P. graminicola was the dominant species present. qPCR samples were able to detect Pythium, but levels were not able to be distinguished from symptomatic versus non-symptomatic plants. The highest recovery occurred at the V8 sample timing and recovery was lowest at the V2-V3 timing. As data continues to be collected, we are interested in moving to field to field comparison of abundance to test feasibility of diagnostic tool development. For Objective 3, previous year results showed that stalk mechanics was a better indicator of end-of-season yield and mechanics. We again used a device called the Plant Pusher to measure the flexural stiffness of the stalks - a measure of the stalk's ability to resist bending. This year we focused on gathering early season mechanics as a predictive indicator of performance. This approach was successful as we found that, both non-symptomatic and symptomatic, plants showed a high correlation in their early and late season mechanics. In other words plants that were the weakest at ~V4 were still the weakest at R6. In Objective 2, we are interested in understanding infection interactions across root-types. We have found that agar plugs limit success of plate based assays and began the process of developing a method for production and release of zoospores from P. graminicola. This work had been the focus of a M.S. student project and a methods publication is in preparation for submission in 2025. The newly developed method will allow for improved plate based assays in remaining project years. We have also constructed a smaller set of rhizoboxes to begin experiments analyzing the transition from embryonic to post-embryonic root growth. Greenhouse trials are planned for spring 2025 to assess variation in isolate aggressiveness in small pot assays prior to selection of an isolate for continued rhizobox screening.

Publications


    Progress 03/01/23 to 02/29/24

    Outputs
    Target Audience:Audiences that will benefit from this work will include: extension educators at state and county level growers of maize and other crpps impacted by Pythium crop consultants, allied industry, and famers reached through grower meetings, field days, publications, and social media Changes/Problems:No major problems were encountered in the first project year. We are pleased with the data that was collected and we will use this data to optimize and refine experimental approaches for year two to maximize project resources. What opportunities for training and professional development has the project provided?This project has provided training for one graduate student that is developing skills in pathology, microscopy, and plant mechanical testing. How have the results been disseminated to communities of interest?Growers: A field demonstration was held on August 9 as part of the Carvel Research and Education Center summer field day in Georgetown, DE. Additionally, at the 2023 Mid-Atlantic Crop School a "Crash Course on Corn Disease Identification" was conducted in Ocean City, MD to educate growers about common diseases of corn in the Mid-Atlantic region, with a focus on how to detect and manage diseases including Pythium root rot. Academic and Federal Researchers: three extension and research seminars have been given in the past year by Drs. Koehler and Sparks to disseminate first year findings with other researchers at professional meetings and universities. Academic Research Seminars: E. Sparks at University of Minnesota, Department of Agronomy and Plant Genetics. "Engineering Root Systems for Climate Resilience", Feb 8, 2024. Extension Presentations: A.K. Betts. 2024. Tar Spot and Corn Disease Update, MD Mid-Shore Agronomy Meeting, Denton, MD A.K. Betts. 2024. 2023. Corn Disease Management and Research Update. Delaware Ag Week Corn Agronomy Session, Harrington, DE What do you plan to do during the next reporting period to accomplish the goals?On-farm trials will be continued to gain additional data on distribution of Pythium in qPCR soil samples and Plant Pusher stalk biomechanics. We will continue to expand plate based assays, microscopy screening, and rhizobox utilization to study Pythium localization in roots.

    Impacts
    What was accomplished under these goals? To complete project objectives plots were established in a grower collaborator field. Symptomatic plants were marked at the V2-V3 growth stage along with a neighboring symptomless plant for tracking through the season. For Objective 1, soil samples were collected at varying depths and from symptomatic v. symptomless plants for qPCR analysis of pathogen abundance. As seen in preliminary data, P. graminicola was the dominant species present. qPCR samples were able to detect Pythium, but differences were not observed by sampling depth. The amount of Pythium recovered increased at the VT/R1 timing compared to V2-V3 and the temporal observations may be expanded in future years. For Objective 3, using the paired sets identified for qPCR soil collection, stalk biomechanics were monitored using two devices. For stalk biomechanics, a device called the Plant Pusher was used to measure the flexural stiffness of the stalks - a measure of the stalk's ability to resist bending. For root biomechanics, a device called Sorghum and Maize Under Rotation Force (SMURF) was used to measure the root system stiffness - a measure of the root's ability to resist rotation. When correlating with end of season yield estimates, the Plant Pusher was most informative for potential early season observations that could inform management decisions. In future years, focus will be placed on sampling with the Plant Pusher. Objective 2 focuses on quantification of Pythium infection on root-type specific growth. Plate based assays have been under testing to reliably germinate corn and inoculate with Pythium. We started the process by inoculating with plugs and will be moving towards testing with a calibrated zoospore suspension. Microscopy screening is underway testing multiple stains to identify optimal contrast. Additionally, rhizobox testing is under way to optimize artificial inoculation.

    Publications