Project Details:

Title:
Seedling Diseases: Biology, Management and Education

Parent Project: This is the first year of this project.
Checkoff Organization:North Central Soybean Research Program
Categories:Soybean diseases, Education, Environmental stress
Organization Project Code:225640
Project Year:2016
Lead Principal Investigator:Jason Bond (Southern Illinois University at Carbondale)
Co-Principal Investigators:
Leonor Leandro (Iowa State University)
Gary Munkvold (Iowa State University)
Alison Robertson (Iowa State University)
Christopher Little (Kansas State University)
Martin Chilvers (Michigan State University)
Berlin Nelson (North Dakota State University)
Albert Tenuta (Ontario Ministry of Agriculture-Food & Rural)
Kiersten Wise (Purdue University)
Febina Mathew (South Dakota State University)
Ahmad Fakhoury (Southern Illinois University)
Dean Malvick (University of Minnesota)
Loren Giesler (University of Nebraska)
Tony Adesemoye (University of Nebraska at Lincoln)
Sydney Everhart (University of Nebraska at Lincoln)
Heather Kelly (University of Tennessee-Institute of Agriculture)
Show more
Keywords:

Contributing Organizations

Funding Institutions

Information and Results

Click a section heading to display its contents.

Project Summary

Soilborne seedling and root diseases of soybean significantly reduce yields in the North Central region of the United States. Seedling diseases rank among the top 4 pathogen threats to soybean, because their insidious nature makes them difficult to diagnose and control. It is nearly impossible to predict when they will take a heavy toll, until it happens. The challenges and failures of managing soilborne diseases and pathogens of soybean and other crops are based in part on limitations in knowledge and methods.

This project will address critical limitations in identifying and managing seedling diseases. Producers and industry will see benefits in the form of rapid diagnostics and management recommendations. This benefit will also help industry in their assessments in pesticides and germplasm development. Producers will also see their check-off funding being maximized by the synergy of this team, the USB seedling disease project and the USDA-NIFA oomcyete project. This project complements the USB seedling disease project.

Research themes of the USB seedling disease project are to:
• Determine the effect of temperature, soil type/texture and pH on infection by seedling disease
• Characterize the species complex associated with soybean iron chlorosis in field conditions
• Metagenomics analysis to improve our understanding of seedling biology and the environmental conditions that favor disease
• Establishing baseline inter- and intra-field variability for seedling pathogens
• Impact of cover crops on causal agents of seedling disease
• Outreach and Education

We propose to leverage the USB and OSCAP project to develop additional diagnostic tools to the genus and species level that rapidly identify soybean pathogens to improve seedling disease management. We also propose to develop and conduct metagenomics analysis to improve our understanding of seedling biology and the environmental conditions, including soil factors that drive disease. During the first cycle of the project, we used specific genetic barcodes to identify fungal species isolated from diseased soybean seedlings. We currently have a collection of around 3,000 isolates. The vast majority of these isolates are expected to be pathogenic on soybean and to be involved in the incidence and development of seedling diseases. We are also currently working on developing qPCR probes specific to the fungal species that were found to be most prevalent in the first three years of the project. In addition, we propose to enhance our understanding of various aspects of the biology of the interaction between seedling pathogens and soybean, which will enable us to develop more effective management options for improved stand establishment and yield.

Project Objectives

Objective 1: Development and deployment of a panel of QPCR probes to identify and quantify fungal seedling pathogens of soybean (A. Fakhoury-SIU, Cilvers-MSU, and D. Malvick-UMN)
Objective 2. Curate the collection of fungal pathogens collected during the first phase of this project (A. Fakhoury-SIU)
Objective 3: Improve understanding of the biology of Rhizoctonia solani as seedling pathogen of soybean
Objective 4. Improve understanding of the biology of Fusarium sp. as seedling pathogen of soybean
Objective 5: Improve understanding of the biology of Pythium as a seedling pathogen of soybean
Objective 6: Evaluate the effect of multiple pathogen interactions on seedling disease (A. Robertson and G. Munkvold-ISU)
Objective 7: Impact of seed treatments on the interaction of seedling pathogens (A. Fakhoury and J. Bond-SIU)
Objective 8: Communicate research results with farmers and stakeholders (K. Wise-Purdue and others)

Project Deliverables

Objective 1-2:
• Development of a QPCR panel to detect and quantify 5-10 fungal and oomycete pathogenic species.
• Optimization and validation of the panel in at least two laboratories with 3 different QPCR platforms.
• Development of standard operating procedures for the easy adoption of the panel by other users.
• Maintenace of a collection of ~3,000 isolates of fungi collected from diseased soybean seedlings.
• Development and testing of long-term storage techniques for the different fungal species in the collection.
• Development and maintenance of a searchable database of collection of isolates.

Objective 3:
• Establish collection of R. solani isolates from soybean fields in underrepresented states, including new production areas towards the west
• Report results of R. solani fungicide sensitivity immediately using a Google Fusion interactive map of counties with outbreaks (individual field locations will not be disclosed) – see example here: http://tinyurl.com/pram-google-fusion
• Determine R. solani anastomosis groups recovered from soybean seedlings and soil and identify the dominant anastomosis group.
• Develop 2-3 peer-reviewed publications on fungicide sensitivity, anastomosis group diversity, and genetic analysis of dominant anastomosis groups. Results will also be disseminated at grower meetings, field days, crop production clinic, online in CropWatch, and other Extension publications.
• Determine if useful levels of resistance or tolerance to Rhizoctonia root rot exist in northern varieties and breeding lines, and determine how soil populations of this pathogen relate to disease development in soybean

Objective 4:
• Improved understanding of Fusarium species causing seedling disease on soybeans
• Identification of a resistant germplasm to more than one Fusarium species that are capable of causing damping off and root rot.
• Improved understanding of Fusarium species from soybeans can affect corn and vice-versa; this will have influence on disease management practices (crop-rotations) in future.
• Test at least 2 common seed treatment active ingredients against a large collection of Fusarium proliferatum isolates that originate from diseased seedlings and seeds from Kansas.
• Screen 20-30 entries in MG III, MG IV, and MG V (et al.) from the Kansas State University breeding program (and other public programs) for resistance to F. proliferatum using a high-throughput rolled-towel pathogenicity assay.
• Publish at least 1 journal article reporting sensitivity of F. proliferatum to seed treatment active ingredients and/or reactions of breeding germplasm to this pathogen.

Objective 5:
• Determine fungicide sensitivity of = 250 isolates (82 species * 3 isolates per species)
• Determine fungicide sensitivity to chemistries = 2 (mefenoxam, ethaboxam)
• Screen chemistries at temperatures = 2 (55F, 75F)
• Improved understanding of Pythium-soybean interaction
• Improved understanding of the effect of cold temperatures and Pythium spp. on stand establishment of treated soybean
• Data regarding effect of cold (<50F) temperatures at varying intervals after planting on the emergence of 2 to 3 soybean varieties that vary in resistance to Pythium
• Data regarding effect of cold (<50F) temperatures at varying intervals after planting on seedling diseases caused by two species of Pythium
• Data regarding effect of cold (<50F) temperatures at varying intervals after planting on the efficacy of two commercial seed treatments.
• Fungicide sensitivity (EC50) data for seedling pathogens of soybean

Objective 6:
• Improved understanding of seedling disease pathogen complex
o Data on what species are often associated in the seedling disease complex
• Improved understanding of interactions between seedling pathogens and their contribution to seedling disease
o Emergence and disease data associated with the interaction of three or more Pythium species
o Emergence and disease data associated with the interaction of three or more Fusarium species
o Emergence and disease data associated with the interaction of two or more Pythium and two or more Fusarium species

Objective 7:
• Data will be generated to characterize the effect of 2-3 seed treatments on the population of fungal species in the rhizosphere and their ability to infect soybean plants.
• Greenhouse protocols will be developed to test the effect of 2-3 seed treatments on the collective ability of 3-4 fungal species to infect soybean seedlings.
• Results from greenhouse experiments will be compared and contrasted to those from field experiments.
• A manuscript will be prepared to publish the data learned from the research. Data will also be shared with researchers and other constituencies through presentations.

Objective 8:
• Provide high-quality Extension materials for soybean seedling diseases:
o This will include two full length publications, 1 set of disease scouting cards, 3 web-based videos and 1 slide sets to help farmers and agribusiness professionals to understand seedling diseases and make informed decisions on best management practices.

Progress of Work

Update:
Objective 1: We have developed qPCR assays targeting the following most frequently found fungal species associated with seedling diseases in soybean, as determined in the first phase of the USB-NCSRP project. Currently assays are being optimized and tested for specificity against non-target species.

Objective 2: We have finished cataloguing 3000 fungal isolates that are now stored and maintained at SIU. A searchable site is still under construction and will be publically available soon.

Objective 3: In May and June of 2015, we surveyed for seedling disease in 14 different counties, an area of approximately 24,500 square miles and representing the major soybean production areas in Nebraska. From each field, we collected 10 to 20 soil samples and (when present) symptomatic seedlings from a total of 20 grower fields. A total of 195 soil and plant samples were collected, with 75 processed in the Adesemoye Lab and 120 independently processed in the Everhart Lab.

Objective 4:Most F. proliferatum isolates are more resistant to fludioxonil that those of other pathogenic species of Fusarium. However, F. proliferatum isolates that were resistant to fludioxonil were not resistant to azoxystrobin. Most germplasm screened using F. proliferatum in the rolled towel assay (30C, pH 6.5) resulted in medium to high severity ratings. However, some materials are highly resistant to this fungus (e.g., AR AES R09-430, Bayer HBK RY4721, Midland 3884NR2, NK S39-T3, and Nutech 7414). Among the 2014 KSVT germ plasm entries, a positive relationship appears to exist between F. proliferatum disease severity and that observed for SDS (R2 = +0.52) and SCN (R2 = +0.13) in the field.

Representative isolates of Pythium lutarium, P. oopapillum, P. sylvaticum, and P. torulosum were retrieved from the OSCAP culture collection housed at the MSU Chilvers lab and sent to ISU Robertson lab for fungicide sensitivity testing. The OSCAP culture collection is comprised of isolates from across 12 US states and Ontario, Canada. The Chilvers lab at MSU has been developing a high-throughput fungicide sensitivity assay. Testing and validation of the assay has been completed for mefenoxam sensitivity and additional rounds of testing are being conducted for ethaboxam sensitivity determination.

Objective 5: The pathogenicity and fungicide sensitivity of three representative isolates of each species from each state in the NC region was evaluated at 55 F and 73 F. All species from all states, except AR, were more sensitive (>50% mycelia inhibition) to metalaxyl and ethaboxam than to azoxystrobin. Isolates of P. lutarium from IA, ND, and NE were less sensitive to azoxystrobin at 23°C compared to 13°C. At 13°C isolates of P. oopapillum from all states, except AR, were less sensitive to all fungicides compared to 23°C. Isolates of P. sylvaticum from IA, ND, NE, SD, and WI were less sensitive to all fungicides at 23°C compared to 13°C. At 13°C isolates of P. torulosum from IA, IN, ND, NE, and WI were less sensitive to all fungicides tested compared to 23°C.A significant three-way interaction was detected between cold stress, inoculation and seed treatment (P<0.0001). In general, emergence of untreated seeds was reduced more than 70% when subjected to periods of cold stress (P<0.05). Emergence was improved with seed treatments (P<0.0001).

Objective 6a: A preliminary analysis of the data from the two seedling disease projects has been done. Hierarchical clustering was used to identify groups of isolates frequently recovered together from the same field. When data were analyzed including all fungi and oomycetes, regardless of pathogenicity, six distinct clusters were identified. Within each cluster were fungal and oomycete pathogens and non-pathogens. When data were reanalyzed with only pathogenic species, four distinct clusters were present. Cluster 3 contained only Fusarium species, while cluster 2 contained several Pythium species as well as Phytophthora sojae, Phytophthora sansomnea and F. verticiliioides. The remaining two clusters contained both Fusarium and Pythium species.

Objective 7: Impact of seed treatments on the interaction of seedling pathogens
A greenhouse experiment has been established at SIU to test the effect of seed treatments on seedling pathogens. In a first experiment, Fusarium oxysporum, Rhizoctonia solani were used to inoculate the soil one day using infected sorghum seeds. Next, treated seeds were planted and covered with a thin layer of soil. Roots will be collected 3 weeks after planting and qPCR assays will be conducted to quantify each pathogen.

Objective 8:
Scouting card of common seedling diseases/disorders
Full length publication on soybean seedling diseases
17,500 publications downloaded/distributed
Updated soybean fungicide seed treatment efficacy table
Two seed treatment publications in development
Three videos released, Hosted on SRII, One additional video in development

View uploaded report Word file

Update:
Objective 1: We have developed qPCR assays targeting the following most frequently found fungal species associated with seedling diseases in soybean, as determined in the first phase of the USB-NCSRP project. Assays continue to be optimized and tested for specificity against non-target species, and new assays are being evaluated.

Objective 2: We have finished cataloguing 3000 fungal isolates that are now stored and maintained at SIU. A searchable site is still under construction and will be publically available soon.

Objective 3: In 2015 and 2016, symptomatic seedlings and soil samples were collected across NE soybean production regions. Isolates were recovered and identified based on morphology and sequencing of the ITS regions. Pathogenicity was determined for all 35 recovered Rhizoctonia isolates. Among the 35 isolates, 22 were identified as R. solani and 13 as R. zeae. The most prevalent AG of R. solani was AG-4 for which 14 isolates were recovered. Assay results indicated a wide range of virulence among isolates. The most virulent isolate was R. solani AG-4 strain G1927, which exhibited cross-virulence to both corn and wheat. Results have been communicated in on-farm visits, webinars with farmers/consultants, a CropWatch technical publication, a departmental seminar.

We have continued to refine and evaluate inoculation methods and have identified different levels of virulence among isolates of R. solani that can influence disease severity in soybean.  Multiple soybean varieties were evaluated in greenhouse and field trials for resistance and tolerance to R. solani. Potential differences in resistance and/or tolerance were identified in both trials.  

Pathogenicity of Fusarium species causing soybean seedling disease (F. Mathew, SDSU)
To determine Fusarium species associated with soybean in South Dakota, a survey of 100 commercial soybean fields was conducted in 2015 covering 29 counties. Ten plant samples were collected from each field that was arbitrarily selected at vegetative growth stages VE (plant emergence) to V4 (four unrolled trifoliate leaves and five nodes). A total of 1000 root samples were collected. The fields sampled had either corn, soybean or sunflower as the previous crop.

Pathogenicity of Fusarium isolates
To determine the aggressiveness of Fusarium spp. recovered, 59 representative Fusarium isolates were evaluated. For proof of pathogenicity, Fusarium isolates representing ten species were re-isolated from the inoculated plants, to comply with Koch’s postulates.

Survey, and isolation of Fusarium spp.
F. oxysporum was the most recovered from the fields (24.5%) followed by F. acuminatum (21.3%) and F. graminearum (15.5%) (Fig. 1). F. equiseti and F. subglutinans were the least prevalent in the fields sampled at 0.4% and 1% respectively.

Objective 4: Screening for aggressiveness was conducted using laboratory and greenhouse assays. Overall, all of the F. proliferatum isolates tested significantly decreased seed germination in laboratory (p < 0.001) assays. In greenhouse assays, most F. proliferatum isolates tested were also able to reduced seed vigor (p < 0.001) when compared with mock-inoculated control.Overall, the effects of seedborne F. proliferatum isolates on soybean seed germination decreases as the inoculum potential in contact with the seeds decreases.

Objective 6: Analysis of the data from the two seedling disease projects has been done.  Hierarchical clustering was used to identify groups of isolates frequently recovered together from the same field. When data were analyzed including all fungi and oomycetes, regardless of pathogenicity, six distinct clusters were identified.  Within each cluster were fungal and oomycete pathogens and non-pathogens. When data were reanalyzed with only pathogenic species, four distinct clusters were present.  Cluster 3 contained only Fusarium species, while cluster 2 contained several Pythium species as well as Phytophthora sojae, Phytophthora sansomnea and F. verticiliioides. The remaining two clusters contained both Fusarium and Pythium species.

Objective 7: Impact of seed treatments on the interaction of seedling pathogens (Fakhoury and Bond, SIU)
A greenhouse experiment has been established at SIU to test the effect of seed treatments on seedling pathogens. In a first experiment, Fusarium oxysporum, Rhizoctonia solani were used to inoculate the soil one day using infected sorghum seeds. Roots were collected 3 weeks after planting and qPCR assays were conducted to quantify each pathogen.

Objective 8: Education and Outreach (K. Wise – Purdue)
Scouting card of common seedling diseases/disorders
Full length publication on soybean seedling diseases
17,500 publications downloaded/distributed
Updated soybean fungicide seed treatment efficacy table
Two seed treatment publications in development
Three videos released, Hosted on SRII, One additional video in development 


View uploaded report Word file

Final Project Results

Benefit to Soybean Farmers

Performance Metrics

Project Years