Project Details:

Title:
Seedling Diseases: Biology, Management and Education

Parent Project: Seedling Diseases: Biology, Management and Education
Checkoff Organization:North Central Soybean Research Program
Categories:Soybean diseases, Education, Environmental stress
Organization Project Code:NCSRP
Project Year:2019
Lead Principal Investigator:Jason Bond (Southern Illinois University at Carbondale)
Co-Principal Investigators:
Gary Munkvold (Iowa State University)
Alison Robertson (Iowa State University)
Christopher Little (Kansas State University)
Martin Chilvers (Michigan State University )
Febina Mathew (South Dakota State University)
Ahmad Fakhoury (Southern Illinois University)
Dean Malvick (University of Minnesota)
Tony Adesemoye (University of Nebraska at Lincoln)
Sydney Everhart (University of Nebraska at Lincoln)
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Keywords: Important, Yield Loss, Seedling Disease, Early Season, Devastating, Replant, Management

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

Brief Project Justification and Rationale — Need, state-of-the-art, opportunity for farmers and the soybean industry:
Soilborne seedling and root diseases of soybean significantly reduce yields in the North Central region of the United States.

The 4th year of the proposed work is needed to complete several complex objectives. Several trials examining the efficacy and role of seed treatments for the management of oomycetes (Pythium and Phytophthora) are being conducted in field and lab experiments, and will lead to improvements in soybean disease management.
Our studies of Rhizoctonia diseases clearly indicate that northern-adapted soybean cultivars and breeding lines differ in susceptibility to the prevailing type of R. solani found in Minnesota and other areas. Additional funding will enable further identification and characterization of the disease resistance in field, greenhouse, and laboratory studies, which will likely lead to improved resistance and disease management.

Our work will determine the pathogenicity of Rhizoctonia isolates, which has already identified a surprising number of Rhizoctonia zeae that are pathogenic to soybean. Thus, receiving funding for a fourth year will allow completion and publication of this work.

Additionally, novel insight into the sensitivity of this pathogen to commonly used fungicide
will be gained. An additional year of funding will allow application of genetic markers to characterize the population structure and biology of this pathogen, which is important for understanding the establishment and spread of the pathogen within and between fields.

Currently molecular markers are not available for use in breeding programs to screen soybean varieties for resistance to any virulent species of Fusarium causing seedling disease and root rot (e.g. F. graminearum). The underlying goal of our research is to understand candidate genes conferring resistance to F. graminearum in soybean so that the breeders can develop resistant varieties for the farmers.

Producers will also see their check-off funding being maximized by the synergy of this team, the USB seedling disease 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 variabilityfor seedling pathogens

Impact of cover crops on causal agents of seedling disease

We propose to leverage the USB 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 3,000+ isolates. The 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-SlU, Chilvers-MSU, and D.
Malvick-UMN)
Objective 2: Curate the collection of fungal pathogens collected during the first phase of this project (A. Fakhoury-SlU)
Objective 3: Improve understanding of the biology of Rhizoctonia solani as seedling pathogen of soybean
Objective 3a: Characterize R. solani anastomosis groups affecting soybean seedlings throughout the U.S. (S. Everhart and T. Adesemoye-UN)
Objective 3b: Monitor shifts in fungicide sensitivity in R. solani populations (S. Everhart and T. Adesemoye-UN)
Objective 3c: Identification and characterization of resistance and tolerance to Rhizoctonia root rot (D. Malvick-UMN)
Rhizoctonia root rot has been a major problem in Minnesota in recent years as well as in other North central states.
Objective 4. Improve understanding of the biology of Fusarium sp. as seedling pathogen of soybean
Objective 4a: Pathogenicity of Fusarium species and identifr resistant germplasm
Objective 5: Improve understanding of the biology of Pythium as a seedling pathogen of soybean
Objective 5a Compare pathogenicity and fungicide sensitivity of Pythium lutarium, oopapillum, sylvaticum, torulosum and other species across the North Central region
Objective 5b: Evaluate the effects of low temperature stress on soybean seedling susceptibility to disease and the contribution of seed treatments (A. Robertson-ISU)
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

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. SOP’s have been developed for the Phytophthora assays as part of the OSCAP project, these SOP’s can be adapted for additional assays.
• Maintenance 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 Rhizoctonia isolates from soybean fields in underrepresented states, including new production areas towards the west.
• Determine R. solani anastomosis groups recovered from soybean seedlings and soil and identify the dominant anastomosis group.
• Determine pathogenicity of Rhizoctonia isolated from soybean fields
• Develop 1-2 additional peer-reviewed publications on fungicide sensitivity, anastomosis group diversity, and pathogenicity of Rhizoctonia. Results will also be disseminated at grower meetings, field days, crop production clinic, online in CropWatch, and other Extension publications.
• Determine if early maturity group soybean germplasms vary in response to Rhizoctonia root rot and identify those with different levels of susceptibility

Objective 4:
• Improved understanding of Fusarium species causing seedling disease on soybeans
• Identification of a resistant genotypes to more than one Fusarium species that can cause 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
o Improved understanding of the effect of cold temperatures and Pythium spp. on stand establishment of treated soybean – experiments done; manuscript in progress
o 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 -
o Data regarding effect of cold (<50F) temperatures at varying intervals after planting on seedling diseases caused by two species of Pythium
o Data regarding effect of cold (<50F) temperatures at varying intervals after planting on the efficacy of two commercial seed treatments experiments done; manuscript in progress

Objective 6:
• Improved understanding of seedling disease pathogen complex
o Data on what species are often associated in the seedling disease complex experiments done; manuscript in progress
• 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 experiments done; data analysis and interpretation in progress

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, 3 web-based videos and 1 slide set to help farmers and agribusiness professionals to understand seedling diseases and make informed decisions on best management practices.

Progress of Work

Updated April 20, 2019:
Objective 1: Development and deployment of a panel of QPCR probes to identify and quantify fungal seedling pathogens of soybean (A. Fakhoury-SIU, M. Chilvers-MSU, and D. Malvick-UMN)
Chilvers Lab - We have collaborated with Dr. Tim Miles (MSU) and Dr. Frank Martin (USDA) on the validation of an isothermal RPA assay for genus level detection of Phytophthora. The testing of this assay will provide information to the diagnostic companies for commercialization of the kits. Which would allow wider deployment and use of this type of assay which can be used in the field with relatively simple equipment. The assay components are provided in lyophilized for and are simple to use, with detection/identification being achieved in a matter of minutes. We are currently gathering the last of the data for this part of the project to be written up and for companies to use in their decision making process, with respect to how to best path to commercialization.

Fakhoury lab – we have added another universal assay targeting both Phytophtora and Pythium genera. The assay has been optimized and tested for efficiency and sensitivity. Multiplexing option has been eliminated due to primer-dimer formation between different assays, in addition to the selectivity of some assays where high abundant isolates were easily detected, whereas rare isolates tend to fade in the background.. We have validated the probe panel on a set of seedlings inoculated with a mix of seedlings pathogens (including Fusarium species and Rhizoctonia), and the assays were successful in detecting the inoculated pathogens. In the past quarter, efficiency and sensitivity of the developed assays was successfully tested on a variety of soil and root samples from diseased seedlings soybean

A manuscript is under preparation entitled “A probe panel assay for the detection and quantification of seedlings pathogens in Soybean fields” is under preparation and will be submitted during 2019.

Objective 2: Curate the collection of fungal pathogens collected during the first phase of this project (A. Fakhoury-SIU)
The website that describes each isolate collection and allows for retrieval request is under construction.

Objective 3a: Characterize R. solani anastomosis groups affecting soybean seedlings throughout the U.S. (S. Everhart and T. Adesemoye-UNL)
Our results have expanded the collection of Rhizoctonia root and stem rot isolates, adding a total of 114 Rhizoctonia isolated from soybean fields in 2015, 2016, and 2017, with additional survey underway in 2019. Thus far, we have identified Rhizoctonia zeae (75), R. solani AG-4 (26), and 13 other R. solani identified as one of the following anastomosis groups: AG1-1 IA, AG-B, AG-3, AG-5, AG-K, and AG-2-1. Our work is further characterizing the level of pathogenicity of these isolates and has identified a surprising number of Rhizoctonia zeae that are pathogenic to soybean. Currently, we are leveraging funding from other sources to perform GC-MS and determine if toxins produced by R. zeae showing aggressiveness to soybean are the same as R. solani characterized previously.

This survey has provided novel information, which shows that Rhizoctonia zeae (Waitea circinata var. zeae) is an important pathogen of soybean when evaluated at higher temperatures than R. solani. We have identified Rhizoctonia zeae and Rhizoctonia solani AG-4 as the two most prevalent groups among a total of more than 100 Rhizoctonia isolated. In 2019, we are increasing our survey collection and will characterize these isolates using population genetic markers that are under development in Objective 3b. Thus far, we have presented information to growers about conditions that may favor Rhizoctonia infection as well as information on disease management.

Objective 3b: Monitor shifts in fungicide sensitivity in R. solani populations (S. Everhart and T. Adesemoye-UN)
Fungicide sensitivity assays are nearly complete, with results from the entire both experimental replicates showing that Rhizoctonia zeae has a broad range of fungicide sensitivity to prothioconazole, sedaxane, and fludioxonil. For example, average EC50 from the two prothioconazole experiments were 0.174 ppm and 0.172 ppm. Average EC50 from the two sedaxane experiments were 0.067 ppm and 0.065 ppm. Consistency of results demonstrated reproducibility of the experiments. Assays for determining EC50 for azoxystrobin using various combinations of compounds to repress alternate oxidative pathways thus far have shown no effect and in planta assays in the greenhouse are currently underway. Results to date are consistent with previous studies that suggest Rhizoctonia zeae is completely insensitive to azoxystrobin fungicide, which is currently one of most common fungicides used due to the expected high specificity of action. The greenhouse studies will be critical for determining the appropriate chemical control recommendations.

Our population analysis is also underway. We obtained whole-genome sequence data for five R. zeae isolates. We identified 1,594 candidate SSR markers and designed primers for 40 candidate loci that will be screened this summer for polymorphisms using a selection of R. zeae isolates. It is important to identify polymorphic markers so that isolates in a population can be differentiated from each other. Polymorphic SSR loci to characterize the population structure of these populations across the region. We obtained isolates of Rhizoctonia zeae from Dr. O. O. Ajayi-Oyetunde and Dr. C. Bradley, collected from several North Central states and will initiate new sampling efforts in 2019 that will specifically seek to expand our R. zeae. These genetic markers will be applied to assess population structure of isolates obtained throughout the region, which will also allow us to gain deeper insight into the biology of this relatively understudied soybean pathogen.

Publications in 2018 to present:
1. Adesemoye, A. O. 2018. Root and Soilborne Diseases Update. CropWatch July 2, 2018.
2. Adesemoye, A. O. 2018. Soilborne and early seedling pathogens and delayed planting in corn and soybean. CropWatch May 3, 2018.
3. Ajayi, O.O., S.E. Everhart, P.J. Brown, A.U. Tenuta, A.E. Dorrance, and C. Bradley. 2019. Genetic structure of Rhizoctonia solani AG-2-2IIIB from soybean in Illinois, Ohio, and Ontario. Phytopathology. Accepted pending revision.
4. Gambhir, N., S. Everhart, S. Kodati, & A. Adesemoye. 2018. Fungicide Resistance: Risk and Management. SoybeaNebraska., Spring 2018, Page 22.
5. Kodati, S., A. Adesemoye, N. Gambhir, & S. Everhart. 2018. Rhizoctonia Diseases in Soybean. SoybeaNebraska, Spring 2018, Page 23.

Presentations in 2018 to present:
6. Everhart, S.E. and Adesemoye, A.O. 2018. An update on the project presented during the 2018 Crop Production Clinics at the Sandhills Convention Center, North Platte, NE on January 11, 2018.
7. Gambhir, N., Kodati, S., Adesemoye, A.O., and Everhart, S.E. 2018. Fungicide sensitivity of Rhizoctonia spp. isolated from soybean fields in Nebraska. Poster at ICPP Meeting in Boston, MA.
8. Gambhir, N., Kodati, S., Adesemoye, A.O., and Everhart, S.E. 2018. Fungicide sensitivity of Rhizoctonia zeae from soybean and corn in Nebraska. Presentation at International Rhizoctonia Workshop: Rhizoctonia at crossroads: research advances and challenges, Boston, MA.
9. Kodati, S. 2018. Characterization, anastomosis grouping and pathogenicity of Rhizoctonia spp. on multiple plant hosts in Nebraska. Department of Plant Pathology Seminar Series, UNL, December 3, 2018.
10. Kodati, S. and Adesemoye, A. O. 2018. Emerging understanding of the pathogenesis of Rhizoctonia zeae in row crops. ICPP-APS Joint Conference holding August 1 to 5 in Boston, MA.
11. Kodati, S. and Adesemoye, A.O. 2018. Diversity and pathogenicity of Waitea circinata on row crops. Presentation at International Rhizoctonia Workshop: Rhizoctonia at crossroads: research advances and challenges, Boston, MA.
12. Presentation during 2018 Nebraska Crop Management Conference at the Younes Conference Center, Kearney, NE on January 24 and 25, 2018.

Objective 3c: Identification and characterization of resistance and tolerance to Rhizoctonia root rot (D. Malvick-UMN)
The primary goals of this project are to: (i) determine if northern soybean germplasms vary in reaction to Rhizoctonia root & stem rot and identify those with low susceptibility, (ii) compare methods for assessing reaction to R. solani, (iii) determine if R. solani isolates from soybean vary in AG group and virulence, and (iv) determine if isolates vary in susceptibility to fungicides. Significant differences were detected among northern cultivars and breeding lines for reaction to R. solani in greenhouse and field studies, suggesting that some soybean germplasms in northern maturity groups differ in susceptibility to Rhizoctonia diseases. Additional greenhouse studies are underway and preparations are being made for field studies in 2019. The predominant anastomosis group of R. solani that we have detected infecting soybean in MN is AG 2-2 IIIB, and these isolates vary in aggressiveness on soybean seedlings. Fungicide sensitivity studies ae underway with multiple isolates, and the isolates vary in sensitivity to different fungicidal groups. Studies are ongoing to further characterize fungicide sensitivity and cultivar reaction to diverse isolates using different methods assay methods. This project is providing results needed to improve management of this disease.

Objective 4a: Pathogenicity of Fusarium species and identify resistant germplasm (F. Mathew-SDSU)

Publications:
o Okello, P. N., Petrovic, K., Singh, A. K, Kontz, B., and Mathew, F. M. 201X. Characterization of species of Fusarium cause root rot of soybean (Glycine max L.) in South Dakota, USA. Can. J. Plant Pathol. XX: 000-000. (Manuscript in preparation for submission by June 2019).
o Okello, P. N., and Mathew, F. M. 2019. Cross pathogenicity studies show South Dakota isolates of Fusarium acuminatum, F. equiseti, F. graminearum, F. oxysporum, F. proliferatum, F. solani, and F. subglutinans from either soybean or corn are pathogenic to both crops. Plant Health Prog. 20: 44-49.

o Two hundred and forty-seven accessions from the USDA soybean germplasm collection in Maturity Groups (MG) 00 to V were screened for their resistance to a single isolate of F. graminearum using the inoculum layer method with two susceptible checks, Williams 82 and Asgrow 1835. Disease severity caused by the F. graminearum isolate was evaluated 21 days’ post-inoculation on a 1-to-5 rating scale and expressed as relative treatment effects (RTE). Eight soybean accessions (PI437949, PI438292, PI612761A, PI438094B, PI567301B, PI408309, PI361090 and P188788) were observed to be significantly less susceptible to F. graminearum when compared to Williams 82 and Asgrow 1835.
o Performance measure - The eight accessions may be used in breeding programs as sources of resistance to F. graminearum for development of resistant soybean cultivars, which the soybean growers can use to protect yield from the pathogen.
o Future studies will focus on identifying markers associated with F. graminearum resistance using mapping strategies.
o The Ph.D. graduate student (Paul Okello) who was partially funded by NCSRP seedling diseases project is defending his thesis on April 22, 2019 and expect to graduate in May 2019.

Objective 4b. Improve understanding of the biology of Fusarium sp. as seedling pathogen of soybean (K. Little-KSU)
I. Soybean seedling-borne Fusarium proliferatum sensitivity to azoxystrobin. F. proliferatum isolates obtained from soybean seedlings in Kansas show a range of reactions to azoxystrobin, a common active ingredient in seed treatment fungicides. Approximately 14% of F. proliferatum isolates tested in Kansas appear to have some level of tolerance to the fungicide. EC50s range from 1.2 to 3.0 ug of a.i./ml for the tolerant isolates and <0.01 to 0.33 ug a.i./ml for the sensitive isolates, which is log order(s) less concentration required to reduce growth for the sensitive isolates.

II. Rolled-towel test for germination and seedling development after seed treatment with azoxystrobin. Mock-inoculated seed and F. proliferatum-inoculated seed were pretreated with azoxystrobin at a 1 g a.i./100 kg seed equivalent. Interestingly, seeds are still killed by the sensitive isolate after fungicide treatment because the seed are imbibed with such a high level of inoculum (2 x 10^5 conidia/ml for 10 min). But, it appeared that seedling health is improved (healthier hypocotyls) in the azoxystrobin-sensitive treatment. The azoxystrobin-tolerant isolate does not respond to fungicide treatment and is therefore much more aggressive in the rolled-towel assay. More necrotic/stunted seedlings are found in the azoxystrobin-tolerant isolate treatment.

III. Screening soybean germplasm for resistance to seedling disease. In past updates, we have reported on soybean screening using different species of Fusarium. Now, we have pursued screening of a wide range of germplasm with F. proliferatum in order to find entries some level of general resistance to this seedling pathogen. During the course of this work we have developed (and are refining) a seedling quality scale (S.D.S.) as a means to estimate seedling disease severity and general seedling health. Screening of a subset of germplasm from the K-State soybean breeding program has shown that 9/115 genotypes are resistant (>4 S.Q.S.) to the pathogen (although these must re-tested to ensure that they are not "escapes"). A total of 39/115 genotypes showed an intermediate reaction (2 to 4 S.Q.S.). And, the remaining 67/115 were susceptible (< 2 S.Q.S.).

Objective 5: Improve understanding of the biology of Pythium as a seedling pathogen of soybean (A. Robertson-ISU and M. Chilvers-MSU)
Our research evaluating the effect of cold stress on soybean seedling disease caused by P. sylvaticum (see peer-reviewed manuscripts submitted below) did not take into account soil moisture. Preliminary trials were done in the growth chamber to include soil moisture. At high soil moisture, emergence of soybean was reduced. Inoculation with P. sylvaticum further reduced emergence.

The following papers regarding our work have been published:
1. Serrano, M. and Robertson, A.E. 2018. The effect of cold stress on damping off of soybean caused by Pythium sylvaticum. Plant Dis. 102: 2194-2200
2. Serrano, M., McDuffee, D. and Robertson, A.E. 2018. Seed treatment reduces damping-off caused by Pythium sylvaticum on soybeans subjected to periods of cold stress. Can. J. Pl. Path. https://doi.org/10.1080/07060661.2018.1522516

Chilvers Lab: Manuscript describing the high-throughput fungicide sensitivity assay has been accepted pending minor revisions. The assay has been taught to a number of other labs to aid in speeding the process of fungicide sensitivity testing, and will be widely available once the manuscript is published. An additional manuscript describing Pythium and Phytophthora species sensitivity to mefenoxam and ethaboxam has also been accepted pending minor revisions, this manuscript clearly demonstrates the need for multiple chemistry seed treatments for the management of mixed oomycete populations, this data will be of value to farmers in making seed treatment decisions.

The high-throughput fungicide assay will enable improved monitoring for fungicide resistance amongst oomycete (Pythium and Phytophthora species), which aids in ensuring seed treatments in use are effective. The study of fungicide sensitivity of oomycetes to ethaboxam and mefenoxam demonstrates the need for both chemistries when control of a broad spectrum of oomycetes is necessary, and this information can potentially be used to further prescribe seed treatments based on a knowledge of causal organisms.

Objective 6: Evaluate the effect of multiple pathogen interactions on seedling disease (A. Robertson and G. Munkvold-ISU)
The following paper was accepted for publication:
Lerch, E. and Robertson, A.E. XXXX. Effect of co-inoculations of Pythium and Fusarium species on seedling disease development of soybean. Can. J. Pl. Path.

Objective 7: Impact of seed treatments on the interaction of seedling pathogens (A. Fakhoury and J. Bond-SIU)
Fewer modifications were introduced to isolate the effect of each pathogen and in combination on plant health. Fusarium species were evaluated individually and under interaction in the following scheme: A; B; C; A+B; A+C; B+C and A+B+C (whereby A= F. oxysporum; B= F. proliferatum; C= F. sporotrichioides). Root length, surface area and projected area data were collected for each inoculation scheme.

Our results have shown that Fusarium proliferatum to be more aggressive than the other two species Fusarium oxysporum and F. sporotrichioides based on root morphology and pathogen density. On the other hand, F. oxysporum, and F. proliferatum data suggested that they have an additive (synergistic) effect when causing root rot on soybean. Rhizosphere soil tightly attached to roots and rhizome were collected for quantitative PCR. At a later stage of this set of experiments, fungicide seed treatments will be incorporated as an additional variable affecting the interaction between the different isolates and soybean. In the past quarter, additional seedlings disease pathogens were tested in dual plate assays. Fusarium species were screened against Pythium irregulare; a strong antagonism was evident between fusaria and Pythium species. A greenhouse assay is being developed to test the combined effect of these organisms on soybean seedlings. In parallel, we have developed an artificial media system to visualize the root architecture and its development as affected by seedling pathogens in a 3D view. The media contains all the required nutrients and the pictures will be analyzed using a MATLAB script. The assay will allow us to document closely using 3D imaging the modes of pathogen infection, spread and interaction with other pathogens in presence of the root system.
Insights collected from these assays will be used to synthesize an artificial core microbiome that includes both pathogenic an non-pathogenic species that can play a systemic role in seedlings disease suppression.

Objective 8: Communicate research results with farmers and stakeholders (K. Wise-UK and others)
We published the 2019 soybean seed treatment efficacy table in March. https://cropprotectionnetwork.org/resources/publications/fungicide-efficacy-for-control-of-soybean-seedling-diseases

View uploaded report Word file

View uploaded report 2 PDF file

Updated January 15, 2020:

View uploaded report PDF file

View uploaded report 2 PDF file

View uploaded report 3 PDF file

View uploaded report 4 PDF file

Final Project Results

Updated January 15, 2020:
Objective 1: Development and deployment of a panel of QPCR probes to identify and quantify fungal seedling pathogens of soybean (A. Fakhoury-SIU, M. Chilvers-MSU, and D. Malvick-UMN)
The Chilvers lab has developed diagnostic detection assays for soybean seedling and root rot diseases, Phytophthora root rot and soybean sudden death syndrome. The Phytophthora assay is currently being validated for potential commercial production. For soybean sudden death syndrome an assay for Fusarium virguliforme has been widely deployed and is being used by diagnostic labs for rapid identification of SDS infected plants. The Chilvers lab is continuing work on this assay as a soil risk prediction assay for SDS. In addition, an assay was developed and published for Fusarium brasiliense, which was recently reported by our group for the first time in the U.S. Interestingly, we have found this pathogen extensively in dry bean fields in Michigan.

The Fakhoury lab developed and validated a probe panel targeting a set of pathogens causing seedling diseases including Fusarium species and Rhizoctonia. The assays were successful in detecting the pathogens in different matrices including soil samples and infected plant roots. The developed probe panel can be used for the fast identification and accurate quantification of key seedlings disease pathogens from different matrices (soil, roots and stem) and provide a powerful decision tool for farmers and researchers.

Objective 2: Curate the collection of fungal pathogens collected during the first phase of this project (A. Fakhoury-SIU and M. Chilvers-MSU)
The Fakhoury lab maintained and characterized the collection 3,000+ fungal pathogens and other organisms isolated from soybean. If funds are identified to continue curating this collection, the collection should continue to allow for research discoveries into the future.

The Chilvers lab has maintained an extensive oomycete isolate collection from the OSCAP-NIFA/NCSRP/USB projects and has redistributed isolates from this collection back to multiple PIs throughout the U.S. The collection has also formed the basis for screening for species fungicide sensitivity and has provided insights into fungicide efficacy and soybean seedling disease management.

Objective 3a: Characterize R. solani anastomosis groups affecting soybean seedlings throughout the U.S. (S. Everhart and T. Adesemoye-UNL)
An average of 18 soybean fields per year were in 2015, 2016, and 2017 and a grand total of 957 soil and plant samples were collected. From these, more than 115 Rhizoctonia were isolated and identified to species / sub-species using gene sequencing. Our results showed that Rhizoctonia zeae (Waitea circinata var. zeae) is an important pathogen of soybean. More than 100 Rhizoctonia spp. were isolated and identified. Rhizoctonia zeae and R. solani AG-4 were determined to be the two most prevalent groups, which is different than what a recent study in Illinois found and may be attributable to the reputation of R. zeae as a more aggressive pathogen of grasses. Our greenhouse studies showed that R. zeae is able to cause disease on soybean seedlings. Although we observed no change in stand count, infected seedlings had reduced plant biomass. Young plants infected with R. zeae tend to be weaker, which would increase susceptibility to other pathogens in field conditions. Since R. zeae causes disease symptoms at temperatures higher than R. solani, disease management recommendations may need to be revised (especially with respect to recommended fungicides, see Obj. 3b).

Objective 3b: Monitor shifts in fungicide sensitivity in R. solani populations (S. Everhart and T. Adesemoye-UN)
Our laboratory studies showed current commercial fungicides (prothioconazole, sedaxane, and fludioxonil) are effective and no fungicide resistance was observed. However, a crucial finding was that R. zeae is completely insensitive to azoxystrobin fungicide, which is currently one of most common fungicides used owing to its expected high specificity of action. Thus, any use of azoxystrobin is not expected to have an effect on R. zeae. Population structure of Rhizoctonia zeae is being characterized (see technical report), which will enable a deeper insight into the biology and mode of spread of this pathogen. This information has been presented multiple times to the scientific community, establishing the importance of this pathogen in soybean and the fungicides that can effectively manage this disease.

Objective 3c: Identification and characterization of resistance and tolerance to Rhizoctonia root rot (D. Malvick-UMN)
The primary goals of this objective were to: (i) determine if northern soybean germplasm vary in reaction to Rhizoctonia root & stem rot and identify those with low susceptibility, (ii) compare methods for assessing reaction to R. solani, (iii) determine if R. solani isolates from soybean vary in AG group and virulence, and (iv) determine if isolates vary in susceptibility to fungicides.

Additional field and growth chamber studies were completed to determine reaction of northern soybean germplasm to various isolates of R. solani. Disease severity was moderate to high in the two fields locations in MN, and the germplasm varied in reaction/susceptibility to R. solani. An additional study was completed to assess the reaction of soybean to different methods of inoculation. The consistency and severity reaction of soybean to R. solani was influenced by method of inoculation, which in turn can influence the level of susceptibility measured to R. solani. Studies of isolate virulence were completed, demonstrating that isolates vary significantly in virulence/aggressiveness to soybean, which influences the level of disease that develops in different soybean breeding lines and varieties. We completed studies to characterize fungicide sensitivity in a group of 35 isolates of R. solani from soybean and sugarbeet (grown in rotation with soybean) in Minnesota. Results with azoxystrobin were inconsistent and inconclusive, as had been reported by other researchers. All isolates tested were sensitive to sedaxane, penthiopyrad, and pyraclostrobin, although the level of sensitivity varied among isolates and fungicides.

Objective 4a: Pathogenicity of Fusarium species and identify resistant germplasm (F. Mathew-SDSU)
Two hundred and forty-seven accessions from the USDA soybean germplasm collection in Maturity Groups (MG) 00 to V were screened for their resistance to a single isolate of F. graminearum using the inoculum layer method with two susceptible checks, Williams 82 and Asgrow 1835. Disease severity caused by the F. graminearum isolate was evaluated 21 days’ post-inoculation on a 1-to-5 rating scale and expressed as relative treatment effects (RTE). Eight soybean accessions (PI437949, PI438292, PI612761A, PI438094B, PI567301B, PI408309, PI361090 and P188788) were observed to be significantly less susceptible to F. graminearum when compared to Williams 82 and Asgrow 1835.

The eight accessions may be used in breeding programs as sources of resistance to F. graminearum for development of resistant soybean cultivars, which the soybean growers can use to protect yield from the pathogen.

Objective 4b. Improve understanding of the biology of Fusarium sp. as seedling pathogen of soybean (K. Little-KSU)
I. Soybean seedling-borne Fusarium proliferatum and other Fusarium spp. for sensitivity to fludioxonil and azoxystrobin.
Fludioxonil and azoxystrobin were tested against a wide range of Fusarium spp. including F. proliferatum (see below). F. proliferatum grew better on fludioxonil than isolates of F. oxysporum collected from seedlings in Kansas. The reaction of F. proliferatum isolates to fludioxonil was more variable than the reaction to azoxystrobin. F. proliferatum isolates show a range of reactions to azoxystrobin, a common active ingredient in seed treatment fungicides. Approximately 14% of F. proliferatum isolates tested in Kansas appear to have some level of tolerance to the fungicide. EC50s range from 1.2 to 3.0 ug of a.i./ml for the tolerant isolates and <0.01 to 0.33 ug a.i./ml for the sensitive isolates, which is log order(s) less concentration required to reduce growth for the sensitive isolates.

II. Rolled-towel test for germination and seedling development after seed treatment with azoxystrobin.
Mock-inoculated seed and F. proliferatum-inoculated seed were pretreated with azoxystrobin at a 1 g a.i./100 kg seed equivalent. Interestingly, seeds are still killed by the sensitive isolate after fungicide treatment because the seed are imbibed with such a high level of inoculum. But, it appeared that seedling health is improved (healthier hypocotyls) in the azoxystrobin-sensitive treatment. The azoxystrobin-tolerant isolate does not respond to fungicide treatment and is therefore much more aggressive in the rolled-towel assay. More necrotic/stunted seedlings are found in the azoxystrobin-tolerant isolate treatment. In addition to fludioxonil and azoxystrobin, we have tested Fusarium isolates against captan using a seedling quality test (see the Seedling Quality Scale below). Captan improved seedling quality (“health”) by 50% for seeds inoculated with F. oxysporum, compared to an average of 14-33% for seedlings inoculated with other Fusarium spp. Likewise, fludioxonil improved seedling quality for seeds inoculated with F. graminearum by 75% compared to 0-33% for other Fusarium spp.

III. Screening soybean germplasm for resistance to seedling disease.
Using the rolled-towel assay developed during this project, F. proliferatum, F. oxysporum, and F. soloni isolates were more pathogenic than control and were the most pathogenic species in pathogenicity assays. Commercial entries in the Kansas Soybean Variety Trials have shown a range of resistance and susceptibility. Two isolates of F. graminearum from corn were pathogenic to soybean KS3406. Isolates of F. thapsinum from sorghum were found to be pathogenic to soybean KS3406 as well. Therefore, cross-pathogenicity likely exists in a number of Fusarium spp. And will deserve further experimentation.

We have pursued screening of a wide range of germplasm with F. proliferatum in order to find entries some level of general resistance to this seedling pathogen. During the course of this work we have developed a seedling quality scale (S.Q.S.) as a means to estimate seedling disease severity and general seedling health. Screening of a subset of germplasm from the K-State soybean breeding program has shown that 9/115 genotypes are resistant to the pathogen (although these must re-tested to ensure that they are not "escapes"). A total of 39/115 genotypes showed an intermediate reaction. And, the remaining 67/115 were susceptible.

1.Approximately 14% of Fusarium proliferatum isolates from Kansas exhibit tolerance (>1.2 ug a.i.) to the common seed treatment active ingredient fludioxonil. Response to azoxystrobin is variable. In both cases, tolerant and sensitive F. proliferatum isolates exist.
2.Development of assays including the “rolled-towel pathogenicity assay” and “seedling quality scale” for estimating seedling viability and health in response to Fusarium pathogens. These assays have been used to identify resistant germplasm in Kansas breeding program and Kansas Soybean Variety Trial.
3.After seed treatment, Captan improves seedling quality/health by 50% in response to F. oxysporum. Fludioxonil improves seedling quality/health by 75% in response to F. graminearum. Captan is not the best active ingredient for use to control every Fusarium spp.
4.Cross-pathogenicity experiments have shown that isolates of F. graminearum and F. thapsinum from Kansas corn and sorghum fields, respectively, are pathogenic to soybean.

Objective 5: Improve understanding of the biology of Pythium as a seedling pathogen of soybean (A. Robertson-ISU and M. Chilvers-MSU)
The Robertson lab evaluated the effect of cold stress on soybean seedling disease caused by P. sylvaticum (see peer-reviewed manuscripts submitted below) did not take into account soil moisture. Preliminary trials were done in the growth chamber to include soil moisture. At high soil moisture, emergence of soybean was reduced. Inoculation with P. sylvaticum further reduced emergence.

The Chilvers lab developed and published a high-throughput assay for assessing oomycete fungicide sensitivity. The high-throughput assay enables the screening of multiple chemistries or isolates for fungicide efficacy/sensitivity. The manuscript describing the assay is also being adapted for publication into an online Pythium methods book.

Objective 6: Evaluate the effect of multiple pathogen interactions on seedling disease (A. Robertson and G. Munkvold-ISU)
The Chilvers lab screened USDA germplasm collections for resistance to select oomycete seedling pathogens (publication in progress), and assisted the MSU breeding program in screening elite germplasm to identify lines and resistance loci.

Objective 7: Impact of seed treatments on the interaction of seedling pathogens (A. Fakhoury and J. Bond-SIU)
Our results have shown Fusarium proliferatum to be more aggressive than Fusarium oxysporum and F. sporotrichioides based on root morphology and pathogen density. On the other hand, F. oxysporum, and F. proliferatum data suggested that they have an additive (synergistic) effect when causing root rot on soybean. Rhizosphere soil tightly attached to roots and rhizome were collected for quantitative PCR. Fusarium species were screened against Pythium irregulare; a strong antagonism was evident between fusaria and Pythium species. A greenhouse assay is being developed to test the combined effect of these organisms on soybean seedlings.

We also developed an artificial media system to visualize the root architecture and its development as affected by seedling pathogens in a 3D view. The media contains all the required nutrients and the pictures were analyzed using a MATLAB script. The assay allows us to document closely using 3D imaging the modes of pathogen infection, spread and interaction with other pathogens in presence of the root system. Insights collected from these assays were used to synthesize an artificial core microbiome that includes both pathogenic an non-pathogenic species that can play a systemic role in seedlings disease suppression.

Objective 8: Communicate research results with farmers and stakeholders (K. Wise-UK and others)
We published the 2019 soybean seed treatment efficacy table in March. https://cropprotectionnetwork.org/resources/publications/fungicide-efficacy-for-control-of-soybean-seedling-diseases

Publication List:
1. Adesemoye, A. O. 2018. Root and Soilborne Diseases Update. CropWatch July 2, 2018.
2. Adesemoye, A. O. 2018. Soilborne and early seedling pathogens and delayed planting in corn and soybean. CropWatch May 3, 2018.
3. Ajayi, O.O., S.E. Everhart, P.J. Brown, A.U. Tenuta, A.E. Dorrance, and C. Bradley. 2019. Genetic structure of Rhizoctonia solani AG-2-2IIIB from soybean in Illinois, Ohio, and Ontario. Phytopathology. Accepted pending revision.
4. Gambhir, N., Kodati, S., Adesemoye, A.O., and Everhart, S.E. 2018. Fungicide sensitivity of Rhizoctonia spp. isolated from soybean fields in Nebraska. Poster at ICPP Meeting in Boston, MA.
5. Gambhir, N., Kodati, S., Adesemoye, A.O., and Everhart, S.E. 2019. Fungicide sensitivity and population structure of Rhizoctonia zeae isolated from soybean and corn in the North Central U.S. Poster at the APS Annual Meeting held in Cleveland, OH.
6. Gambhir, N., S. Everhart, S. Kodati, & A. Adesemoye. 2018. Fungicide Resistance: Risk and Management. SoybeaNebraska., Spring 2018, Page 22.
7. Kodati, S. 2019. Diversity and Pathogenicity of Rhizoctonia spp. from Different Host Plants in Nebraska. University of Nebraska-Lincoln, Ph.D. Dissertation. ProQuest, in press.
8. Kodati, S., A. Adesemoye, N. Gambhir, & S. Everhart. 2018. Rhizoctonia Diseases in Soybean. SoybeaNebraska, Spring 2018, Page 23.
9. Kodati, S., Gambhir, N., Everhart, S., and Adesemoye, A. O. (2017). Prevalence and pathogenicity of Rhizoctonia spp. from soybean in Nebraska. A poster presentation during the American Phytopathological Society (APS) Annual meeting (poster #546-P), which held at San Antonio, Texas. August 5-9, 2017.
10. Kodati, S. and Adesemoye, A. O. 2018. Emerging understanding of the pathogenesis of Rhizoctonia zeae in row crops. ICPP-APS Joint Conference holding August 1 to 5 in Boston, MA.
11. Lerch, E. and Robertson, A.E. XXXX. Effect of co-inoculations of Pythium and Fusarium species on seedling disease development of soybean. Can. J. Pl. Path.
12. Noel, Z.A., McDuffee, D., Chilvers, M.I. submitted May 17, 2019. Influence of soybean tissue and oomicide seed treatments on oomycete isolation. Plant Disease.
13. Noel, Z.A., Chang, H.-X., Chilvers, M.I. Submitted Apr 29, 2019, Accepted Nov 2019. Variation in soybean rhizosphere oomycete communities from Michigan fields with contrasting disease pressures. Applied Soil Ecology.
14. Noel, Z.A., Rojas, A.J., Jacobs, J.L., Chilvers, M.I.. 2019. A high-throughput microtiter fungicide phenotyping platform for oomycetes using Z’-factor. Phytopathology 109:1628-1637 https://doi.org/10.1094/PHYTO-01-19-0018-R .
15. Noel Z.A., Sang, H., Roth, M.G., Chilvers, M.I. 2019. Convergent evolution of C239S mutation in Pythium spp. B-tubulin coincides with inherent insensitivity to ethaboxam and implications for other Peronosporalean oomycetes. Phytopathology https://doi.org/10.1094/PHYTO-01-19-0022-R .
16. Okello, P. N., Petrovic, K., Singh, A. K., Kontz, B., and Mathew, F. M. 201X. Characterization of species of Fusarium cause root rot of soybean (Glycine max L.) in South Dakota, USA. Can. J. Plant Pathol. XX: 000-000. (Accepted for publication 27-Aug-2019 (TCJP-2019-0154).
17. Okello, Paul N., "Species of Fusarium Causing Root Rot of Soybean in South Dakota: Characterization, Pathogenicity, and Interaction with Heterodera Glycines" (2019). Electronic Theses and Dissertations. 3251. https://openprairie.sdstate.edu/etd/3251.
18. Pimentel, M., Arnao, E., Warner, A., Subedi, A., Rocha, L., Srour, A., Bond, J., and Fakhoury, A.Trichoderma isolates inhibit Fusarium virguliforme growth, reduce root rot, and induce defense-related genes on soybean seedlings. Plant Disease XXXX.
19. Roth, M.G., Oudman, K.A., Griffin, A., Jacobs, J.L., Sang, H., Chilvers, M.I. 2019. Diagnostic qPCR assay to detect F. brasiliense, a causal agent of soybean sudden death syndrome and root rot of dry bean. Plant Disease https://doi.org/10.1094/PDIS-01-19-0016-RE .
20. Serrano, M. and Robertson, A.E. 2018. The effect of cold stress on damping off of soybean caused by Pythium sylvaticum. Plant Dis. 102: 2194-2200.
21. Serrano, M., McDuffee, D. and Robertson, A.E. 2018. Seed treatment reduces damping-off caused by Pythium sylvaticum on soybeans subjected to periods of cold stress. Can. J. Pl. Path. https://doi.org/10.1080/07060661.2018.1522516.
22. Srour, A., Ammar, H., Subedi, A., Pimentel, M., Cook, R., Bond, J., and Fakhoury, A. Microbial communities associated with long-term tillage and fertility treatments in a corn-soybean cropping system. Frontiers in Microbiology XXXX.

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The project resulted in the development of diagnostic detection assays for soybean seedling and root rot diseases, Phytophthora root rot and soybean sudden death syndrome. The Phytophthora assay is currently being validated for potential commercial production. For soybean sudden death syndrome an assay for Fusarium virguliforme has been widely deployed and is being used by diagnostic labs for rapid identification of SDS infected plants. Additional assays were developed and validated to detected a mix of seedlings pathogens (including Fusarium species and Rhizoctonia). The developed probe panels can be used for fast identification and accurate quantification of key seedlings disease pathogens from different matrices (soil, roots and stem) and provide a powerful decision tool for farmers and researchers.

We maintained and characterized a collection of 3,000+ fungal pathogens and other organisms isolated from soybean. This effort led to the identification and characterization of new soybean pathogens and biocontrol agents that could be used as control measures in the future. In addition, this led to additional research into the crop practices that may increase the density of these beneficial organisms in production fields. If additional research funding is identified the collection will be maintained and should continue to allow for research discoveries into the future.

We investigated the oomycete (water mold) species causing disease of soybean seedlings. We have undertaken survey studies to identify the prevalence and pathogenicity of these species. The isolate collection from the survey has been used to determine fungicide sensitivity of these species to improve management decisions. Key findings include identifying the most abundant and pathogenic species, determining that temperature affects species aggressiveness, with some species being more pathogenic at cool temperatures and others at warm temperatures. We have developed diagnostic assays to assist in diagnosing diseased soybean seedlings and roots. A high-throughput fungicide sensitivity assay was developed and made available for others to use. Using this fungicide sensitivity assay we determined that most species are sensitive to mefenoxam, however there are distinct differences within groups of these species to some new fungicides such as ethaboxam.

Two hundred and forty-seven accessions from the USDA soybean germplasm collection were screened for their resistance to F. graminearum. Eight accessions were identified to be used in breeding programs as sources of resistance to F. graminearum for development of resistant soybean cultivars, which the soybean growers can use to prevent yield loss caused by the pathogen.

Significant differences were documented for reaction to R. solani among northern maturity groups, cultivars and breeding lines, suggesting that soybean germplasm differ in susceptibility to Rhizoctonia diseases. However, under field conditions favoring severe levels of disease, all germplasm appeared to be susceptible to R. solani, indicating that none of the germplasm tested had high levels of resistance. Under less severe conditions, those germplasm lines with reduced susceptibility to Rhizoctonia root and stem rot likely perform significantly better than others.

Methods for assessing reaction to R. solani. were developed. This is of primary value to researchers. We found that some published methods did not work effectively, and thus we needed to test and identify better methods. Our results demonstrate the relative efficacy of different methods for inoculation of soybean with R. solani, which was valuable for our research and will be useful for future researchers as well. We determined if R. solani isolates from soybean vary in anastomosis group and virulence. We collected isolates of R. solani from infected soybean in a number of fields in Minnesota. The predominant anastomosis group of R. solani that we detected infecting soybean in MN was AG 2-2 IIIB. These isolates vary in aggressiveness which can affect greenhouse and field studies of resistance and disease management. This also suggests that the type of isolates present in a field may influence the level of disease severity that develops in that field.

We determine the sensitivity of 35 R. solani isolates to four fungicides commonly used for managing Rhizoctonia root and stem disease, i.e., sedaxane, penthiopyrad, pyraclostrobin and azoxystrobin. We obtained solid results with the first three, and found inconsistent performance of azoxystrobin as had been reported previously by other researchers. R. solani isolates were more sensitive to SDHI fungicides than QoI fungicides; however, the three fungicides are effective for suppressing the pathogen growth and likely remain effective for managing R. solani in the field.

We communicated results with over 30 scientific and popular press articles and presentations. In addition, the 2019 soybean seed treatment efficacy table was released. This is the only table of its kind and is an unbiased resources for the efficacy of seed treatments against seedling pathogens. This table is used by farmers, university extension personnel, crop consultants, seed and crop advisors and the chemical industry.

Benefit to Soybean Farmers

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 of pesticides and germplasm development.

Specific Benefits:
The commercialization of the Phytophthora assay will improve the in-field diagnosis capability of CCA’s and diagnostic services to improve identification of soybean seedling diseases and improved management.

The developed probe panel for fungi can be used for the fast identification and accurate quantification of key seedlings disease pathogens from different matrices (soil, roots and stem) and provide a powerful decision tool for farmers and researchers.

Eight soybean accessions were identified to be used in breeding programs as sources of resistance to F. graminearum for development of resistant soybean cultivars, which the soybean growers can use to protect yield from the pathogen.

Research has led to manuscripts that clearly demonstrate the need for multiple chemistry seed treatments for the management of mixed oomycete populations, this data will be of value to farmers in making seed treatment decisions.

Insights collected from thee assays will be used to synthesize an artificial core microbiome that includes both pathogenic an non-pathogenic species that can play a systemic role in seedlings disease suppression.

We published the 2019 soybean seed treatment efficacy table in March. This seed table is widely used by the industry and farmers for selecting the best seed treatments.

Performance Metrics

Project Years