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:2017
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)
Kiersten Wise (Purdue University)
Febina Mathew (South Dakota State University)
Ahmad Fakhoury (Southern Illinois University)
John Rupe (University of Arkansas)
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)
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Keywords: Seedling Diseases

Contributing Organizations

Funding Institutions

Information and Results

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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 project and results from the 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 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
Objective 2. Curate the collection of fungal pathogens collected during the first phase of this project
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
Objective 7: Impact of seed treatments on the interaction of seedling pathogens

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 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.

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 – completed, final data being collected for publication.

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: A manuscript describing the Phytophthora detection assays has been prepared for submission and is undergoing final co-author reviews. As part of the OSCAP project a training was conducted in both qPCR and isothermal detection assays for diagnosticians and researchers.

Several assays have been developed that appear promising alone and in combination for selective detection of a group of key fungal seedling pathogens of soybean. The Fakhoury lab has developed several probe-based assays for the detection and quantification of top 10 fungal species frequently found associated with seedling diseases in soybean, as determined in the first phase of the USB-NCSRP project.

Objective 2: The Fakhoury lab has finished cataloguing 3000 fungal isolates that are now stored and maintained at SIU. Long term storage methods have been optimized. A searchable site is still under construction and will be publically available by the end of 2017. Curated sequences are currently being submitted to GENBANK.

Objective 3: Population genetic analysis of isolates from Illinois, Ohio, and Ontario has been performed on SNP data obtained using Genotyping By Sequencing (GBS; in collaboration with Carl Bradley and former lab). A publication is currently in preparation and will be submitted to Phytopathology later this year.

Objective 4a: About 503 Fusarium isolates were recovered from 1000 soybean roots surveyed in 100 fields covering 29 counties in South Dakota. F. subglutinans and F. equiseti were least recovered (<2%). DNA sequencing to confirm the identity of the Fusarium species recovered is on-going. This will be completed in the coming months.

To evaluate soybean germplasm for resistance to four Fusarium species (F. proliferatum, F. graminearum, F. sporotrichioides, and F. subglutinans), the soybean genotypes seeds were obtained from the United States National Plant Germplasm System. A susceptible soybean cultivar Asgrow 1835 (Monsanto, St. Louis, MO Company) was used as the check.

Objective 4b: Fludioxonil and azoxystrobin were tested against a wide range of Fusarium spp. including F. proliferatum (FPR). In general, FPR has greater cumulative growth across a range of fludioxonil concentrations than does other seedling pathogens including F. oxysporum, indicating that FPR is more resistant to this chemical. Nearly 30% of the FPR isolates tested showed relative resistance to fludioxonil compared to the other isolates. Screening of 118 entries from the 2014 Kansas Soybean Variety Trial with a pathogenic isolate of FPR showed five entries that had significantly lower disease severity ratings than all the other entries.

Objective 5: Isolates collected through the OSCAP project were maintained at MSU and a subset of isolates was sent to ISU for fungicide sensitivity screening. 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. In general, all species from all states behaved as expected but there were a few isolates that were different. A manuscript is in progress.

At MSU data sets are being collected on Phytopythium species to finalize the comparison of the high-throughput oomycete fungicide sensitivity assay to standard poison plate assays. Figures for a manuscript describing the assay are being finalized.

At ISU, a growth chamber experiment in a five-way factorial design with two cold stress temperatures (4ºC and 10 ºC), two cold stress timings (24 and 96 hours after planting), three cold stress durations (24, 48 and 96 hours), and two levels of seed treatments (Intego SuiteTM and untreated) was conducted in cups inoculated with P. sylvaticum or a non-inoculated control. Data from this study confirm cold stress soon after planting may increase the risk of reduced crop stands and suggests seed treatments effectively protect seedlings when adverse conditions are expected soon after planting.

Objective 6: A cup assay was used to evaluate the effect of the interaction between Pythium sylvaticum, P. irregulare, Fusarium graminearum and F. oxysporum on soybean seedling disease development. Preliminary data analysis showed seedling disease was more severe in cups inoculated with Pythium compared to those inoculated with Fusarium. No difference in disease development was observed for the Pythium species tested, but more severe disease development occurred on seedlings inoculated with F. graminearum compared to those inoculated with F. oxysporum.

Objective 7: A greenhouse experiment has been established to test the effect of seed treatments on seedling pathogens. In a first experiment, F. oxysporum, R. solani were used to inoculate the soil using infected sorghum seeds. In a second experiment, Fusarium species were selected since they were the most common pathogens isolated and their density was significantly higher.

View uploaded report Word file

Update:

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 is collaborating with Dr. Frank Martin and Dr. Tim Miles in the validation of Phytophthora genus specific markers that will have applicability across species and hosts. In addition, preliminary Pythium genus specific markers have been designed and are undergoing additional testing. A manuscript describing the use of a qPCR assay for quantification of F. virguliforme and identification of risk factors for SDS development within a field was submitted to Phytopathology. The manuscript was accepted pending revisions.

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 52 Rhizoctonia isolated from soybean fields in Nebraska, with an additional 31 Rhizoctonia isolated in the 2017 season. Thus far, we have identified Rhizoctonia zeae (23), R. solani AG-4 (20), R. solani AG-3 (2), R. solani AG-2 (1), R. solani AG 1-IB (4), and AG-B (2). 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.

Objective 3b: Monitor shifts in fungicide sensitivity in R. solani populations (S. Everhart and T. Adesemoye-UN)

Fungicide sensitivity assays are currently underway for Rhizoctonia isolates using the plate dilution method. Screening will be done for four fungicides with different modes of action: propiconazole (DMI), fludioxonil (Phenylpyrrole), thiabendazole (TBZ), and penflufen or sedaxane (SDHI). Results from this work will use isolates previously collected in the North Central states and new isolates collected from Nebraska, to provide a comprehensive overview of sensitivity across the region.

Publications and scientific presentations:
• Kodati, S., Eskelson, M. J., and Adesemoye, A. O. 2017. Cross-pathogenicity of Rhizoctonia spp. isolated from multiple hosts to corn, soybean, and wheat. A PowerPoint presentation made during the Annual Meeting of the North Central Division meeting of American Phytopathological Society, Champaign, IL. June 14-16, 2017.
• 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.
• Adesemoye, A. O. presented a seminar in Lincoln, NE on September 29, 2017 titled: “Harnessing components of the root microbiome for integrated management of soilborne plant diseases” and findings from this study was part of the discussion. The seminar was part of the UNL Department of Agronomy and Horticulture fall seminar series, which was well publicized.
• Adesemoye, A.O., S. Kodati, and R. Werle. 2016. Herbicide Injury and Pathogen Infection on Soybean Seedlings. CropWatch June 17, 2016
• Kodati, S., and A.O. Adesemoye. 2016. Characterization, Anastomosis Grouping and Pathogenicity of Rhizoctonia solani on Multiple Plant Hosts in Nebraska. Department of Plant Pathology Seminar Series. Nov. 7, 2016.

Objective 3c: Identification and characterization of resistance and tolerance to Rhizoctonia root rot (D. Malvick-UMN)

The Malvick lab has been investigating resistance and tolerance to Rhizoctonia root rot of soybean caused by Rhizoctonia solani in greenhouse and field trials. The central goal of this project is to determine if early maturity group soybean germplasms vary in response to this disease and to identify those with different levels of susceptibility. Replicated field and greenhouse studies were conducted to determine if randomly chosen soybean cultivars and breeding lines respond differently to R. solani in growth or yield. Cultivars and lines responded with significant differences in plant height and stand count in the greenhouse and with significant differences in stand count and yield in the field for inoculated vs. noninoculated treatments. These initial studies suggest that many soybean cultivars can respond differently to Rhizoctonia root and stem rot, which could relate to yield in the field under conditions favorable for this disease. Additional studies are being completed in the field and are underway in the greenhouse to validate these results under varying conditions.

Outputs:
• Malvick, D., and Floyd, C. 2017. Characterization of response to Rhizoctonia solani among soybean cultivars in field and greenhouse environments. Poster presented at the Annual Meeting of the North Central Division of the American Phytopathological Society in Champaign, IL. June 2017.
• Presented results from this project at a field workshop and created demonstration plots to demonstrate the effects of Rhizoctonia and it's interactions with planting date on soybean stand and growth
Objective 4 Objective 4. Improve understanding of the biology of Fusarium sp. as seedling pathogen of soybean

Soybean germplasm screening for resistance to multiple species of Fusarium (F. Mathew-SDSU)
Soybean genotype seeds belonging to maturity groups 0 and I were screened for resistance against four Fusarium species [F. graminearum isolate FUS052 (McCook County, SD), F. proliferatum isolate FUS026 (McCook county, SD), F. sporotrichioides isolate FUS002 (Clay County, SD) and F. subglutinans isolate FUS035 (Sanborn County, SD).
The Fusarium isolates were recovered from commercial soybean fields in South Dakota and the inoculum layer method (Bilgi et al. 2008) was used in the greenhouse screening.
The plant introductions (PIs) with origins from 15 countries, were obtained from the USDA Soybean Germplasm Collection, Urbana, IL and from Iowa State University, Ames.
A susceptible soybean cultivar Asgrow 1835 (Monsanto, St. Louis, MO Company) was used as the check.

Disease severity was measured as lesion length on plant tap root caused by the Fusarium isolates and was evaluated at 14 days after inoculation.
All the Fusarium isolates caused both pre and post-emergence damping off on most of the PIs. Soybean genotypes in which the fungal pathogens caused less than 35% mortality were subjected to analysis of variance using R (v2.11.1; R core team 2012; https://www.rstudio.com/). and means separated by Fisher’s protected least significant difference (LSD) at P = 0.05.
• A total of 160 soybean genotypes belonging to maturity group 0 (54) and 1 (106) were screened against F. graminearum. There was observed significant difference (P < 0.05) on lesion length caused by F. graminearum on the roots of soybean genotypes belonging to maturity group 0. 15 genotypes belonging to maturity 0 has statistically shorter lesion length compared to the check at P = 0.05.
• 227 soybean genotypes belonging to maturity group 0 (104) and 1 (123) were screened against F. proliferatum. 85 genotype belonging to maturity group 0 had statistically shorter lesion length than the susceptible check at P = 0.05 compared to 43 genotype belong to maturity group I.
• 115 soybean genotypes belonging to maturity group 1, have been screened against F. sporotrichioides. 86 genotypes caused less than 35% mortality and none caused lesion length that was significantly different from the susceptible check at P = 0.05.
• 73 soybean genotypes belonging to maturity group 1 have been screened against F. subglutinans of which 48 caused less than 35% mortality. 21 genotypes caused lesion length that were statistically different from the susceptible check at P = 0.05.
• One genotype (PI 437343) had a shorter lesion (< 28mm) caused by the four Fusarium species. This genotype may be used to develop soybean breeding line and commercial cultivar with resistance to the four species of Fusarium.

Objective 4. Improve understanding of the biology of Fusarium sp. as seedling pathogen of soybean (K. Little-KSU)

1. Part of understanding the biology of Fusarium spp. as seedling pathogens of soybean is the detection of pathogens in seed and seedlings that have the potential to cause disease. In particular, we are focusing on F. proliferatum at KSU. DNA was extracted from seed samples from three soybean genotypes from five locations in Kansas. ITS primers and nested primers were used to amplify fungal sequences using Illumina MiSeq. Overall, F. proliferatum was the most abundantly amplified Fusarium sp. from soybean seed. 2. So far, we have concentrated on the interaction of F. proliferatum with fludioxonil. Isolates of F. proliferatum were collected from soybean seeds and seedlings in Kansas and tested against increasing concentrations of fludioxonil. In our tests, isolates with EC50 > 100 ug/ml a.i. were considered resistant to fludioxonil. Approximately 25% of F. proliferatum isolates were found to be resistant to fludioxonil with EC50s between 115-447 ug/ml a.i. Additionally, we have tested isolates of F. oxysporum and F. fujikuroi against fludioxonil. Unlike F. proliferatum, only 6.7% of F. fujikuroi isolates showed resistance to fludioxonil. 3. Germplasm screening using the rolled towel test has been used to evaluate entries for resistance to F. proliferatum in the Kansas Soybean Variety Trial. For example, two varieties that appear to have moderate resistance to F. proliferatum are LG C3333RX and Midland 9373R2.

Objective 5: Improve understanding of the biology of Pythium as a seedling pathogen of soybean (A. Robertson-ISU and M. Chilvers-MSU)

At ISU, a manuscript entitled “Seed treatment reduces damping off caused by Pythium sylvaticum on soybeans subjected to periods of cold stress” was submitted to the Canadian Journal of Plant Pathology. A second manuscript titled “The effect of cold stress on damping-off of soybean, seed exudation and sporangia germination of Pythium sylvaticum” will be submitted for peer –review in November.

At MSU, a training of Dr. Robertson’s graduate students was conducted on the use of our high-throughput fungicide sensitivity assay. Manuscripts describing guidelines for accurate fungicide sensitivity estimation and the high throughput fungicide sensitivity assay were submitted for publication.

Publications and presentations:
• Publication. Noel, Z.A., Wang, J. Chilvers, M.I. Submitted Jun 20, 2017, accepted pending minor corrections. Guidelines for accurate EC50 estimation for fungal and oomycete plant pathogens. Plant Disease
• Publication. Noel, Z.A., Rojas, A.J., Jacobs, J.L., Chilvers, M.I. Submitted, requires revision prior to resubmission Sep, 2017. Development and evaluation of a high-throughput fungicide sensitivity assay for oomycetes. Phytopathology
• Presentation. MSU soybean variety performance trial tour. Soybean and corn disease discussion. Allegan, MI. Sep 21, 2017
• Presentation. Soybean, and corn disease update. Alma, MI. Aug 30, 2017
• Presentation. MSPC reporting. Plant Pathology Farm, East Lansing, MI. Aug 30, 2017
• Presentation. Manage soybean disease to protect yield. AgroExpo, MI. Aug 16, 2017
• Presentation. Southwest Michigan Row Crops Field Day 2017, Vicksburg, MI. Aug 2, 2017

Objective 6: Evaluate the effect of multiple pathogen interactions on seedling disease (A. Robertson and G. Munkvold-ISU)

A manuscript discussing the interaction of two Pythium species with two Fusarium species on damping off of soybeans is being prepared.

Objective 7: Impact of seed treatments on the interaction of seedling pathogens (A. Fakhoury and J. Bond-SIU)

A new graduate student was hired to work on the interaction experiments.

Objective 8: Communicate research results with farmers and stakeholders (K. Wise-UK and others)

Albert and Kiersten met with the CPN group in Chicago in October and drafted a short communication piece summarizing some of Carl Bradley's work on Rhizoctonia solani AG groups. The draft is in review now, and we hope to distribute it the SRII website and media outlets in early November. Once this draft is final, we will also distribute to the co-PIs to show them an example of the types of papers/outputs we are hoping to summarize from this project.

View uploaded report Word file

Final Project Results

Updated April 7, 2018:
Year End Report to cover October 1, 2016 - December 31, 2017
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: A real time PCR and an isothermal RPA assay for genus and species level detection of Phytophthora, Phytophthora sojae and Phytophthora sansomeana was developed and published. The real-time PCR assay is applicable for use on samples in the lab setting, and most research and diagnostic labs are familiar with this type of assay and have equipment to run the real-time PCR assay. The advantage of this assay is the ability to quantify the pathogen in soil and plant samples. For example the assay could be used to assess the ability of seed treatments to protect from Phytophthora infection, or assess germplasm for the ability to limit infection. The isothermal RPA assays that were developed can also be used in the diagnostic lab with the same equipment, but give a quicker turnaround of less than 20 minutes. The RPA assays also have the advantage of being conducted with much simpler equipment, allowing them to be used in the field to diagnose a plant sample in the field.

Fakhoury Lab: Several assays have been developed that appear promising alone and in combination for selective detection of a group of key fungal seedling pathogens of soybean. we have developed several probe-based assays for the detection and quantification of top 10 fungal species frequently found associated with seedling diseases in Soybean, as determined in the first phase of the USB-NCSRP project. The list of fungal species includes F. oxysporum, F. solani, F. acuminatum, F. equiseti, F. graminearum, F. sporotrichioides, F. proliferatum, T. harzianum, M. elongata and R. solani. In addition, a more general assay targeting all Fungi was developed and showed more specificity and accuracy than available tests.

The primer/probe sets were designed based on the intergenic spacer of the ribosomal RNA (IGS) and translation elongation factor alpha gene (EF1). The designed primer/probe assays were first tested in silico against a constructed database collected in this project. Furthermore, the specificity of the qPCR assays has been tested against at least twenty non-target fungal species, all of which turned out to be specific. All of the assays showed optimal amplification efficiency and a limit of detection below 0.1 pg of the target pathogen DNA. A manuscript summarizing our efforts is currently in preparation. Currently we are working on including several of these assays in a multiplex RT-qPCR method which would reduce time, technical variation and cross contamination while providing the same level of sensitivity observed in singleplex reactions..

Objective 2: Curate the collection of fungal pathogens collected during the first phase of this project (A. Fakhoury-SIU)
The Fakhoury lab has finished cataloguing 3000 fungal isolates that are now stored and maintained at SIU. Long term storage methods have been optimized. A searchable site is still under construction and will be publically available by the end of 2017. Curated sequences are currently being submitted to GENBANK. Additional bioinformatics tools and features are being tested before final release.

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 52 Rhizoctonia isolated from soybean fields in Nebraska, with an additional 31 Rhizoctonia isolated in the 2017 season. Thus far, we have identified Rhizoctonia zeae (23), R. solani AG-4 (20), R. solani AG-3 (2), R. solani AG-2 (1), R. solani AG 1-IB (4), and AG-B (2). 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.

Objective 3b: Monitor shifts in fungicide sensitivity in R. solani populations (S. Everhart and T. Adesemoye-UN)
Fungicide sensitivity assays are currently underway for Rhizoctonia isolates using the plate dilution method. Screening will be done for four fungicides with different modes of action: propiconazole (DMI), fludioxonil (Phenylpyrrole), thiabendazole (TBZ), and penflufen or sedaxane (SDHI). Results from this work will use isolates previously collected in the North Central states and new isolates collected from Nebraska, to provide a comprehensive overview of sensitivity across the region.

Objective 3c: Identification and characterization of resistance and tolerance to Rhizoctonia root rot (D. Malvick-UMN)
The Malvick lab has been investigating resistance and tolerance to Rhizoctonia root rot in northern soybean germplasms in greenhouse and field trials. Replicated field and greenhouse studies were conducted to demonstrate that different soybean cultivars and breeding lines respond differently to R. solani in survival, growth, and yield. Varieties and lines responded with significant differences in plant height and stand count in the greenhouse and with significant differences in stand count and yield in the field for inoculated vs. noninoculated treatments. We have continued to refine and evaluate greenhouse inoculation methods, and our results suggest that different inoculation methods are more effective than others at detecting resistance/tolerance to R. solani .

Objective 4a: Pathogenicity of Fusarium species and identify resistant germplasm (F. Mathew-SDSU)
Screening soybean germplasm (performed in March to August 2017) = For F. graminearum, of the 160 soybean genotypes that were screened for resistance, the fungus caused significantly shorter lesions on 15 genotypes when compared to the susceptible check at P = 0.05. For F. proliferatum, of the 227 genotypes that were screened for resistance, the fungus caused significantly shorter lesions on 85 genotypes belonging to maturity group 0 and 43 genotypes belong to maturity group I compared to the susceptible check at P = 0.05. For F. sporotrichioides, of the 115 soybean genotypes, the pathogen did not cause significantly shorter lesions on any of the genotypes when compared to the susceptible check at P = 0.05. For F. subglutinans, the pathogen caused significantly shorter lesions on 21 genotypes when compared to the susceptible check at P = 0.05.

Cross-pathogenicity of Fusarium causing disease on soybean and corn (performed in November 2017) = Seven Fusarium species were identified causing disease on corn, among which Fusarium graminearum and Fusarium oxysporum were most frequently recovered (=20%). In the greenhouse pathogenicity study, significant differences in aggressiveness (P = 0.05) was observed among the Fusarium species with F. graminearum (FG13) and F. acuminatum (FA8) being the most aggressive. The cross-pathogenicity experiment is in progress to determine the effect of Fusarium isolates from corn on soybean.

Publication
Okello, P. N., and Mathew, F. M. 201X. Interaction between Fusarium and soybean cyst nematode on soybean (Glycine max L.). Plant Dis. (PDIS-10-17-1570-RE; Accepted 16-Dec-2017).

Outreach
Byamukama, E., Strunk, C., Tande, C. and Mathew, F. 2017. Sudden death syndrome of soybean confirmed in South Dakota. Online, iGrow Published September 2017.
Okello, P., Osborne, S., Kleinjan, J., and Mathew, F. 2017. Evaluating fertilizer effect on the interaction between Fusarium proliferatum and soybean cyst nematode on soybean. American Phytopathological Society Annual Meeting, San Antonio, TX. August 5-August 9, 2017 (Poster).
Okello, P., Osborne, S., and Mathew, F. 2017. Effects of N-P-K fertilizer rates on the interaction between Fusarium virguliforme and soybean cyst nematode on soybean. American Phytopathological Society Annual Meeting, San Antonio, TX. August 5-August 9, 2017 (Poster).
Byamukama, E. Mathew, F. Strunk, C. and Tande, C. 2017. Scout for root rots in soybean. Online, iGrow Published June 2017.

Objective 4b. Improve understanding of the biology of Fusarium sp. as seedling pathogen of soybean (K. Little-KSU)
Fusarium spp. are one of the most important pathogen groups on soybeans, their identity and frequency in seeds as well as their importance as seedborne pathogens remains unclear. Thus, the objectives of this work was to characterize: i) the identity and frequency of Fusarium spp. present within 408 soybean seed samples in the state of Kansas during three growing seasons 2010. 2011 and 2012; and ii) to test the pathogenicity of the most commonly encountered seedborne Fusarium spp. on soybean seeds and seedlings under growth chamber and greenhouse conditions using artificially inoculated seeds. A semi-selective medium (PCNB) was used for Fusarium isolation. Identification was based upon morphological characters and PCR. The influence of Fusarium spp. on soybean seed germination and vigor was assessed by pathogenicity assays in laboratory and greenhouse. The three-year screening effort showed that 33% of the seed samples analyzed contained Fusarium spp. at some level. Nine Fusarium species were identified among the infected seed samples. Fusarium semitectum was the most frequently encountered species, followed by F. proliferatum, F. verticillioides, Fusarium acuminatum, F. equiseti, F. thapsinum, F. fujikuroi, F. oxysporum, and F. graminearum were least frequently observed in infected soybean seeds. Regarding pathogenicity, only soybean seeds artificially inoculated with F. proliferatum, F. graminearum, F. fujikuroi, F. oxysporum, and F. thapsinum significantly decreased seed germination (p > 0.001) and vigor (p > 0.001) when estimators of seed quality were compared with mock-inoculated control. No significant reductions of seed quality were observed for seeds artificially inoculated with F. semitectum, F. verticillioides, F. acuminatum, and F. equiseti. Understanding the relationship between pathogenic Fusarium spp. and soybean seeds will contribute to improvements for seed health testing methods, and ensure global food security, quality and production.

Objective 5: Improve understanding of the biology of Pythium as a seedling pathogen of soybean (A. Robertson-ISU and M. Chilvers-MSU)
At ISU, a growth chamber experiment with two cold stress temperatures (4ºC and 10 ºC), two cold stress timings (24 and 96 hours after planting), three cold stress durations (24, 48 and 96 hours), and two levels of seed treatments (Intego SuiteTM and untreated) was done in cups inoculated with P. sylvaticum or a non-inoculated control. Emergence was reduced when the pathogen was present compared to the non-inoculated controls. In untreated seed, cold stress duration reduced emergence, but no difference in timing of cold stress and cold stress temperatures (4ºC and 10 ºC) were detected. The seed treatment improved emergence, reduced root rot severity, and increased shoot weight. There was no significant effect of seed treatment on root weight. Data from this study confirm cold stress soon after planting may increase the risk of reduced crop stands and suggests seed treatments effectively protect seedlings when adverse conditions are expected soon after planting. Two manuscripts describing these studies were prepared and submitted for peer review.

A high-throughput fungicide sensitivity assay for oomycetes (Pythium and Phytophthora) was also developed and a manuscript was submitted for publication, the manuscript is currently being revised. The advantage of the assay is the ability to screen many isolates for fungicide sensitivity or fungicides for their efficacy, as opposed to the current poison plate assays which are more labor and resource intensive. The assay has been utilized to assess the fungicide efficacy of mefenoxam and ethaboxam across 84 oomycete species collected from diseased soybean seedlings. Graduate students and technicians were trained from Iowa State in use of the protocol.

Published abstracts and presentations:
Serrano, M. and Robertson, A. E. 2017. The effect of cold stress on damping-off of soybean caused by Pythium sylvaticum. (Abstr.) Phytopathology 107:S5.1.
Lerch, E. and Robertson, A.E. 2017. Co-inoculation of Pythium sylvaticum and Fusarium oxysporum on soybean seedling disease development. (Abstr.) Phytopathology 107:S5.173
Serrano, M. and Robertson, A.E. Using seed treatments to manage soybean seedling disease. Iowa Soybean Association On-Farm Research Conference, Feb 2017 (~150 attendees)

Objective 6: Evaluate the effect of multiple pathogen interactions on seedling disease (A. Robertson and G. Munkvold-ISU)
A cup assay was used to evaluate the effect of the interaction between Pythium sylvaticum, P. irregulare, Fusarium graminearum and F. oxysporum on soybean seedling disease development under controlled environment conditions. Cups were inoculated with either Pythium alone, Fusarium alone, or co-inoculated with both genera. Non-inoculated cups were used as a control. Preliminary data analysis showed seedling disease was more severe in cups inoculated with Pythium compared to those inoculated with Fusarium. No difference in disease development was observed for the Pythium species tested, but more severe disease development occurred on seedlings inoculated with F. graminearum compared to those inoculated with F. oxysporum.

Objective 7: Impact of seed treatments on the interaction of seedling pathogens (A. Fakhoury and J. Bond-SIU)
At SIU, a greenhouse experiment has been established to test the effect of seed treatments on seedling pathogens. In a first experiment, Fusarium oxysporum, Rhizoctonia solani were used to inoculate the soil using infected sorghum seeds. Next, treated seeds were planted and covered with a thin layer of soil. Roots were collected 3 weeks after planting and qPCR assays are currently being conducted to quantify each pathogen.
In a second experiment, Fusarium species were selected since they were the most common pathogens isolated and their density was significantly higher. Fusarium species included F. sporotrichioides, F. oxysporum, and F. proliferatum isolates. A dual plate assay was conducted to test for competition and antagonism between these species. The dual pate assays confirmed an antagonism between F. oxysporum, F. proliferatum, and F. acuminatum. Following, we carried a second test using different media (Weak PDA and Oatmeal Agar). The results were consistent, showing non-environmental dependence of the interaction between these species. The pathogenicity of each pathogen was next tested separately in a greenhouse setting Subsequently, numerous permutations have been conducted, including pairwise and total combinations of the selected isolates. Disease scores and vigor ratings were taken at 10, 18 and 28 days. No difference in root rot scores and vigor were observed between the different permutations of the selected 3 fusaria. Rhizosphere soil tightly attached to roots and rhizome were collectedf End of Project Final Report
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

Objective 8: Communicate research results with farmers and stakeholders (K. Wise-UK and others)
Albert and Kiersten met with the CPN group in Chicago in October and drafted a short communication piece summarizing some of Carl Bradley's work on Rhizoctonia solani AG groups. The draft is in review now, and we hope to distribute it the SRII website and media outlets in early November. Once this draft is final, we will also distribute to the co-PIs to show them an example of the types of papers/outputs we are hoping to summarize from this project.

View uploaded report Word file

Benefit to Soybean Farmers

Performance Metrics

Project Years