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.

View uploaded report

View uploaded report 2

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.