Incidence of Sclerotinia stem rot (SSR) is highly dependent on specific environmental requirements necessary for infection. In instances where flowering coincides with cooler temperatures (less than 28°C) and extended surface wetness, disease severity can be significant (Grau and Hartman, 1999). The sporadic nature of the disease results because these weather conditions must be present at the time of soybean flowering; if they are absent, then SSR is unlikely to occur. Occasionally, secondary spread of SSR can occur via plant-to-plant contact (Grau and Hartman, 1999). Sclerotinia stem rot incidence is often higher in fields with high yield potential resulting in a dense canopy and in situations where plants are in narrow rows and at high population. In these instances, canopy humidity and wetness can be high, thereby promoting increased incidence of SSR. In the temperate North Central soybean production areas of the United States, SSR can be a significant yield limiting disease. Combinations of management strategies have been utilized to limit losses from SSR. These include cultural practices such as reduced tillage, crop rotation, and canopy management; and chemical control provided thorough coverage and timely application. Objective 1 will address the effect of weather and application timing on fungicide efficacy.

Objective 2 of this proposal will address molecular mechanisms of pathogenesis to identify targets for resistance breeding. Currently, a major problem is that pathogenic development of S. sclerotiorum is complex and not well understood, with most models focused on plant interactions with biotrophs. Nevertheless, it is clear that S. sclerotiorum is able to alter the plant recognition process via the secretion of oxalic acid OA; a key component for the fungus to cause disease) and possibly other molecules. The regulation of reactive oxygen species (ROS) plays a key role in this process. One of the major sources of ROS in plants is the plasma membrane bound NADPH oxidases. RNA sequencing of resistance and susceptible soybean lines following S. sclerotiorum challenge has recently confirmed the importance of soybean NADPH oxidases in disease development. This project is funded by a Wisconsin Soybean Marketing Board grant to Kabbage and Smith.

Overall, resistance to S. sclerotiorum is poorly understood, as specific genetic players involved in resistance (or susceptibility) to this pathogen have not been identified in soybean. The characterization of soybean NADPH oxidases and their role in SSR disease development will provide much needed information on the underlying genetic mechanisms in this disease system. The information gathered from this study can be utilized in marker-assisted breeding or the generation of transgenic soybean lines to provide a sustainable source of Sclerotinia stem rot resistance.

The significance of fungicide resistance as a nationwide problem is highlighted by the 2014 creation of the Soybean Fungicide Resistance Hub on the Plant Management Network. This was established to survey and report discovery of fungicide resistant pathogens throughout the U.S. soybean production area. But the reality is that we still have limited knowledge about mechanisms of resistance emergence. Random sampling from fields is not sufficient to gain insight into factors driving resistance emergence. In addition, there is speculation in the literature (Gressel, 2011) that low doses of fungicides may hasten resistance emergence. This is important to soybean production because low doses of fungicides are likely to occur in the field, whether due to intentional reduction of application rates or unintentionally because of incomplete spray coverage. Objective 3 will investigate fungicide resistance emergence in this system.

Another important goal of this study (objective 4) is to develop much needed, current, economic and outreach materials pertaining to Sclerotinia stem rot of soybean with emphasis on potential management practices for the disease in the North Central Region. Recent advances in this system will be compiled with results from this project to develop a range of extension products to reach farmers with a variety of preferred learning styles. There is a serious need to update how this pathogenic system is viewed.

Updated December 6, 2019:
Progress report (October 01, 2018)

Objective 1. Effect of weather conditions and application timing on fungicide efficacy.

Identifying the optimal timing for fungicide application is crucial in order to maximize the control of Sclerotinia stem rot (SSR), which is caused by Sclerotinia sclerotiorum. Therefore, the impact of canopy closure and soil temperature on apothecia production needs to be investigated to optimize fungicide application timing. The manuscript describing the development of Sclerotinia apothecia and the effect of canopy closure and row spacing is now published in Plant Disease (Fall et al., 2018). The results explain the inconsistent efficacy of fungicide applications based on the soybean growth stage and will be helpful for informing disease models and fine-tuning fungicide application strategies. The findings of canopy closure and row spacing and their impact on Sclerotinia apothecia development were incorporated into the Sclerotinia apothecia risk prediction model.

Overall, our study lays the groundwork for the future use of a canopy closure percentage of 40% and a soil temperature between 20 and 25°C as risk indicators for the production of apothecia, and for the integration of those indicators as initiation parameters into the recently developed apothecia production models.

Multistate fungicide trials data from 2018 are currently being analyzed. The 2017 results were integrated in a large meta-analysis of fungicide efficacy. These studies were used to test 10 popular active ingredients, and seven common application timings. Active ingredient was found to significantly affect disease severity index (DIX) reduction and yield benefit (P < 0.0001). Application timing was also found to significantly affect disease reduction (P < 0.0001) and yield benefit (P = 0.0009). The moderator variable of non-treated disease severity, while not significant (P = 0.07), was useful in evaluating the impact of different active ingredients on soybean yield benefit. These studies were also used in nonlinear regression analyses to determine the effect of disease severity index (DIX) on soybean yield. A three-parameter logistic model was found to best describe soybean yield loss (pseudo-R2 = 0.309). In modern soybean cultivars, yield loss due to SSR does not appear to occur until 20-25% DIX and considerable yield loss (-697 kg ha-1 or -10 bu a-1) is observed beginning at 68% DIX. Further analyses determined an 80-95% probability for return on investment using various products and programs. These studies will help growers select cost effective fungicide programs for use in integrated management of SSR in soybean. This manuscript is under consideration in Phytopathology (Willbur et al., XXXX).

Overall, In the North Central region, a single application of BOSC (Endura, BASF, Research Triangle Park, NC) or two applications of PICO (Aproach, DuPont, Wilmington, DE) are standard recommendations for SSR management. In this study, these active ingredients along with LACT (Cobra, Valent U.S.A., Walnut Creek, CA) consistently resulted in the highest reductions in SSR and/or yield benefits, under high disease pressure. The active ingredients FLUO + FLUT (Fortix, Arysta Lifescience, Cary, NC), TETR (Domark, Isagro USA, Morrisville, NC), and THIM (Cerexagri-Nisso, King of Prussia, PA) were consistently found to have among the lowest efficacies and yield benefits. LACT was among the more efficacious active ingredients in high disease pressure situations, and its relatively low cost compared to other products, resulted in high estimated value to the farmer. Economical disease management balances efficacy and cost; therefore, BOSC, a highly efficacious active ingredient, was not as profitable in this analysis because of its high cost.

In this period we have also validated our white mold prediction model to better time fungicide applications (Willbur et al., 2018). This model was used in the development of a free smart phone app, Sporecaster, designed to help farmers predict the need for a fungicide application to control white mold in soybean. We are please to report that this app is available for free use and it uses university research to turn a few simple taps on a smartphone screen into an instant forecast of the risk of apothecia being present in a soybean field, which helps growers predict the best timing for white mold treatment during the flowering period.

Publications:

Willbur, J.F., Fall, M.L., Byrne, A.M., Chapman, S.A., McCaghey, M.ga, Mueller, B.D., Schmidt, R., Chilvers, M.I., Mueller, D.S., Kabbage, M., Giesler, L.J., Conley, S.P., and Smith, D.L. (2018) Validating Sclerotinia sclerotiorum apothecial models to predict Sclerotinia stem rot in soybean (Glycine max) fields. Plant Disease. doi.org/10.1094/PDIS-02-18-0245

Willbur, J.F., Mitchell, P.D., Fall, M.L., Byrne, A.M., Chapman, S.A., Floyd, C.M., Bradley, C.A., Chilvers, M.I., Kleczewski, N.M., Malvick, D.K., Mueller, B.D., Mueller, D.S., Kabbage, M., Conley, S.P., and Smith, D.L. (2018) Meta-analytic and economic approaches for evaluation of fungicide impact on Sclerotinia stem rot and soybean yield in the North Central U.S. Phytopathology. XX:XX-XX.

Fall, M.L., Willbur, JF., Smith, D.L., Byrne, A.M., Chilvers, M.I. Spatiotemporal distribution pattern of Sclerotinia sclerotiorum apothecia is modulated by canopy closure and soil temperature in an irrigated soybean field. Plant Disease. Volume 102, 9:1794-1802

Fall, M.L., Boyse, J.F., Wang, D., Wilbur, J.F., Smith, D.L., Chilvers, M.I. 2018. Case study of an epidemiological approach dissecting historical soybean Sclerotinia stem rot observations and identifying environmental predictors of epidemics and yield loss. Phytopathology 108(4):469-478 https://doi.org/10.1094/PHYTO-12-16-0446-R


Objective 2: The role of soybean NADPH oxidases in Sclerotinia stem rot disease development

In previous reports, we discussed that the silencing of a specific soybean genes of NADPH oxidases (termed GmRBOH-VI) led to enhanced resistance to S. sclerotiorum and drought tolerance in soybean. These genes appear to be hijacked by this pathogen to achieve pathogenic success. Thus, taking a way these soybean factors from the pathogen could result in an effective strategy to genetically control SSR.

We have completed the generation of transgenic plants that are silenced in these genes, and the transformation is now complete. We were able to generate approximately 70 transgenic plants. We have collected a first round of seed from all the transgenic plants. The next step will be to test the performance of the transgenics against a range of soybean diseases and abiotic stresses to evaluate their potential wide deployment. An article was published this summer describing this work. We have also filed a patent with the Wisconsin Alumni Research Foundation (WARF). This patent was selected for the WARF innovation award.

Patent filing:
Patent P170294: Silencing of specific soybean NADPH oxidases confers drought tolerance and resistance to Sclerotinia Stem Rot

Publications:
Ranjan, A., Jayaraman, D., Grau, C., Hill, J. H., Whitham, S. A., Ané, J.-M., Smith, D. L. and Kabbage, M. (2017), The pathogenic development of Sclerotinia sclerotiorum in soybean requires specific host NADPH oxidases. Molecular Plant Pathology. Accepted Author Manuscript. doi:10.1111/mpp.12555

McCaghey Mga, Willbur J, Smith DL, and Kabbage M. (2018) The complexity of the Sclerotinia sclerotiorum pathosystem in soybean: virulence factors, resistance mechanisms, and their exploitation to control Sclerotinia stem rot . Tropical Plant Pathology. In press.

Ranjan Apd, Jain Sga, Piotrowski JS, Ranjan M, Kessens Rga, Westrick NMga, Stiegman Lua, Grau CR, Smith DL, and Kabbage, M. Resistance against the broad host range pathogen Sclerotinia sclerotiorum in soybean involves a reprogramming of the phenylpropanoid pathway and upregulation of anti-fungal activity targeting ergosterol biosynthesis. Plant Biotechnology Journal. Resubmitted 07/27/18.

Objective 3. Fungicide resistance emergence in Sclerotinia sclerotiorum

In the past three field seasons, we created a culture collection of S. sclerotiorum with >1,000 isolates. Specific to this objective, a large number of these isolates originated from field plots that received different fungicide treatments. Isolates were catalogued and preserved in long-term storage for further use. Nine isolates of S. sclerotiorum were exposed to fungicides for a total of 12 generations and the experiment repeated. Fungicides selected for the study were azoxystrobin (QoI), iprodione (dicarboximide), thiophanate methyl (BZI), and boscalid (SDHI). Each of the nine isolates was independently exposed to each of these fungicides for a total of 12 generations and the experiment was repeated. Fungicide sensitivity of each isolate was estimated before and after long-term sub-lethal exposure. Genotyping (SSR and AFLP) was used to determine if sub-lethal exposure increased mutation rates. Our results showed mutated loci of fungicide-exposed isolates had an average 22-fold higher mutation rate. AFLP showed fungicide-exposed isolates (12 of 17) accumulated mutations to group them as a separate group from control isolates. Results were published in PLoS One (Amaradasa and Everhart, 2016).

Whole-genome sequence data was obtained for 55 selected isolates and resulted in ~15X coverage. Point mutations were identified in pre-fungicide exposed (control) and post-fungicide exposed (treatment) individuals. Loci with mutations and missing data in control were censored to identify number of mutations in treatments. No significant differences were found in the number of mutations with respect to treatment group (?2 p-value > 0.05).

Mutations characterized as SNPs or INDELs were found to occur in a ratio of ~3:7. Transitions and transversions were found to occur in a ratio of ~8:2. Also, mutation locations were either genic or intergenic, which were found at a rate of ~2:8. All genic mutations were in genes identified as encoding hypothetical proteins (GenBank). The total number of mutations per chromosome showed no relationship between the number of mutations and length of chromosome. Chromosome 11, although not the longest chromosome, harbored the most number of cumulative mutations. A neighbor joining tree was constructed to show relationships between genomes. We report that most genomes were found to have greater similarity to fungicide-treated genomes than genomes of other isolates. No mutations were shared among all fungicide-exposed genomes. Characterization of structural variants and a publication is currently underway.

Publications:
Gambhir, N., Z.N. Kamvar, and S.E. Everhart. 2017. Effects of sublethal fungicide stress on genomes of Sclerotinia sclerotiorum. APS National Meeting. Phytopathology In press.

Gambhir, N., Z.N. Kamvar, and S.E. Everhart. 2017. Genomic alterations in Sclerotinia sclerotiorum after sublethal exposure to a mitosis-inhibiting fungicide. APS North Central Division Meeting. Phytopathology In press.

Nieto-Lopez, E.H., and S.E. Everhart. 2017. Fungicide sensitivity of Sclerotinia sclerotiorum from soybean in the North Central United States. APS North Central Division Meeting. Phytopathology In press.

Amaradasa, B.S., and S.E. Everhart. 2016. Effects of sublethal fungicides on mutation rates and genomic variation in fungal plant pathogen, Sclerotinia sclerotiorum. PLOS ONE. 11(12): e0168079. DOI 10.1371/journal.pone.0168079

Everhart, S., and B. Amaradasa. 2016. Fungicide stress induces genome mutation in Sclerotinia sclerotiorum. Phytopathology 106:S4.169.

Amaradasa, B., and S. Everhart. 2016. Sub-lethal fungicides induce microsatellite mutation in Sclerotinia sclerotiorum. Phytopathology 106:S4.139.

Gambhir, N., A. Pannullo, S. Campbell, B.S. Amaradasa, R. Jhala, J. Steadman, and S. Everhart. 2016. Comparison of four methods for fungicide sensitivity determination of Sclerotinia sclerotiorum. Phytopathology 106:S4.188.

Amaradasa, B.S., and S. Everhart. 2016. Sub-lethal fungicides induce microsatellite and AFLP marker mutation in Sclerotinia sclerotiorum. Phytopathology 106:S4.184.

Amaradasa, B.S., and S.E. Everhart. 2015. Sub-lethal doses of fungicide induce resistance emergence in Sclerotinia sclerotiorum. Phytopathology. 105:S4.7.

Objective 4: Develop current, grower centric, economic and outreach materials pertaining to Sclerotinia stem rot of soybean with emphasis on potential management practices for the disease in the North Central Region

The extension team assembled for this project continues its effort to communicate our research findings across the tristate area. Extension articles, presentations, winter meetings, and field visits were conducted by our group.

Refereed reports
Byrne, A.M., Chilvers, M.I. 2017. Efficacy of foliar fungicides for white mold management of soybean in 2016a. Plant Disease Management Reports 11:FC030

Byrne, A.M., Chilvers, M.I. 2017. Efficacy of foliar fungicides for white mold management of soybean in 2016b. Plant Disease Management Reports 11:FC029

2017 Presentations, activities and popular press:
1. IPM - Field crop disease update. Dundee, MI 50 participants
2. Dry bean white mold and root rot research. Bean and beet day. Saginaw, MI. Jan 16, 2018. 225 participants
3. IPM - Field crop disease update. Peck 70 participants
4. IPM - Field crop disease update. St Johns 60 participants
5. MABA Fungicide resistance 80 participants
6. Field crop disease management update. Agribusiness meeting, MSU Pavillion, East Lansing, MI. Dec 20, 2017. 320 participants.
7. Soybean and wheat disease management. DF Seeds. Okemos, MI. Dec 13, 2017. 60 participants
8. MSU soybean variety performance trial tour. Soybean and corn disease discussion. Allegan, MI. 20 participants
9. White mold research plot tour. Entrican, MI. Sep 20, 2017. 12 participants
10. Soybean, and corn disease update. Alma, MI. Aug 30, 2017. 30 participants
11. MSPC reporting. Plant Pathology Farm, East Lansing, MI. Aug 30, 2017. 16 participants
12. Manage soybean disease to protect yield. AgroExpo, MI. Aug 16, 2017. 40 participants
13. Southwest Michigan Row Crops Field Day 2017, Vicksburg, MI. Aug 2, 2017. 36 participants
14. Field crop disease update. Corey Seed Crop Shop. Fenton, MI. Mar 16, 2017. 55 participants
15. Dry bean growers meeting for upper peninsula, Michigan. Webcast. Feb 28, 2017. 7 participants
16. Dry bean grower meeting. Ubly, MI. Feb 28, 2017. 57 participants
17. Webcast IPM meeting. Feb 27, 2017. 72 participants
18. IPM, Sanilac. Feb 23, 2017. 102 participants
19. IPM, Dowagiac. Feb 22, 2017. 54 participants
20. IPM, Dundee. Feb 21, 2017. 68 participants
21. Dry bean white mold and root rot disease research. 2017 Bean and Beat Symposium. Saginaw, MI. Jan 17, 2017. 150 participants
22. IPM, Saginaw. Jan 13, 2017. 92 participants
23. IPM, Mt. Pleasant. Jan 12, 2017. 62 participants
24. Michigan Soybean Promotion Committee reporting and information session. East Lansing, MI. Jan 10, 2017. 12 participants
25. Fantastic Fungicides! SouthWest Agricultural Conference. Ridgetown, ON, Canada. Jan 4-5, 2017. 3 sessions total of 270 participants

2017 Extension articles:
Chilvers, M.I. Jul 18, 2017. Sclero-cast: A white mold apothecia risk indicator. MSUE News for Ag

Jensen, B., Liesch, P.J., Nice, G., Renz, M., Smith, D. 2017. Pest Management in Wisconsin Field Crops, University of Wisconsin-Madison, Cooperative Extension (A3646).

Smith, D.L. and Willbur, J. 2017. Wisconsin white mold risk update – August 5. Wisconsin Crop Manager 24(20):103.

Smith, D.L. and Willbur, J. 2017. Wisconsin white mold risk update – July 11. Wisconsin Crop Manager 24(17):88.

View uploaded report

The overreaching goal of this proposal is to provide stakeholders with concrete control measures, and improve awareness and knowledge of sclerotinia stem rot of soybean in the North Central region. It also provides specific information on tools that can be used to introgress resistance into commercial varieties against white mold disease.

Fungicide efficacy, and the effect of weather conditions and application timing on fungicide efficacy:

Farmers continue to be challenged with filtering through marketing information on new or repurposed fungicides for management of Sclerotinia stem rot. We evaluated a standard list of fungicides applied at different timings (R1-R5 growth stages) in each participating state:
lactofen (LACT)
boscalid (BOSC)
picoxystrobin (PICO)
prothioconazole and trifloxystrobin (PROT+TRIF)
boscalid and fluxapyroxad/pyraclostrobin (BOSC+FLUX/PYRA)
fluazinam (FLUA)
fluoxastrobin and flutriafol (FLUO+FLUT)
prothioconazole (PROT)
tetraconazole (TETR) and
thiophanate-methyl (THIM)

Each location collected weather data, disease incidence and severity and yield. An economic analysis was also conducted for each product used and the timing of application to determine the return on investment (ROI) of fungicide application at timings from R1-R5 growth stages.

Detailed apothecia scouting data and spore trapping data were also collected. The data was used to develop a first iteration prediction model for SSR to be used to make decisions on fungicide application. However additional field experiments need to be established in Wisconsin, and in other states such as Michigan, Iowa, and Nebraska to expand upon the dataset and validate model iterations to develop a more robust model. Field sites were established in all participating states. Weather information for the models used to predict SSR, was supplied by USPest.org (http://uspest.org/wea/). USPest.org generates site-specific weather data remotely using open source algorithms. They have allowed us to set up “virtual weather stations” to get site-specific, modeled weather data by simply supplying global positioning system (GPS) coordinates of the research locations. Models were run on a daily basis when the crop was at the R1-R3 growth stages. When high risk for Sclerotinia stem rot is predicted during these growth stages, fungicide was applied. Additionally, application of fungicide was performed in separate plots based on crop phenology. The crop phenology-based program consisted of applying two applications of fungicide (once at R1 and once at R3 growth stages). Apothecial germination data and spore trapping data were also collected to support and validate model iterations.

Takeaways and recommendations:

Lactofen (LACT), boscalid (BOSC), picoxystrobin (PICO), prothioconazole and trifloxystrobin (PROT+TRIF), and boscalid and fluxapyroxad/pyraclostrobin (BOSC+FLUX/PYRA) had the lowest level of disease incidence and were the preferred chemistries in this study.

LACT was among the more efficacious active ingredients in high disease pressure situations, and its relatively low cost compared to other products, resulted in high estimated value to the farmer. Economical disease management balances efficacy and cost; therefore, BOSC, a highly efficacious active ingredient, was not as profitable in this analysis because of its high cost.

Canopy closure percentage of 40% and a soil temperature between 20 and 25°C are better indicators for the production of apothecia, and therefore availability of inoculum than soybean growth stage alone.

Newer statistical methods were used to develop a model that predicts the probability of apothecia of S. sclerotiorum being present in soybean fields during the R1-R3 flowering period. The model uses site-specific, remotely-accessible weather data, and other variables such as crop development stage and canopy closure. In the 2016 and 2017 growing seasons, we found that the model predicted the presence of apothecia with 80% accuracy.

The above model was used to develop a prediction tool in order to time fungicide applications for better efficacy of fungicides. The use of the smart phone application SPORECASTER, developed in this project, will allow for precise detection of risk areas and efficient deployment of fungicides. The app turns a few simple taps on a smartphone screen into an instant forecast of the risk of apothecia being present in a soybean field, which helps growers predict the best timing for white mold treatment during the flowering period.

Other management recommendations:

White mold is best managed by an integrated approach of selecting soybean cultivars with the highest level of resistance and adjusting cultural practices to minimize environmental factors that favor disease development. This approach requires a coordinated plan that matches the level of resistance in a soybean cultivar to expected disease potential and cropping practices that influence crop canopy closure. No single tactic will completely control white mold.

White mold is a disease of high yield potential soybean production. Although several factors are believed responsible for the increased occurrence of white mold, none may be more important than management practices or environmental conditions that promote rapid and complete crop canopy closure. White mold is particularly favored by dense soybean canopies created by plantings in narrow row widths, high plant populations, early planting, high soil fertility, or other management practices that promote rapid and complete canopy closure.

The effect of row width on incidence of white mold and subsequent yield can vary by year and is strongly controlled by annual climatic conditions. Frequently, the yield advantage of narrow row widths, compared to wide widths, is expressed even though the incidence of white mold may be greater in narrow row systems. Increasing row width from a narrow row spacing (6-8”) to a medium spacing (15”) can reduce white mold infections without compromising yields. Lowering seeding rates in narrow row systems is preferable to increasing row widths to a wide row spacing.

Crop rotations that employ non-hosts result in a reduced the incidence of white mold, but some non-hosts are better than others. A preceding crop of small grain, in contrast to corn, has a greater impact on reducing the incidence of white mold. Rotation with non-hosts such as small grains resulted in fewer of apothecia formed under the soybean canopy. The population density of apothecia was greatest in moldboard plow systems compared to no-tillage systems. Fewer apothecia in no-tillage systems is a partial explanation why lower incidence of white mold is observed in no-till fields compared to fields receiving some degree of tillage.

Biological control of white mold has also been researched. Sclerotia can be parasitized by several fungi and these fungi have been investigated as candidates for commercialization. Contans® WG is a commercial biological control product labeled for the control of Sclerotinia sclerotiorum in agricultural soils. Contans® has shown promise as a biological control agent and a potential alternative for chemical fungicides to control white mold. In Wisconsin, the best and most economical times for application are during pre-planting or post-harvest on the stubble of a previously diseased crop. The time between the application of Contans® WG and the typical onset of disease should be as long as possible.

Development of genetic resistance against white mold:

Genetic resistance is the most desirable form of control against any plant pathogen. Unfortunately, commercial soybean varieties with adequate levels of resistance against white mold are currently not available. We have identified a genetic component in soybean that can be manipulated to achieve an adequate level of resistance against this disease. Our results show that a group of genes controlling soybean NADPH oxidase production are specifically induced in the plant following S. sclerotiorum infection, with peak expression at the later stages of the infection process. Thus, it appears that S. sclerotiorum may be co-opting the soybean ROS machinery to its benefit, by modulating the expression of host NADPH oxidases. We used Virus Induced Gene Silencing (VIGS) to turn off, or "silence", this group of genes to see if they are required for disease development. The VIGS system is an established technique in soybean molecular genetics used to study the function of genes, particularly the function of disease resistance genes and defense genes involved in plant-microbe interactions. Using the VIGS system, we were able to achieve a 45-65% reduction in transcript levels in silenced plants compared to the control. The silenced soybean plants were then evaluated for their response to S. sclerotiorum challenge. Five days following inoculation with the pathogen, the control plants showed typical SSR symptoms and began to wilt. In contrast, the silenced plants did not show any wilting symptoms and developed normally. This is a remarkable result that shows silencing of a specific group of soybean NADPH oxidase genes leads to markedly decreased ROS production and enhanced resistance in soybean against infection by S. sclerotiorum. These genes provide a potential target for the generation of SSR-resistant soybean lines. We expanded our screen of silenced plants to include other pathogens and abiotic stress. Surprisingly, we found that these plants were also drought tolerant — possibly because decreased ROS production would limit the oxidative damage and ultimately death of the plant imposed by excessive ROS levels during drought stress. We have now generated transgenic soybeans with the described characteristics, and we are excited to evaluate the efficacy of these new lines in the field against a broad range of diseases and abiotic stresses.

Takeaways and recommendations:

We have identified a group of genes in soybean that can be manipulated to provide resistance against white mold.

Targeting the same genes in soybean confers drought tolerance, thus making it feasible and viable to use a transgenic strategy to address both of these issues.

Transgenic plant were produced as part of this grant, and will be assayed in the field against wide range of biotic and abiotic stresses

The same transgenic approach should be pursued in commercial varieties to test these varieties and eventually release them for public use.


Fungicide resistance emergence in Sclerotinia sclerotiorum:

Broad sampling throughout the soybean producing region was achieved by collecting a minimum of 10 samples from ten field locations in each represented state. GPS coordinates of each field site was recorded for reporting of fungicide sensitivity in each county. Fungicide sensitivity of each isolate was determined using high-throughput fungicide resistance assays in the Everhart Lab. This was performed in a specialized instrument capable of creating a gradient of fungicide concentrations on media in an oversized Petri plate (150 mm). Fungicide sensitivity (EC50) for each isolate was determined for fungicides with different modes of action (QoI, MBC, SDHI, etc). Appropriate controls and technical replications were included.

Our work also characterized the effects of sub-lethal fungicide exposure on resistance emergence and genome evolution. S. sclerotiorum was an ideal model system for this study because of its noted long-term stability in genetic variation and also due to the availability of a fully annotated and optically mapped, published genome. Our approach was to generate a panel of fungicide exposed S. sclerotiorum isolates, to pre-screen for small and large genomic changes using SSR and AFLP genetic markers, and submit selected isolates for whole-genome sequencing to examine extent of genomic change. Fungicides used in this study represented all commercial fungicides currently in use for control of disease caused by S. sclerotiorum in the U.S. and represent chemicals with different modes of action: Azoxystrobin and Pyraclostrobin (QoI’s), Iprodione (DMI), Thiophanate methyl (MBC), and Boscalid (SDHI). Sub-lethal fungicide exposure utilizes the AutoPlate 5000, a specialized instrument that creates a radial gradient of fungicide concentration Petri plate containing PDA. Each isolate is inoculated onto the fungicide gradient and myclial growth from the 50-100% inhibition zone is collected for the next generation, which is repeated a total of 12 times.

Takeaways and recommendations:

At this point, evidence suggest that field populations of S. sclerotiorum have not developed widespread resistance to the common fungicides used.

Results of the current study conclusively show in vitro sublethal fungicide stress induces mutations in the S. sclerotiorum genome, where future studies using whole-genome sequencing may shed more light on genomic damage specific to each class of fungicide and used to examine fungicide-resistant isolates for evidence of such mutagen exposure.

Results were broadly disseminated and reported directly to growers

Whole genome sequencing was performed on isolates exhibiting genomic change (as indicated by SSR / AFLP analysis conducted) and those with induced resistance. Results of SSR/AFLP analysis were submitted for publication.

Compilation of fungicide sensitivity data and trends was performed. Bioinformatic pipelines will be developed for genomic data analysis.