Objective 1 – Understand the role of ethylene on SDS disease development.
This objective focused on measuring the changes in ethylene signaling in soybean in response to infection by F. virguliforme, determining the effect of ethylene inducers and suppressors on SDS development, and examining the gene pathways involved in ethylene-based soybean defense responses to SDS.

Greenhouse experiment
A greenhouse experiment was conducted to determine the effect of an ethylene inducer (ethephon) and two ethylene suppressors (cobalt chloride and MCP) on SDS development and on expression of soybean defense genes. Soybean cultivars Williams 82 and MN1606 were treated 24 h before and 24h after Fv inoculation with either ethephon (ethylene inducer), cobalt chloride (ethylene biosynthesis inhibitor), or 1-MCP (ethylene perception inhibitor). Inoculated plants were grown for 21 days at 24°C in the greenhouse and then evaluated for SDS severity and expression of soybean defense genes.

Soybean plants treated with ethephon developed less severe SDS symptoms compared to water-treated seedlings, whereas those treated with cobalt chloride (ethylene biosynthesis suppressor) or 1-MCP (ethylene perception suppressor) showed the same or higher SDS foliar severity compared to the water treated control. Ethephon application also resulted in activation of genes involved in ethylene biosynthesis, and genes involved in soybean defense responses, such as phenylpropanoid pathway, pathogenesis related proteins and transcription factors. Cobalt chloride and 1-MCP treatments had little or no effect on these genes.

This study demonstrated that the ethylene-signaling pathway plays an important role in resistance against SDS, and that treatment of soybeans with the ethylene inducer, ethephon, enhances soybean defenses and contributes to suppression of SDS in greenhouse conditions.

In-vitro assay
A laboratory experiment was conducted to investigate the effect of ethephon and cobalt chloride on Fv growth and development in culture. PDA media was amended with ethephon at concentrations of 1, 2, and 4 mM, or cobalt chloride concentrations of 0.1, and 1 mM. The control consisted of PDA media amended with water. Fv mycelium plugs were placed in the middle of the plates, and colony diameter and sporulation was measured after two weeks of incubation.

The experiment showed that ethephon reduced Fv growth rate and spore production, while cobalt chloride has no effect. This suggests that a direct negative effect of ethephon on Fv may also contribute to suppression of SDS in treated plants.
Objective 2 - Develop ethylene-based management tools for SDS
For this objective, we focused on testing the effectiveness of the ethylene inducer, ethephon, in reducing SDS in greenhouse and field conditions.

Greenhouse
In the greenhouse experiment, soybeans were treated with ethephon by applying: i) a soil drench at planting (VP), ii) a soil drench at VP followed by a second application at emergence (VP+VE), iii) a soil drench at VP and unifoliate stage (VP+VC), and iv) a soil drench at VP and at first trifoliate stage (VP+V1-V2). The plants were then evaluated for SDS severity and incidence over time.

We found that all ethephon treatments significantly reduced SDS foliar symptom severity by 50-60% compared to the untreated control (P<0.05) in the susceptible cultivar Williams 82. However, SDS severity did not differ among ethephon treatments, suggesting that the single application at planting has the main role in suppressing SDS.

Field trials
Field studies were conducted at two locations in 2015 and 2016 to test if applications of the ethylene inducer, ethephon, could suppress SDS in field conditions. Ethephon (0.1 mM) was applied to soybean plots as: i) a soil drench (in-furrow) at planting, ii) a soil drench at emergence (VE), and iii) a foliar spray at V1-V2. Plant were evaluated for SDS incidence and severity over the season, and yield.

In 2015, application of ethephon at planting or at plant emergence significantly (P<0.05) reduced SDS foliar severity compared to the untreated control. In one location, the overall disease index (DX) was 57% lower in plots exposed to ethephon treatment at VE growth stage compared to untreated plots. At the other location, there was a 75% reduction in SDS foliar severity in plots that received the in-furrow treatment compared to untreated plots. However, yield did not differ significantly among treatments in either location. In 2016, SDS disease pressure was low in both locations, and ethephon applications did not significantly affect SDS or yield. However, the same numerical pattern was observed, where plots treated with ethephon at VE growth stage had the lowest SDS severity.

These results suggest that the use of plant defense inducers could potentially be used as a tool to manage SDS under field conditions. Field trials are continuing in 2017 to further investigate if ethephon can consistently suppress SDS and protect across different environments.


Screening for SDS resistance using triple response tests
In addition to the field trials, a triple response assay was used to screen soybean genotypes for sensitivity to ethylene. This screening method would allow us to determine if there is a correlation between ethylene sensitivity and SDS resistance. Seeds of 8 genotypes, 4 resistant and 4 susceptible to SDS, were treated with ACC (an ethylene precursor) or water and incubated in germination pouches. Ethylene triple response (ETR) parameters (short roots, exaggerated curvature of the apical hook, short and thick hypocotyl) were measured. All soybean genotypes were found be sensitive to ethylene but some genotypes exhibited a stronger response to ethylene than others.

The results obtained suggest that SDS resistant cultivars tend to be more sensitive to ethylene than the most susceptible cultivar tested, but future work is needed to determine if enhanced sensitivity could be used to screen for resistance or develop more resistant varieties.