Studies on the role of ethylene in the soybean - Fusarium virguliforme interaction Noor A. Abdelsamad 1, Gustavo C. MacIntosh2, and Leonor F. Leandro 1 1 Dept. of Plant Pathology and Microbiology, Iowa State University, Ames, IA, 50011 2 Dept. of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, 50011 Introduction Results Results Soybean Sudden death syndrome (SDS) caused by the soilborne fungus Fuarium virguliforme, is an economically devastating disease affecting soybean production in North and South America (Roy et al., 1997). The onset of foliar symptoms usually occurs during reproductive stages, suggesting that ethylene hormone regulation might involved the in Fv-soybean interaction. Ethylene can improve plant resistance against pathogens, particularly those with necrotrophic lifestyle. In other cases ethylene may promote disease susceptibility (Van Loon, 2006). Previous transcriptomic analysis showed that ethylene was accumulated in soybean roots and leaves in response to Fv infection (Radwan et al., 2011, 2013). However, no work has been done to understand the role of ethylene in the soybean- Fv interaction. A better understanding of the role of ethylene in Fv infection is needed to determine if manipulation of this hormone, through chemical suppressors or inducers could be used as a management tool against soybean SDS. The goal of this work is to understand the role of ethylene in soybean SDS disease development The effect of cobalt chloride on SDS development (Fig.2) Soybean seedlings treated with high concentrations of cobalt (1, and 10 mM) showed the same or higher (P<0.0001) SDS foliar and root rot severity as the untreated control, and the effect was only significant in MN1606 (Fig.2D and E). Seedlings treated with low cobalt concentrations (0.01, and 0.1 mM) showed significant (P<0.0001) lower SDS severity than controls in Williams 82 but not in MN1606 (Fig.2D and E). There was no toxic effect of cobalt chloride treatment, as either a foliar sprayed or soil drench, on F. virguliforme population density in soil (Fig.2F). Fig.1. Relative expression of the ethylene oxidase gene (ACO) in response to Fv infection at the indicated soybean growth stages. Values are expression levels normalized to non-infested control plants. Bars marked with different letters are significantly different (P<0.05). A A A A B B The effect of ethephon and cobalt chloride on soybean root development (Table 1) All ethephon concentrations did not show inhibitory effects on soybean root dry weight at any concentration with either application methods. Seedlings that received foliar application of cobalt chloride at low concentrations (0.01, and 0.1 mM) showed significant higher root dry weight compared to all other concentrations, while seedlings treated with the highest cobalt concentration (10 mM) showed a reduction in root dry weight compared to the untreated control (P<0.05) (Table 1). A D a a a a b Objectives b A b b b Measure changes in soybean ethylene expression in response to infection by Fusarium virguliforme Determine the effect of ethylene inducer and suppressor applications on SDS development b B A B b AB B B B E Conclusions Materials and Methods a a a a ab Ethephon treated seedlings showed reduction in SDS foliar and root rot disease severity at all concentrations compared to water treated control. Treatment with low cobalt chloride concentrations, reduced foliar and root rot severity, while high concentrations increased SDS severity. Ethephon and cobalt chloride treatments had no toxic effect of Fv population in soil, suggesting that the reduction in SDS symptoms might be related to induced resistance against Fv. Ethephon application as either a foliar spray or soil drench had no effect on soybean root growth, while cobalt chloride at high concentrations showed an adverse effect. The reduction in SDS development in seedlings treated with low cobalt concentrations, may be due to the activation of plant defense mechanisms against the heavy metal stress caused by cobalt (Poschenrieder et al., 2013). A b b Two experiments were conducted in the ISU greenhouse facility. Two soybean varieties; MN1606 (resistant), and Williams 82 (susceptible) were used in all experiments. Fusarium virguliforme isolate NE 305 was used to prepare inoculum in a sand-cornmeal mix (Arias et al., 2013). The inoculum was mixed with pasteurized soil at a ratio of 1:15. Non-infested soil was used for controls. Experiment 1: ACO (ethylene oxidase) gene expression in Fv infested and non-infested seedlings were quantified at (V2-V3, V4-V5, and R1-R2) growth stages using qPCR. The experiment followed a completely randomized design with 3 reps. Experiment 2: Ethylene inducer (ethephon) and suppressor (cobalt chloride) were sprayed or soil drenched at VC growth stage, one day before infection and one day after infection. The experiment followed a randomized complete block design with 7 reps. Assessments: Foliar and root rot % severity, Fv population density in soil, ACO gene expression, and root dry weight, were assessed 21, 24, and 27 days after infection (DAI). ANOVA was conducted using the PROC GLIMMIX procedure of SAS. b AB b b b NS AB B B C F NS NS Future work Ethephon concentration(mM) Cobalt chloride concentration(mM) Study the crosstalk between ethylene and other phytohormones like, jasmonic acid and salicylic acid on SDS development. Examine the pathways involved in ethylene-based soybean defense response to Fv infection by quantifying key defense genes. Develop ethylene- based management tool under field conditions. Fig.2. Effects of different concentrations of an ethylene inducer (ethephon) and ethylene suppressor (cobalt chloride) on SDS disease development in soybean cultivars MN1606 (resistant), and William 82 (susceptible) soybean cultivars at 27 DAI. (A,B) Effect of ethephon treatment on SDS foliar and root rot severity, respectively. (D,E) Effect of cobalt chloride on SDS foliar and root rot severity, respectively. (C,F) Effect of ethephon and cobalt chloride on Fv population density in soil, respectively. Bars marked with different letters are significantly different (p<0.05). NS = non-significant. Ethylene oxidase (ACO) gene expression (Fig.1.) Seedlings infested with F. virguliforme showed higher ACO gene expression compared to non-infested seedlings, at different soybean growth stages. Resistant cultivar MN1606 showed higher ACO accumulation compared to the susceptible cultivar Williams 82, at early and later vegetative stages (P<0.05). The effect of ethephon on SDS development (Fig.2.) Foliar spray or soil drench of ethephon significantly (P<0.0001) reduced SDS foliar symptoms at all concentrations in both cultivars compared to water treated control (Fig.2A) Williams 82 cultivar showed significant (P<0.0001) reduction in root rot severity at all ethephon concentration while MN1606 showed no differences in root rot (Fig.2B). There was no toxic effect of ethephon treatment, as either a foliar sprayed or soil drench, on F. virguliforme population density in soil (Fig.2C). Results References Table 1. The effect of ethephon and cobalt chloride treatments on root dry weight (g) of Fv infested seedlings Treatment Root dry weight (g) Ethephon (mM) Foliar (ns) Soil drench 0.22 B 0.22 BC 0.1 0.32 A 0.28 AB 0.5 0.27 AB 0.33 A 1 0.26 AB 2 0.29 AB 0.19 C 4 0.25 BC Cobalt chloride (mM) Foliar Soil drench (ns) 0.22 b 0.22 a 0.01 0.34 a 0.24 a 0.31 a 0.23 b 10 0.12 c 0.18 a Roy et al., 1997. Sudden death syndrome of soybean. Plant Dis 81:1100–1111 Van Loon et al., 2006. Ethylene as a modulator of disease resistance in plants. Trends in plant sciences pages 184-191. Radwan et al., 2013. Effect of Fusarium virguliforme phytotoxin on soybean gene expression suggests a role in multidimensional defence. Molecular plant pathology 14(3) 293, 307. Radwan et al., 2011. Transcriptional Analysis of Soybean Root Response to Fusarium virguliforme, the Causal Agent of Sudden Death Syndrome. MPMI Vol. 24, No. 8, 2011, pp. 958–972 Arias et al., 2013. Phytopathology 103 (8):822-832. Acknowledges Funding was provided by the Iowa Soybean Association. Thanks for Jessica Hohenstein from MacIntoch laboratory for helping in gene expression analysis. Means in the same column followed by different letters are significantly different at p < 0.05 (ns = non-significant).