Induction of ethylene inhibits development of soybean sudden death syndrome by inducing defense-related genes and reducing Fusarium virguliforme growth

Abstract

Ethylene is a gaseous hormone that regulates plant responses to biotic and abiotic stresses. To investigate the importance of ethylene in soybean resistance to Fusarium virguliforme (Fv), the causal agent of sudden death syndrome (SDS), soybean cultivars Williams 82 (SDS-susceptible) and MN1606 (SDS-resistant) 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. In both cultivars, plants treated with ethephon showed lower SDS foliar severity compared to the other treatments, whereas those treated with cobalt chloride or 1-MCP showed the same or higher SDS foliar severity compared to the water-treated control. Ethephon application resulted in activation of genes involved in ethylene biosynthesis, such as ethylene synthase (ACS) and ethylene oxidase (ACO), and genes involved in soybean defense response, such as pathogenesis-related protein (PR), basic peroxidase (IPER), chalcone synthase (CHS), and defense-associated transcription factors. Cobalt chloride and 1-MCP treatments had little or no effect on the expression of these genes. In addition, ethephon had a direct inhibitory effect on in-vitro growth of Fv on PDA media. Our results suggest that ethephon application inhibits SDS development directly by slowing Fv growth and/or by inducing soybean ethylene signaling and the expression of defense related genes.

Fig 1. Induction of ethylene biosynthesis genes in response to Fusarium virguliforme infection.

Expression analysis of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes in response to Fusarium virguliforme infection in soybean roots at 2 and 4 days post inoculation (DPI). Soybean seeds of MN1606 (resistant) and Williams 82 (moderately susceptible) were planted in Fv infested soil and roots were collected at 2 and 4 DPI for quantification of ACS and ACO gene expression using real time PCR. The soybean actin gene was used as an internal control. The level of expression is shown relative to that of time zero plants (non-inoculated). Each column represents the mean fold change of six biological samples (3 replicates × 2 runs), and each sample consisted of a pool of two plants. Error bar indicates standard error of the mean (n = 6). * indicates a significant difference (P<0.05) between the two cultivars within the same time point.

Fig 2. Effect of chemical treatments on severity of foliar symptoms of soybean sudden death syndrome in resistant cultivar MN106 and susceptible cultivar Williams 82.

Soybean seedlings were drenched with water (control), ethephon (ethylene inducer) at concentrations of 0.1 mM, 1 mM, and 4 mM, or cobalt chloride (ethylene suppressor) at concentrations of 0.1 mM, and 1 mM, 24 h before and 24 h after transplant into soil infested with Fusarium virguliforme. 1-MCP (ethylene perception suppressor) was sprayed until run off at the same times. SDS symptoms were assessed 21 days post inoculation (DPI). Each bar represents the mean of 21 replicates (7 cups × 3 runs), and the error bar represents the standard error of the mean. Means with different letters are significantly different (P<0.05).

Fig 3. In-vitro effect of ethephon and cobalt chloride on mycelial growth of Fusarium virguliforme.

Mycelia plugs were grown for fourteen days on PDA plates containing water, ethephon concentrations of 1mM, 2mM, and 4mM, or cobalt chloride concentrations of 0.1mM, and 1mM. Colony diameter (A), and number of conidia per ml (B) were measured fourteen days after incubation at room temperature under dark conditions. Each bar represents the mean of 18 replicates (6 plates × 3 runs) and the error bar represents the standard error of the mean. Means with different letters are significantly different (P<0.05).

Fig 4. Effect of chemical treatment on defense gene expression in MN1606.

Expression analysis of soybean genes twenty-four hours after exposer to water (control), 1-methylcyclopropene (MCP), cobalt chloride 1mM (CC), or ethephon 4mM (ETH) in the resistant cultivar MN1606. (A) Genes involved in ethylene biosynthesis and signaling, 1-aminocyclopropane-1-carboxylic acid synthase (ACS), 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), and β-1,3-endoglucanase (PR2), (B) defense-related genes, pathogenesis-related protein 1 (PR1), Chitinase class I (PR3), Intercellular pathogenesis-related protein 10 (PR10), Chalcone synthase (CHS), basic peroxidase (Peroxidase), and ethylene response factor 1 (ERF1). Soybean seedlings were drenched with water (control), ethephon 4 mM, cobalt chloride 1 mM, or foliar sprayed with 1-MCP. Roots were sampled 24 hours after chemical treatment. The soybean actin gene was used as an internal control. The level of expression is shown relative to that of control plants. Each column represents the mean fold change of eight biological samples (four samples × two runs), and each sample consisted of a pool of two plants. Error bar indicates standard error of the mean (n = 8). Columns with different letters are significantly different (P<0.05).

Fig 5. Effect of chemical treatment on defense gene expression in Williams 82.

Expression analysis of soybean genes twenty-four hours after exposer to water (control), 1-methylcyclopropene (MCP), cobalt chloride 1mM (CC), or ethephon 4mM (ETH) in the moderately susceptible cultivar Williams 82. (A) Genes involved in ethylene biosynthesis and signaling, 1-aminocyclopropane-1-carboxylic acid synthase (ACS), 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), and β-1,3-endoglucanase (PR2), (B) defense-related genes, pathogenesis-related protein 1 (PR1), Chitinase class I (PR3), Intercellular pathogenesis-related protein 10 (PR10), Chalcone synthase (CHS), basic peroxidase (Peroxidase), and ethylene response factor 1 (ERF1). Soybean seedlings were drenched with water (control), ethephon 4 mM, cobalt chloride 1 mM, or foliar sprayed with 1-MCP. Roots were sampled 24 hours after chemical treatment. The soybean actin gene was used as an internal control. The level of expression is shown relative to that of control plants. Each column represents the mean fold change of eight biological samples (four samples × two runs), and each sample consisted of a pool of two plants. Error bar indicates standard error of the mean (n = 8). Columns with different letters are significantly different (P<0.05).

Fig 6. Effect of chemical treatments on the expression of soybean defense-related genes in the resistant cultivar MN1606 infected with Fusarium virguliforme.

Soybean seedlings were drenched with water (control), ethephon 4 mM, cobalt chloride 1 mM, or foliar sprayed with 1-MCP twenty-four hours pre and post Fusarium virguliforme inoculation. Roots were sampled at 2 and 4 days after F. virguliforme (DAI). The level of expression of pathogenesis-related protein 1 (PR1), Chitinase class I (PR3), Intercellular pathogenesis-related protein 10 (PR10), Chalcone synthase (CHS), basic peroxidase (Peroxidase), and ethylene response factor 1 (ERF1), is shown relative to that of control plants. Each column represents the mean fold change of eight biological samples (four samples × two runs), and each sample consisted of a pool of two plants. Error bar indicates standard error of the mean (n = 8). Columns with different letters are significantly different (P<0.05).

Fig 7. Effect of chemical treatments on the expression of soybean defense-related genes in the susceptible cultivar Williams 82 infected with Fusarium virguliforme.

Soybean seedlings were drenched with water (control), ethephon 4 mM, cobalt chloride 1 mM, or foliar sprayed with 1-MCP twenty-four hours pre and post Fusarium virguliforme inoculation. Roots were sampled at 2 and 4 days after F. virguliforme (DAI). The level of expression of pathogenesis-related protein 1 (PR1), Chitinase class I (PR3), Intercellular pathogenesis-related protein 10 (PR10), Chalcone synthase (CHS), basic peroxidase (Peroxidase), and ethylene response factor 1 (ERF1), is shown relative to that of control plants. Each column represents the mean fold change of eight biological samples (four samples × two runs), and each sample consisted of a pool of two plants. Error bar indicates standard error of the mean (n = 8). Columns with different letters are significantly different (P<0.05).