Control of Fusarium wilt in wheat seedlings by grain priming with defensin-like protein

Extraction and purification of defensin from Trigonella foenum-graecum seeds

The natural defensin from 50-g powdered Fenugreek (Trigonella foenum-graecum) seeds, FD, was extracted and purified, according to Oddepally and Guruprasad (2015) as follows: Fine flour (100 g) was prepared from the seeds of Fenugreek in a mill. A protein extract was prepared from this flour, using 500 ml of extraction buffer (10 mM Na₂HPO₄, 15 mM NaH₂PO₄, 100 mM KCl, 1.5% EDTA, pH 5.4) for 2 h at 4 °C with constant agitation. This protein extract was centrifuged at 15,000g, and the supernatant was fractionated at 70% relative ammonium sulfate saturation at 4 °C for 18 h. After centrifugation under the same conditions, the precipitate was re-dissolved in distilled water and heated at 80 °C for 15 min in a water bath. This heated protein extract was centrifuged at 3000 rpm for 5 min. The supernatant was recovered and extensively dialyzed against distilled water for 3 days and then recovered by freeze-drying. For peptides, purification was initially performed on a DEAE-Sepharose column (with 100 ml of resin), equilibrated with 20 mM Tris–HCl (pH 8.0), at a flow rate of 60 ml/h. The freeze-dried protein extract (50 mg) was reconstituted in 5 ml of the equilibrium buffer and centrifuged (6000 rpm, 3 min at 4 °C), and the supernatant was loaded onto the column. A non-retained fraction (D1) was eluted in the equilibrium buffer and used as defensin source. The protein content in the purified extract and the crude extract was determined by Lowry et al. (1951) method and subjected to protein electrophoresis.

Culturing of plant pathogenic fungus

Fusarium oxysporum was friendly obtained from Assistant Professor: Manal Tawfik, Assistant Professor of Mycology, Botany Department, Faculty of Science, Zagazig University, Egypt.

The fungus spores were prepared by growing the organism on Hordeium grains medium. Five hundred-milliliter flasks, containing 100-g washed Hordeium grains and 80-mL tap water, were autoclaved at 121 ± 1 °C for 15 min. The autoclaved Hordeium grains were inoculated with the fungus under aseptic conditions and incubated at 25 ± 1 °C for 21 days. The medium was mixed and rubbed together to release mycelium and spores from Hordeium grains. The mycelium and spores were taken in sterile water and used for inoculation of plants. Calculate spores concentration using a hemocytometer. Prepare 10⁶ spores/ml suspensions in autoclaved distilled water for inoculation (Hegazy 2008).

Pot experiment

Wheat (Triticum aestvium L.; cultivar Sakha 96) grains were obtained from the Agricultural Research Center, Giza, Egypt.

Pot experiments were carried out in the greenhouse of the Botany Department, Faculty of Science, Zagazig University, Egypt. Plastic pots (20-cm diameters) were filled with about 2 kg of soil (peat moss soil) each. The pots were divided into two groups. The first remained as it is, while the second was infected by 100 ml of the prepared F. oxysporum spore suspension.

Grains were surface sterilized by 0.1% mercuric chloride solution followed by rinsing with distilled water. A group of the seeds was soaked in the purified extracted defensin solution for 12 h then dried again to get rid of excess moisture (defensin-primed group), and the other group was kept dry (non-primed group). Each group was planted in the plastic pots (seven grains in each pot). Pots were arranged on the greenhouse benches and kept under natural photoperiod (12 to 13 h), temperature (28 ± 4 °C) and irrigated weekly. Samples were collected after 10 days (1st stage) and 20 days (2nd stage). The experiment was done in triplicate.

Markers of oxidative stress

H₂O₂ content in 1-g fresh leaves was determined according to the method of Alexieva et al. (2001).

MDA (malonyldialdehyde) determination in 1-g fresh leaves has followed the method described by Li (2000).

Antioxidant activity

Five-gram fresh leaves were homogenized in 0.05 M cold phosphate buffer (pH 6.5) containing 1 mM EDTA, Na₂ and centrifuged at 3800g for 10 min. The supernatant was completed to a total known volume (5 ml) and used as the protein and enzyme source. The residue was carefully washed with distilled water and centrifuged several times. The wall fraction was then kept with 10 ml of 1 M NaCl for 1 h to release cell wall-bound POX and centrifuged at 3800g for 10 min; the supernatant was used as the source of wall-bound POX (Saroop et al. 2002).

SOD activity was measured by the nitro blue tetrazolium (NBT) reduction method (Beyer and Fridovich 1987). PPO was estimated according to Kar and Mishra (1976). Soluble and cell wall-bound (POX) was determined according to Saroop et al. (2002). The enzymes’ activity was expressed as U mg⁻¹ protein min⁻¹.

Free and cell wall-bound phenols were extracted from 1-g leaves of both control and treated plants according to the method of Campbell and Ellis (1992).

Proline content was determined in 1 g of Fresh leaves according to the method of Bates et al. (1973).

Determination of hydrolysis processes

Metabolic activity marker (shikimic acid) content

Shikimic acid concentration in 1 g of fresh leaves was determined according to Zelya et al. (2011). The frozen plant leaves or calli were ground (0.2 g) before 1 ml/100 mg biomass of 0.25 M HCl was added. The extracts were shaken (2 min.) and then centrifuged at 3800g for 30 min. The supernatant (50 μl) reacted with 0.5 mL of a 1% solution of periodic acid. After 3 h at room temperature, 0.5 ml of 1 M sodium hydroxide and 0.3 ml of 0.1 M glycine were added per sample. Samples were centrifuged again and absorbance measured at 380 nm. The amount of shikimic acid in the test sample was calculated using the standard curve.

Hormones

The method of hormone extraction was essentially similar to that adopted by Shindy and Smith (1975) and described by Hashem (2006). To estimate the amounts of acidic hormones abscisic acid (ABA), the plant hormone fractions and standard ones were methylated according to Vogel (1975) to be ready for gas chromatography (GC) analysis. Flame ionization detector was used for the identification and determination of acidic hormones using Hewlett Packard Gas Chromatography (5890) fitted and equipped with HP-130 mx 0.32 mm × 0.25 mm capillary column coated with methyl silicone. The column oven temperature was programmed at 10 °C min⁻¹ from 200 °C (5 min) to 260 °C and kept finally to 10 min. Injector and detector temperature were 260 and 300 °C, respectively. Gas flow rates were 30, 30, and 300 cm s⁻¹ for N₂, H₂, and air, respectively, and flow rate inside column was adjusted to 2 ml min⁻¹. Jasmonic acid (JA) was determined, according to Kramell et al. (1997) using NUCLEODEX beta-PM, 200 mm and 4 mm ID column, flow rate adjusted at 1 ml min⁻¹ and detected at UV 210 nm. Standards of ABA and JA were used. Peak identification was performed by comparing the relative retention time of each peak with those of ABA and JA standards. Peak area was measured by triangulation, and the relative properties of the individual components were therefore obtained at various retention times.

Statistical analysis

All results were analyzed by SPSS software (version 14). Data was expressed as mean ± SD. Comparison of mean values of the sample and the control was done, using paired T test. P < 0.05 was considered to be significant (Levesque 2007).

Extraction and purification of defensin

The SDS page ensured that the purified protein used in the research was a single band with 10 kDa molecular weight (Fig. 1). The protein content in the crude extract was 158.928 mg protein g⁻¹ dry weight. The protein content in the purified defensin was about 0.492 mg protein g⁻¹ dry weight. This result is correlated to Oddepally and Guruprasad (2015).

Effect of defensin priming on antioxidant machinery in wheat seedlings during Fusarium wilt control

Seed priming is an interesting alternative way in disease management. The results of the present study ensured that priming of the wheat grains with defensin improved the biochemical response of the wheat seedlings against the infection of F. oxysporum. The activities of the antioxidant enzymes (SOD, PPO, soluble and cell wall-bound POX) in the FDI and I group were significantly higher than that in the C group at 10th day. At the 20th day, the enzymes’ activity remained in the FDI group significantly higher than that in the control, while in the I group their activities significantly decreased compared to that at the 10th day (Fig. 3). This activation of the antioxidant system could reflect a defense response to the cellular damage caused by pathogen toxins (Chandrakar et al. 2018). This role was clearly showed in the results of the MDA content, one of lipid peroxidation (LPO) products. Obtained results showed that MDA significantly increased continuously in the I group but in the FDI groups insignificantly increased at 10th day then decreased at 20th day (Fig. 2). This increase in LPO may be related to increase in the ROS against the antioxidant system, which loses its activity with increasing the stress time. It has been found that plants exposed to various stress exhibit an increase in LPO due to the generation of ROS (Han et al. 2016). As indicated and clarified in H₂O₂ content in the results, H₂O₂ content increased significantly under infection either in the case of defensin-primed plants (FDI) or non-primed plants (I) at the 10th day (210.9 and 212.14%, respectively). H₂O₂ content in the FDI group significantly decreased at the 20th day. The content decreased significantly to be only about 17.5% more than that of the control (C). In the I group, the content of H₂O₂ increased significantly to be 245.25% more than that of the C (Figs. 2 and 3). The increase of H₂O₂ content, as a result of stress, can be explained by activation of NADPH-dependent oxidases and cell wall-bound peroxidase as stress response, such as reported by Agarwal and Jha (2010) who reported that during stress, there is an activation in the transcription of NADPH-dependent oxidases genes. In FDI group, the H₂O₂ content significantly increased at 10th day and then decreased at 20th day. This increase is attributed to the fact that H₂O₂ can act as a signal molecule involved in acclamatory signaling triggering tolerance against various stress (Miller et al. 2010).

H₂O₂ content decreased again with time in the FDI group but increased in the other treatment; as in the FDI group antioxidant network (enzymatic and non-enzymatic), it was active, which is constantly acting on the alert for rising H₂O₂ concentrations and provides effective scavenging for it. H₂O₂ is involved in cell wall reinforcement by increasing protein cross-linking and incorporation of phenolics in the cell wall (lignification), which is an effective defense mechanism to both biotrophics and necrotrophics (Han et al. 2016). This is correlated to the results in Fig. 4, which showed the changes in the content of the non-enzymatic antioxidant compounds (free soluble or cell wall-bound phenol) or proline. In FDI and I groups free soluble phenol and proline contents were significantly higher than that in the C group at both days. In the case of cell wall-bound phenols, content in the FDI was significantly higher than that in the control either at 10th or 20th day, while in the I group, its content was significantly higher than that in the control at 10th day and then decreased at 20th day to be lower than that in the control. This ensured the dual role of H₂O₂ stress reported by Miller et al. (2010).

Effect of defensin priming on metabolic status in wheat seedlings during Fusarium wilt disease

The increase in soluble sugar and soluble phosphate in FDI content serves the carbon fluxes from primary to secondary metabolic pathways. The stimulation of secondary metabolic pathways ensured by the results of shikimic acid content is shown in (Fig. 5). Shikimic acid content in the FDI was significantly lower than the content in the control plants. Under infection conditions, the shikimic acid content significantly decreased either in the FDI or I group. Its content in the FDI group was 81.6% lower than the content in the control. In the case of the I group, its content was 19.3% lower than that in the control. This accumulation of the shikimic acid related to the lower content of soluble carbohydrates and soluble phosphates. Thus, there were not enough sources for carbon fluxes for production of phenols through shikimic acid pathway. This explanation agrees with Cipollini et al. (2003) who reported that defense system impose metabolic costs on the plants by lowering the primary metabolites used in the fitness and growth of the plants. Therefore, only resistant plants can defend themselves through increasing the primary metabolites (soluble carbohydrates) to be sufficient for fit growth and formation of defensive secondary compounds.

Effect of defensin priming on hormonal content in wheat seedlings during Fusarium wilt control

Phytohormones are critical to the ability of plants to defend themselves against pathogens by modulating a wide range of adaptive responses (Di et al. 2016). The present work showed that ABA content in the FDI group was higher than the content in the control only at 10th day and decreased to be lower than its content in the control at 20th day. On the other hand, in the I group, the ABA content was higher than its content in the control either at the 10th day or the 20th day (Fig. 6). The difference in the ABA content in the samples with the time is accumulating evidence suggests that for the development of plant resistance, short-term increase in the ABA level is critical during the early stages of plant interaction with stress. This regulates defense responses through its effects on callose deposition, production of reactive oxygen intermediates, and regulation of defense gene expression (Agarwal and Jha 2010). At the same time, high ABA concentrations maintained for a long time reduced the efficiency of defense systems controlled by salicylic, jasmonic acids, and ethylene. This assured that ABA is involved in plant defense mechanisms against pathogens as a regulatory element (Taki-Nakano et al. 2014).

The increase in JA content during stress is well documented as defense signal. Both H₂O₂ and JA are primary signaling molecules during the cellular response involved in saponin biosynthesis mediated by oligogalacturonic acid (OGA), which also leads to the H₂O₂-mediated upregulation of JA (Schaller and Stintzi 2009). Jasmonic acid induces glutathione, an important antioxidant for redox balance. Increased expression of nuclear factor erythroid 2-related factor 2 (NrF2) was also observed, which reduced the ROS level induced by H₂O₂ (Taki-Nakano et al. 2014). This explained the significant increase in JA content and decrease in the H₂O₂ content at the 20th day in the FDI plants. Decreasing of JA with time in the case of the I group can be attributed to the antagonistic effect of the accumulation of ABA in the tissues, while this antagonism not occurred in the FDI plants. Schaller and Stintzi (2009) reported that stress conditions alter oxylipin profiles, particularly the allene oxide synthase branch of the oxylipin pathway, responsible for production of jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (12-OPDA). ABA signal pathway use the 12-OPDA which cause its unavailability for JA biosynthesis. In Arabidopsis, ABA has been shown to antagonize the JA signaling pathway, and this antagonism is considered responsible for the enhancement of disease susceptibility by ABA against the soilborne fungus F. oxysporum (Anderson et al. 2004).