- Open Access
Control of Fusarium wilt in wheat seedlings by grain priming with defensin-like protein
© The Author(s) 2018
- Received: 7 June 2018
- Accepted: 7 August 2018
- Published: 17 August 2018
The exogenous application of natural plant resistance inducer is a new interesting way for disease management. From this point of view, this study investigated the effect of wheat grain priming with defensin on the resistance of the grown plants against the Fusarium wilt. The results showed that priming enhanced the antioxidant system represented in antioxidant enzymes (superoxide dismutase, polyphenol oxidase, soluble and cell wall-bound peroxidase) and non-enzymatic antioxidant compounds (proline, free soluble and cell wall-bound phenols) in the plants. This results in decreasing the H2O2 content and lipid peroxidation in these plants, which protects plants from the oxidative stress. Defensin priming not only improved the antioxidant system but also improved the hydrolysis processes. Under infection, the protease activity in the primed plants decreased compared to its highly significant increase in the non-primed group. Defensin priming also increased the activity of the phosphatases and amylase which increased the soluble phosphate and sugar. This served the high metabolic activity of the defensin-primed plants. The highly significant decrease in the shikimic acid content in the defensin-primed group ensured its high metabolic activity. All these changes are initiated by the regulation of defense signal molecules such as jasmonic and abscisic acids. This helped the plants grown from defensin-primed grains to overcome the Fusarium infection.
- Antioxidant compounds
- Jasmonic acid
- Abscisic acid
- Shikimic acid
The exogenous application of natural plant resistance inducer is a new interesting way for diseases management. It is more effective and safe for the environment and so human health. The use of natural products can enhance the response against the attack of a wide range of pathogens (Oliveira et al. 2016).
Octadecanoid pathway is one of the important defense mechanisms against pathogens. Through this pathway, jasmonic acid is formed activating the expression of several defense genes. Also, H2O2 is formed during this pathway. H2O2 is considered to be the first defense-signaling molecule in plants. A number of other reactive oxygen species (ROS) constantly produced under stress. The increase in ROS production is highly dangerous on cellular macromolecules such as lipids, proteins, and nucleic acids. Several antioxidant enzymes and compounds are enabled to fight the dangerous effect of the ROS. However, this defensive system is often insufficient to fight severe stress conditions. From this point of view, finding exogenous additives to counter ROS-induced injury symptoms and enhance stress tolerance in plants and their parts is highly important need (Chandrakar et al. 2018).
Defense-related enzymes play an important protective role in plants against pathogen invasion. Many studies have reported that superoxide dismutase (SOD), peroxidase (POX), and polyphenol oxidase (PPO) are from the most important defense-related enzymes in plants. SOD, POX, and PPO are representative antioxidant enzymes and are important components of defense against membrane lipid peroxidation and oxidative stress during pathogen invasion. Beside these antioxidant enzymes there are another non-enzymatic antioxidant compounds such as phenols and proline. The soluble phenols play an important role in redox regulation in plant tissues and can act as antimicrobial compounds. Also, the esterification of phenols in the cell wall (lignification) is an effective defense mechanism against pathogen invasion spread (Han et al. 2016). Proline is very important amino acid in plants. It has many functions under stress such as osmolyte, an antioxidative molecule and a signaling molecule (Hayat et al. 2012). Shikimic acid is the known precursor of aromatic amino acids and many secondary metabolites important in resistance mechanism such as phenols specially the lignin (Nadernejad et al. 2013).
The abscisic acid (ABA) is well known as stress hormones. It regulates many metabolic processes involved in plants under stress conditions. ABA initiates defense mechanisms, such as controlling the closing and opening of stomata and expression of defense-related genes (Agarwal and Jha 2010).
Plant defensins are small, cysteine-rich proteins (45–54 amino acids). They have been separated from many plant species and tissues. They have a lot of different functions. Mainly, they have antifungal activity; also, they have a role as an antibacterial agent, zinc tolerance, and blocking of ion channels, as well as inhibition of proteases (Oddepally and Guruprasad 2015).
Fusarium oxysporum is a soilborne fungal pathogen. It causes major economic losses by inducing necrosis and wilting symptoms in various crop plants. Mainly, farmers fight the Fusarium by the use of chemical fungicides. These are injurious to the environment, and their efficiency is often limited by pathogenic variability. Hence, improving safe and effective new strategies for fighting the soilborne pathogens is an urgent need (Swarupa et al. 2014). From this point of view, this present study aimed to introduce a natural, safe, and effective strategy to fight the Fusarium wilt. The effect of wheat grains priming with defensin (natural defense peptide) on the biochemical response of wheat seedlings to fight the Fusarium wilt was evaluated.
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).
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).
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
The amylase activity was measured according to Johnson (2007) and expressed as the amount of starch hydrolyzed min−1 mg protein−1.
Carbohydrates were estimated in 1 g of fresh leaves according to phenol-sulfuric acid method (Dubois et al. 1956).
Organic phosphorus hydrolysis
The activities of the two enzymes were assayed depending on the method of Tominaga and Takeshi (1974). One nkat of enzyme activity was defined as 1 nmol p-nitrophenol liberated min−1 and specific activity as nkat mg−1 protein.
For determination of the total phosphorus content, 5 g of dried powder of plants was digested in a mixture of concentrated nitric acid, sulfuric acid, and perchloric acid at the ratios 10:1:4, respectively. The volume was made up to a constant volume with distilled water according to the method of Chapman and Pratt (1978). Phosphorus content in the digested samples was determined according to Murphy and Riley (1958). Results were expressed as milligrams per gram dry weight.
Protease activity was measured in an azocasein assay (Mel et al. 2000). Specific enzyme activity was expressed as change in optical density mg−1 protein min−1.
The total protein content was identified according to the method of Lowry et al. (1951).
Total free amino acids were estimated in 1 g of fresh leaves according to the method of Lee and Takahashi (1965).
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.
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.
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
Effect of defensin priming on antioxidant machinery in wheat seedlings during Fusarium wilt control
Effect of defensin priming on metabolic status in wheat seedlings during Fusarium wilt disease
Effect of defensin priming on protein hydrolysis (soluble protein, protease activity and free amino acids) in plants under Fusarium infection conditions
Soluble protein (mg g−1 fwt)
Protease activity (change in optical density mg−1 protein min−1)
Free amino acids (mg g− 1 fwt)
31.09 ± 0.078
32.67 ± 0.092
0.0093 ± 0.097
0.0095 ± 0.271
2.5 ± 0.148
2.89 ± 0.109
19.28 ± 0.165*
13.09 ± 0.109*
0.024 ± 0.196*
0.032 ± 0.192*
11.98 ± 0.265*
18.2 ± 0.213*
36.9 ± 0.056*
38.94 ± 0.208*
0.00756 ± 0.083*
0.00708 ± 0.098*
1.56 ± 0.095
1.230 ± 0.109*
30.98 ± 0.023
31.27 ± 0.107
0.0097 ± 0.309
0.0098 ± 0.192
3.09 ± 0.104
3.15 ± 0.056
Effect of defensin priming on carbohydrates hydrolysis (soluble carbohydrates and amylase activity) in plants under Fusarium infection
Soluble carbohydrates (mg glucose/g fwt)
Amylase activity (mg starch hydrolyzed/min/mg protein)
65.98 ± 0.0483
68.78 ± 0.0893
3.26 ± 0.0945
4.52 ± 0.1467
34.98 ± 0.1982*
26.03 ± 0.0724*
2.05 ± 0.0659*
2.12 ± 0.1342*
76.42 ± 0.0738*
77.23 ± 0.0684*
4.92 ± 0.1003*
6.63 ± 0.0583*
73.78 ± 0.0389*
75.95 ± 0.1982*
3.95 ± 0.0678*
6.27 ± 0.0451*
Effect of defensin priming on organic phosphate hydrolysis (soluble phosphate and acid and alkaline phosphatases activity) in plants under Fusarium infection
Soluble phosphate (mg/g dry wt)
Alkaline phosphatase activity (nkat/mg protein)
Acid phosphatase activity (nkat/mg protein)
13.72 ± 0.068
15.04 ± 0.192
37.04 ± 0.098
39 ± 0.07795
42.18 ± 0.049
44.89 ± 0.098
4.68 ± 0.1925*
3.21 ± 0.0933*
19.37 ± 0.088*
17.03 ± 0.094*
22.09 ± 0.157*
19.03 ± 0.169*
17.14 ± 0.344*
18.58 ± 0.210*
42.46 ± 0.196*
43.98 ± 0.105*
47.98 ± 0.119*
50.05 ± 0.103*
13.82 ± 0.139
15.52 ± 0.311
37.58 ± 0.307
38.78 ± 0.079
42.89 ± 0.249
45 ± 0.0958
Effect of defensin priming on hormonal content in wheat seedlings during Fusarium wilt control
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).
The overall conclusion is that priming wheat grains with defensin resulted in growing plants able to defend themselves against F. oxysporum infection. This is ensured by comparing the biochemical changes in primed plants (FDI) and non-primed plant (I) infected with Fusarium. These changes were analyzed at two stages of disease establishment, the first at 10th day (the first record for symptoms at the I group appearance) and the second stage at 20th day (after which the I group plants died). The biochemical changes showed that defensin priming caused in regulating the signal molecules such as ABA and JA, and this, in turn, activates the antioxidant machinery. This antioxidant machinery succeeded in ameliorating the toxic effect of the reactive oxygen species represented in the decrease in H2O2 content and so decrease in the lipid peroxidation. This decreasing in the reactive oxygen species had a role in proteases inhibition, which increased the protein content and decreased the free amino acids content. Besides, the defensin priming improved the activities of the amylase and phosphatases which increased the content of soluble sugars and phosphate. These act as the main energy requirements in the cells; this serves the highly metabolically active status of the primed plants which ensured the low content of the shikimic acid.
I would like to express my great gratitude to my supervisor Professor Hegazy S. Hegazy, Professor of Physiology, for his effort, time, and patience given. Also, I would like to thank Assistant Professor Manal Tawfik, Professor of Mycology, for her effort in the culturing of the plant pathogenic fungus (Fusarium oxysporum) used in this study.
This paper is self-funded, and I did not take any fund from any organization or person.
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