Methods
Materials
Seeds of wheat (Triticum aestivum L. var. Yunmai 53) and faba bean (Vicia faba L. var. 87-147) were tested as plant materials and were obtained from the Yunnan Academy of Agricultural Sciences (Kunming, China). Analytical grade salicylic acid (SA) was purchased from the China Pharmaceutical Group Shanghai Medical Instrument Co., Ltd. (Shanghai, China), and the standard phenolic acids used for HPLC analysis were p-hydroxybenzoate, vanillic acid, syringic acid, ferulic acid, benzoic acid, salicylic acid and cinnamic acid, which were purchased from Sigma-Aldrich (St. Louis, MO, USA). The pectin and cellulose used in this study were purchased from Tokyo Chemical Industry (Tokyo, Japan) and Sigma-Aldrich, respectively. The pathogenic fungus was FOF that was isolated from infected faba bean soil and stored at 4ºC in the Plant-Microbe Laboratory at Yunnan Agricultural University, China. Spore suspensions of FOF were obtained from 14-day-old cultures on potato dextrose agar medium (PDA). The pathogenic mycelia were scraped into sterile water with a sterile L-shaped glass rod and incubated at 28 ℃ for 7 d. The spore suspension was filtered using two layers of gauze and served as the inoculum to infect faba bean.
Experimental design
Greenhouse Experiments
The hydroponic experiment was conducted in a glass greenhouse at Yunnan Agricultural University, China, from September to December 2017. The experiment was designed as a two-factor experiment, in which one factor was different concentrations of salicylic acid, including C0 (0 mg·L-1, CK), C1 (50 mg·L-1), C2 (100 mg·L-1) and C3 (200 mg·L-1). The other factor was two planting patterns (monocropping of faba bean [M]; intercropping of faba bean with wheat [I]). Each treatment was conducted four times, resulting in 32 pots (4 replicates × 2 planting patterns × 4 concentrations). The detailed planting pattern is shown in Fig. 1. The experiments were conducted under 24 h pump ventilation.
Faba bean and wheat seeds were soaked at room temperature for 24 h, germinated at 25 ℃ and then sown in sterile quartz sand that had been moistened with Hoagland nutrient solution. After the true leaves emerged, six faba bean plants were transplanted into 2 L containers that contained different concentrations of SA. The faba bean plants of the M and I system were inoculated with an FOF spore suspension of 1×106 cfu·mL-1 near the roots after two days of transplantation. The seedlings were maintained under natural light and temperature conditions (26/19 ℃ day/night) in a greenhouse, and the relative humidity ranged from 70% to 85%. The nutrient solution was replaced every 2 d.
Laboratory experimental design
A laboratory experiment was conducted to verify the effect of exogenous SA on the pathogenicity of FOF. The concentration of SA was consistent with the design of greenhouse experiment. Various concentrations of SA were treated by high temperature steam sterilization and added to steam-sterilized PDA media to determine their effects on FOF. Four replicates per treatment were conducted.
Analytical methods
Collection and analysis of root exudates
Cleaned roots were placed in containers that contained 300 mL of 0.005 mmol·L-1 CaCl2, which was covered by a black membrane to avoid contamination and light. The root exudates were collected under light ventilation for 4 h (09:00 -- 12:00) and then placed back in the container. A volume of 50 mL of the collected liquid was added to a centrifuge tube, microbial activity was inhibited by adding a drop of concentrated phosphoric acid. The root exudates were lyophilized, and storedat -20 ℃. Each treatment was conducted in quadruplicate.
The lyophilized powder of root exudates was dissolved in deionized water, diluted to 1 mL and filtered using a 0.45 μm Millipore membrane (Burlington, MA, USA). The contents of phenolic acid in root exudates were analyzed using High Performance Liquid Chromatography (Agilent 1260 Infinity, Agilent Technologies, Carpinteria, CA, USA). The analytical conditions were as follows: Kinetex column, 2.6 μm, 4.6×100 mm; temperature of column: 30 ℃; injection volume: 10 μL; DAD detector wavelength: 280nm; velocity of flow: 0.5 mL·min-1; mobile phase: methanol (A) and 0.1% (v/v) phosphoric acid solution (B), which were used as mobile phases with a gradient elution (B: 80% (0min) →5% (15.0min) →5% (18.0 min) →80% (18.5 min) →0% (20.0 min) → stop (25.0 min)). All solvents were spectral grade HPLC. The types of phenolic acids were determined by the retention time, and the contents of phenolic acids were calculated using external standards.
Determination of faba bean growth indices
The samples were collected 45 days after transplantation. Three faba bean plants were randomly selected from each replicate for study. The leaf number per plant, maximum leaf length, maximum leaf width, height, main root length, shoot dry weight and root dry weight were measured.
Assessment of the incidence of Fusarium wilt
Three faba bean plants were randomly selected from each replicate. The severity of disease on individual plants was rated on a level from 0 to 5: 0 indicates no infection; 1 indicates initial symptoms of Fusarium wilt; 2 indicates that the base of the stem or the root had lesions, although they were not contiguous; 3 indicates that 1/3–1/2 of the stem base or root exhibited lesions, discoloration or wilt, and the lateral roots were significantly reduced; 4 indicates that the base of the stem was surrounded by lesions, or most of the roots were discolored and wilted, and 5 indicates that the plants had died or totally wilted. The incidence of Fusarium wilt on faba bean and the disease index were calculated using the following formulae:
Incidence (%) = (Number of diseased plants / total number of plants studied) ×100
Relative control efficacy (%) = [(AM - AI)/AM] × 100 (Where A represents the disease index; M represents the monocropping system, and I represents the intercropping system)
Determination of antioxidant enzymes and membrane lipid peroxidation
Fresh root samples from each treatment and replication were used to determine the activities of peroxidase (POD) and catalase (CAT) and content of malondialdehyde (MDA) as described by Li et al. (2000) [34].
A 1.0 g sample was ground with 2.9 mL of cold extraction buffer (0.05 mol·L-1 phosphate buffer, pH 7.8), and the crude extract was transferred to centrifuge tubes, which were centrifuged at 3000 rpm for 10 min. The supernatant was transferred to a 25 mL volumetric flask; the precipitate was extracted twice with 5 mL phosphate buffer, and the supernatant was transferred to the same volumetric flask. The POD activity was measured using guaiacol as a substrate. A reaction mixture was prepared by combining 2.9 ml of 0.05 mol· L-1 phosphate buffer, 1.0 ml of 2% H2O2, 1.0 ml of 0.05 mol·L-1 guaiacol and 0.1 mL of enzyme solution. The reaction mixture was immediately immersed in a 37 ℃ water bath for 15 min and was then rapidly transferred to cooling water. The absorbance at 470 nm was recorded at 1 min intervals for 5 min. The results are shown as A470 per minute per gram of fresh roots (U·g-1·min-1).
A total of 1.0 g of root material was ground to a homogenate with phosphate buffer (pH 7.8). The homogenate was centrifuged at 4000 rpm for 15 min, and the supernatant was transferred to a volumetric flask and assayed for enzyme activities. The activity of CAT was determined using a titration of potassium permanganate. The reaction mixture contained 2.5 mL phosphate buffer and 2.5 mL 0.1 mol·L-1 H2O2. The solution was incubated at 30 ℃ for 10 min, and 2.5 mL 10% H2SO4 was immediately added. The solution was titrated with a standard solution of 0.1 mol·L-1 KMnO4 until the solution turned pink for at least 30 min. The CAT activity was expressed in milligrams of H2O2 degraded per gram of fresh roots in 1 min (mg·g-1·min-1).
A 1.0 g root sample was homogenized in 5 mL of 5% trichloroacetic acid (TCA), and the homogenate was centrifuged at 3000 rpm for 10 min after grinding. The supernatant was used to determine the content of MDA using thiobarbituric acid (TBA). A volume of 2 mL of the supernatant was added to 2 mL 0.6% TBA. The mixture was boiled for 15 min and then centrifuged after cooling. The absorbance values at 450, 532 and 600 nm were measured. The content of MDA is shown as the amount of substance per gram of fresh roots (μmol·g-1).
Determination of the activities of pathogenesis-related proteins
The activities of chitinase and β-1,3-glucanase were measured using kits purchased from Sino Best Biological Technology Co, Ltd. (Shanghai, China). One unit of chitinase activity was defined as the amount of chitin that produced 1 mg of N-acetyl-D-(+)-glucosamine per gram of tissue per hour. One unit of β-1,3-glucan enzyme activity was defined as the amount of enzyme required to produce 1 g of reducing sugar per gram of tissue per hour.
In vitro test of FOF
Determination of FOF growth
A 9-mm agar hole punch taken from a 7-d-old PDA culture was placed in the center of the plate and incubated at 28 ℃ for 7 d with 1 mL of SA at concentrations of C0, C1, C2 and C3. The colony diameter was measured in three different directions on each plate after incubation for 3 and 7 d.
FOF was grown in 30 mL conical flasks consisting of 30 ml potato dextrose broth and inoculated with a 9-mm agar plug from a 7-d-old PDA culture. The cultures were incubated at 28 ℃ in a rotatory shaker (170 rpm) for 7 d. The fungal biomass (dry weight) was determined after filtration and drying at 80 ℃ for 12 h when a constant weight was achieved.
Extraction and quantification of mycotoxin
FOF was inoculated in Richard’s medium, which consists of 10 g of KNO3, 0.02 g of FeSO4, 5 g of KH2PO4, 2.5 g of MgSO4, 34 g of glucose, brought to 1 L with distilled water amended with SA. One ml spore suspension of 1×106 cfu·mL-1 was added to Richard’s medium and cultured at 28 ℃ for 7 days. Eight strains were removed with a 9-mm diameter punch, transferred to a 250 mL conical flask containing 125 mL of culture medium and incubated at 28 ℃ on a rotary shaker (180 rev/min) for approximately 15 days. The culture medium was centrifuged at 5000 rpm for 10 min and filtered with a 0.45 μm Millipore membrane (Burlington, MA, USA) to remove mycelia and spores. The supernatant was collected and autoclaved to obtain the crude FOF toxin solution.
The crude toxin solution was mixed with an equal volume of ethyl acetate, shaken for 2 min and incubated at room temperature for 30 min. The organic phase was collected and centrifuged at 4000 rpm for 10 min. The supernatant was dried and condensed at < 40 ℃. The entire dried residue was re-dissolved in 5 mL of ethyl acetate, and the absorbance was measured at 269 nm. The content of FA was expressed as mg·L-1.
Measurement of pathogenesis-related hydrolytic enzyme activities
The enzyme-producing culture medium utilized a synthetic medium formula that contained 1% of pectin and cellulose, 0.2 g of MgSO4 · 7H2O, 0.4 g of KH2PO4, 0.2 g of KCl, 1 g of NH4NO3, 0.01 g of FeSO4, 0.01 g of ZnSO4, and 0.01 g of MnSO4 in a total volume of 1 L of distilled water. A volume of 25 mL of this medium was transferred to 100 mL conical flasks and inoculated with FOF fragments that were 9 mm in diameter. The culture medium was incubated at 28 ℃ and 200 rpm for 7 days. It was then collected and centrifuged at 4000 rpm for 10 min. The supernatant was filtered through a 0.45 μm filter. The filtrate served as the crude enzyme solution, which was stored at 4 ℃ until use.
The activities of pectinase and cellulase were determined using 3,5-dinitrosalicylic acid (DNS). A total of 3.15 g of DNS was added to 500 mL of water while stirring for 5 s. The solution was heated to 45 ℃. A volume of 100 mL of 0.2 g·mL-1 sodium hydroxide solution was then gradually added while stirring until the solution became transparent. It is imperative that the solution not exceed 48 ℃ during the addition of the sodium hydroxide. A total of 91.0 g of sodium nitrate potassium tartrate, 2.5 g of phenol and 2.5 g of anhydrous sodium sulfite were added and heated to 45 ℃ in a water bath, while 300 mL of water was added with constant stirring until the material had completely dissolved. Finally, the solution was cooled to room temperature, and distilled water was added to a final volume of 1 L. The solution was stored in the dark at room temperature for 7 days before use. A unit of pectinase was defined as the amount of enzyme required to produce 1 μmol of galacturonic acid, and a unit of cellulase was the amount of enzyme required to produce 1 μmol of glucose per min.
Statistical analysis
All the data were analyzed using Microsoft Excel 2010 (Microsoft Corp., Redmond, WA, USA) and SPSS ver. 20.0 (SPSS Inc., Chicago, IL, USA). The least significant difference test was used to determine differences between the treatments at a significance level of P≤0.05.