Multiskan spectrum microplate spectrophotometer

The test was conducted with some modifications (Chen et al., 2019 (link)). The P. digitatum spore suspensions (1 × 10⁵ CFU mL^–1, 5% PDB) were cultured for 12 h at 25°C, and then incubated with tahantin treatment (0, 1, 2, 4, and 8 μmol L^–1) at 25°C for 2 h and 6 h, respectively. Cell lysis was performed by adding 0.4 g glass beads in each mixture (600 μL). The sample was then placed on ice and vortexted for 30 s periodically for 15 min, until the mixture was clear. After centrifugation at 10,000 × g for 20 min at 4°C, the 100 μL supernatant was stained with an equal volume of Coomassie Brilliant Blue (G-250, C8420, Solaribo, China) for 5 min. The mixture was measured at 595 nm using the Multiskan Spectrum microplate spectrophotometer (BioTek Instruments, Inc., United States). The soluble protein contents were determined via a Bradford assay using BSA as the standard. The experiment was repeated three times.

The fungicidal activities of the peptide BP21 was determined by dose–response curves as previously described (López-García et al., 2002 (link); Wang et al., 2018 (link)). BP21 was added to a final concentration of 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 μM, respectively. In all experiments, three replicates were prepared for each treatment. The growth of the fungi was determined by measuring OD₆₀₀ using a Multiskan Spectrum microplate spectrophotometer (BioTek Instruments, Inc., United States) at 48 h after mixing with BP21. The minimum inhibitory concentration (MIC) of the peptide BP21 for three fungi was defined as the peptide BP21 concentration that completely inhibited growth in all the experiments carried out.

The assay was referenced to the technique of Tang et al. [14 (link)] with appropriate modifications. The G. citri-aurantii genomic DNA was diluted to 50 μg mL⁻¹ with sterile water, and mixed with a gradient concentration of peptides at 1:1 (v/v) for 30 min in the darkness at room temperature. The final peptides concentrations were set as 0, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 μmol L⁻¹. The spectra of the mixture were determined in the range of 230–400 nm by Multiskan Spectrum microplate spectrophotometer (BioTek Instruments, Inc., Vermont, VT, USA).

The efflux of K⁺ of cytoplasmic components is an important indicator of the increasing permeability of the cell membrane. PAF56 solutions (10 or 100 μmol L⁻¹) used in each treatment group were prepared, while controls without PAF56 were treated similarly. The concentration of extracellular potassium in the supernatant and release of cytoplasmic constituents were measured by flame atomic absorption spectroscopy (Shimadzu AA6300, Kyoto, Japan) and using a Multiskan Spectrum microplate spectrophotometer at 260 nm (BioTek Instruments, Inc., Winooski, VT, USA), respectively [12 (link),14 (link),15 (link)]. The mycelia were collected after shaking at 25 °C for 2 d and washed before resuspension in sterilized distilled water (for the measurement of extracellular potassium concentration) or phosphate buffer (0.05 mol L⁻¹, phosphate, pH 7.0) (for the measurement of the release of cytoplasmic constituents). The peptides were added at concentrations of 10 or 100 μmol L⁻¹. The concentration of free K⁺ in the suspensions and absorbance values at 260 nm in the supernatant were measured after treatment at 0, 3, 6, 9, 12, 24, and 48 h. Each treatment was performed in triplicate, and the control lacked peptides.

4′,6-diamidino-2-phenylindole (DAPI) staining method was used to quantify the nucleic acids of G. citri-aurantii, the experiment was done by drawing on the method of Wang et al. [25 (link)] with adjustments as appropriate. The G. citri-aurantii conidial suspension at a concentration of 1 × 10⁶ CFU mL⁻¹ was treated with peptides in shaker (150 r min⁻¹) at 25 °C for 0.5, 2, 4, 6 and 12 h. Then, stained with 15 μg mL⁻¹ DAPI under controlled darkness for 10 min. The fluorescence density of nucleic acid was individually determined with a Multiskan Spectrum microplate spectrophotometer (BioTek Instruments, Inc., Vermont, VT, USA). The excitation wavelength of DNA was 364 nm and RNA was 400 nm, and the emission wavelengths were always 460 nm. The reference control group was the same solution but without peptides. Each treatment was replicated 3 times.

The kinetic parameter of PiGSTd1 was determined based on the CDNB (1-chloro-2,4-dinitrobenzene) method (Li et al., 2018 (link)). Briefly, 0.4 μg of the purified PiGSTd1 was added into 200 μL acetate–phosphate buffer (pH 7.5) containing 50 mM GSH and a series of CDNB (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mM) at 35°C in a transparent 96-well plates, and the absorbance at 340 nm in 0–1 min was recorded in a Multiskan Spectrum Microplate Spectrophotometer (BioTek, Shoreline, WA). Heat-inactivated PiGSTd1 was used as the negative control. The Km and Vmax were calculated by the linear regression of a double reciprocal plot (Balakrishnan et al., 2018 (link)). To optimize the reaction pH and temperature of PiGSTd1, the assays were conducted at fixed concentrations of GSH (1 mM) and CDNB (0.5 mM) with varying acetate–phosphate buffer (pH = 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0) and reaction temperature (20, 25, 30, 35, 40, 50, 55, and 60°C for 30 min). All determinations were performed three times.