Capsicum annuum, L.

Mohammadbagheri, Leila, Nasr-Esfahani, Mehdi, Abdossi, Vahid & Naderi, Davood, 2021, Genetic diversity and biochemical analysis of Capsicum annuum (Bell pepper) in response to root and basal rot disease, Phytophthora capsici, Phytochemistry (112884) 190, pp. 1-10 : 7-8

5.3. Disease severity assessment on inoculated C. annuum

The C. annuum plants at 40-day-old seedling stage of were inoculated with P. capsici isolates, as described by Candole et al. (2012), Dunn et al. (2014) and Nasr Esfahani et al. (2012; 2014). Percent disease ∑ RT× 100 severity (PDS) in each replication was calculated using = S × N formula, 2 weeks post inoculations by counting wilted C. annuum plants, where, T is the total number of underground stems in each category; R is the disease severity scale; N is the total number of underground stems tested; S is the highest number in the scale ( Forghani et al., 2021; Tehrani et al., 2020).

5.4. Bio-mass analysis

Bio-mass analysis was determined by measuring the following biomass parameters: Root Fresh Weight (RFW), Root Dry Weight (RDW), Stem Fresh Weight (SFW), Stem Dry Weight (SDW), Stem Diameter (SD), Root Diameter (RD), Stem length (SL), Root Length (RL), Root Volume (RV) and Leaf length (LL) ( Liu et al., 2020a, 2020b; Zhang et al., 2018, 2019). The mean squares of variance analysis and mean comparison of the individual effect of inoculation treatment for susceptible and resistant C. annuum genotypes were evaluated ( Hashemi et al., 2019, 2020; Yang et al., 2020; Zhang et al., 2012a).

5.5. Statistical analysis

Data were transformed to arcsine square-root and then subjected to analysis of variance (ANOVA, P <0.01), and the means were compared by Duncan’ s multiple range test using SAS software version 9.2. The evaluated genotypes were categorized in four groups: resistant, partially resistant, susceptible, and partially susceptible ( Nasr Esfahani et al., 2012, 2014). Disease rating was scored based on a scale of 0–5, where: 0 = no disease symptoms, 1= <10 % of the wilted plants; 2 = 11≤ to 25 %; 3 = 26≤ to 50 % and 4 = 51≤ to 100 % of the wilted plants (National Institution of Agriculture Botany (NIAB) UK; Anon 1985).

5.6. Genomic DNA extraction for genetic diversity analysis

Genomic DNA extraction for genetic diversity analysis was from the leaf samples taken from the 1-month-old apical leaves of C. annuum plants following CTAB method ( Ghasemi et al., 2014; Tsaballa et al., 2015; Zou et al., 2019). Three samples were collected from each C. annuum genotype and pooled for DNA extraction to make high-resolution mapping practical with the DNA markers. DNA quality and quantity were checked on Agarose gel (1.0 %) and TBE 1X buffer and nano-drop device, respectively, and were stored at 20 ◦ C. For ISSR, 21 UBC primers were used for PCR, polymerase chain reaction (Supplementary Table 1 ). PCR was performed as described by Lijun and Xuexiao (2012), Naderi et al. (2020) and Moghaddam et al. (2020). The amplification products were separated by gel electrophoresis in 1.5–2% Agarose gel and were photographed using gel documentation system (Alpha Imager, 2200; Gholamaliyan et al., 2021).

The electrophoretic pattern was visually analyzed and DNA bands were scored as present (1) and or absent (0) of the related bonds. The obtained matrix was fed into the NTSYS-pc software package ( Nasehi et al., 2019; Rohlf, 1993; Wan et al., 2020), and the genotypes were grouped. Principal Coordinate Analysis (PCA) of molecular data was also performed using NTSYS-pc to demonstrate multiple dimension distribution of the C. annuum genotypes.

Correlation between resistant, bio-mass parameters molecular markers and enzyme activities was evaluated using similarity coefficient and the SPSS 16.0 software package. The data were analyzed in a completely randomized design and the means were compared using LSD test and SAS 9.1 software ( Hashemi et al., 2019).

5.7. Evaluation of defense-related enzyme activities

Leaf tissue (0.5 g) from each C. annuum genotype inoculated to P. capsici isolate was ground in liquid N 2, and then freeze dried. For estimation of the enzymes activity, the extracted enzyme was processed according to Moghaddam et al. (2019) and Monazzah et al. (2018). Leaf tissue from each genotype was homogenized in 0.1 mmol l 1 potassium phosphate buffer (pH 7.5), containing 1 mmol l 1 ethylenediaminetetraacetic acid (EDTA), PMSF 2 mmol l 1, Triton x-100 0.1 % and 1 % polyvinyl polypyrrolidone (w/v) at 4 ◦ C. The data were analyzed in a completely randomized design and the means were compared using LSD test and SAS 9.1 software ( Hashemi et al., 2019).

5.7.1. Evaluation of peroxidase (POX) enzyme activities

The peroxidase (POX) mixture activity was determined from tissue extract, 3.9 ml potassium phosphate buffer (100 mmol l 1; pH 6) and 1 ml pyrogallol solution (5 % w/v), and incubated at 20 ◦ C for 10 min. Then, 1 ml of 0.5 % H2O2 was added to the reaction and recorded at 420 nm continuously. The activity of POX was expressed as pyrogallol oxidized min 1 mg 1 protein in l lmol l 1 ( Moghaddam et al., 2020; Monazzah et al., 2018).

5.7.2. Evaluation of superoxide dismutase (SOD) enzyme activities

Superoxide dismutase (SOD) activity was analyzed using the method of Monazzah et al. (2018); 3-ml reaction solution tubes consisted of 40 mmol l 1 phosphate buffer (pH 7.8), 0.1 mmol l 1 EDTA, 2 lmol l 1 riboflavin, 75 lmol l 1 NBT, 13 mmol l 1 methionine and tissue extract, were kept under fluorescent lamp of 30 W for 20 min. The mixture was scored at 560 nm, and recorded in unit mg 1 protein ( Ghaebi et al., 2019; Moghaddam et al., 2020; Nasr Esfahani et al., 2020).

5.7.3. Evaluation of polyphenol oxidase (PPO) enzyme activities

Activity of Polyphenol oxidase (PPO) was recorded using the procedure of Raymond et al. (1993). The solution contained 0.1 ml of tissue extract, a buffer of 2.5 ml of 0.2 mol l 1 sodium phosphate (pH 6.8) and 0.2 ml of 20 mmol l 1 pyrogallol. The result was recorded at 430 nm continuously, and was expressed as pyrogallol oxidized min 1 mg 1 protein 1 in lmol l 1 ( Nasr Esfahani et al., 2020; Xu et al., 2020a, 2020b).

5.7.4. Evaluation of catalase (CAT) enzyme activities

Activity of Catalase (CAT) was determined as described by Monazzah et al. (2018). The mixture was of 20 ml of protein extract, a buffer (pH 7.0) of 50 mmol l 1 potassium phosphate and 15 mmol l 1 H2O2. The results were scored at 240 nm, and presented as the units of H2O2 breaks down min 1 mg 1 protein in l mol ( Monazzah et al., 2018; Nasr Esfahani et al., 2020).

5.7.5. Evaluation of phenylalanine ammonia-lyase (PAL) enzyme activities

Activity of Phenylalanine ammonia-lyase (PAL) was evaluated as the procedure defined by Martinez et al. (2016). The mixture consisted of 0.1 ml of tissue extract, 1 ml of the extraction buffer, 0.4 ml of double distilled water (ddH2O), and 0.5 ml of 10 mmol l 1 l-phenylalanine, then kept at 37 ◦ C for 60 min, and the reaction was ceased by addition of 0.5 ml of 6 mol l 1 HCl. The produced trans-cinnamic acid was removed by ethyl acetate (5 ml). Solid residue was diffused in 3 ml of NaOH (0.05 mol l 1) after solvent evaporation. The results were recorded at 290 nm for determination of cinnamic acid presence as min 1 mg 1 protein in lmol ( Moghaddam et al., 2020; Monazzah et al., 2018; Nasr Esfahani et al., 2020).

5.7.6. Evaluation of β-1,3-glucanase enzyme activities

To obtain the enzymatic extract of β-1,3-glucanase, the pre-weighed infected and non-infected leaves were homogenized in 1.5 ml of 0.05 M (pH 5.5) Na-acetate buffer. The homogenate was centrifuged at 14 000 g for 20 min at 4 ◦ C. To determineβ-1,3-glucanase activity, spectrophotometry at 500 nm was used to assess the occurrence of a catalyzed reaction, using laminarin (Sigma L-9634) as the substrate and the di-nitrosalicylic acid (DNS) method (Miller, 1959). The β-1,3-glucanase enzyme activity was expressed as the amount of glucose as min 1 mg 1 of soluble protein ( Moghaddam et al., 2019).

5.7.7. Evaluation of phenolic content activities

Total phenolic content measurement was obtained as follows. Stem tissue (0.5 g) was homogenized in 2 ml methanolic HCl and then were transferred to a water bath at 50 ◦ C for 3 h. The mixture was centrifuged at 13 000 g for 20 min. The final supernatant was used to measure total phenolic content. The total phenolic content in sunflower stem was estimated by the Folin–Ciocalteu method (Kaur and Kapoor, 2002). One ml of supernatant was mixed with 250 μl of 25 % Folin–Ciocalteu reagent after 3 min, 1 ml of 400 mmol/l sodium carbonate was added. The mixture was kept for 1 h in the dark, and absorbance was measured at 725 nm. The concentration of total phenolics was calculated from the gallic acid calibration curve. The content of total phenolic compounds was expressed as ug of gallic acid equivalent per g fresh weight ( Monazzah et al., 2018).