Plant material and irradiation treatment
Seeds of sesame cultivar Giza 32 used in this study were obtained from the Agricultural Research Center, Giza, Egypt. Healthy, uniform, and dry seed samples (each 58 g) were exposed to gamma radiation using a Co-60 (Cobalt 60) package irradiator system (Figure 1) with 1 and 4 cm of sample dimensions (Fig. 2 c) at a dose rate of 131 kBq/sec in the Nuclear Physics Department, Faculty of Science, Assiut University. Seed irradiation was performed in the stationary method of operation with varying dose rates of 4.22, 8.45, 12.68, and 16.9 Gy at room temperature and atmospheric pressure, depending on the position and the distance from the radiation source (Table 1). In radiation doses plan No. 1 (Fig. 2 a), four seed samples were sealed in a cylindrical block shield of lead and placed at a 2 cm applied distance, with the bottom of the medical rob at the axis of the cylindrical block shield. In the radiation doses plan No. 2 (Fig. 2 b), each one of the four seed samples was separately sealed in a cylindrical block shield to get radiation exposure from the radioactive source placed at a height of 4 cm. After every 12 h, one of the four seed samples of each radiation doses plan was removed from the radiation exposure system. Finally, all irradiated seed samples were carefully transferred to sterile containers and maintained at 5 °C until use.
Pathogen source, material, and inoculation
Virulent isolate No. 5 of the fungus M. phaseolina (Mp) used in this study was isolated from the root and stem of diseased sesame plants showing charcoal rot symptoms collected from various fields in Assiut Governorate, Egypt, where it was identified as the causal pathogen of charcoal rot disease when tested on sesame Giza 32 cultivar, according to the previous work published by Ahmed et al. (2023). For the preparation of Mp inoculum from the stuck culture, the fungus was cultured in 9 cm Petri dishes containing sterilized potato dextrose agar (PDA) medium supplemented with 50 mg streptomycin sulfate L-1 medium and incubated in the darkness at 28 °C for 7 days. After that, small portions of the Mp colony containing microsclerotia were suspended in 100 µl sterile distilled water to separate microsclerotia from mycelia. Microsclerotia were then cultivated into new PDA plates incubated at 28 °C. Finally, autoclave toothpicks were inserted into Mp colonies growing on the PDA plates for 7 days to harvest microsclerotia. The microsclerotia-containing toothpicks were then injected into micro-tubes and maintained at 4 °C (Edmunds, 1964). Then, 1 g weighted microsclerotia was immediately suspended in 300 ml of 0.01% agarose solution to get the freshly used fungal inoculum (Reyes Gaige et al., 2010) containing 7 × 104 sclerotia ml-1 determined using a hemocytometer.
Greenhouse experiments
Greenhouse experiments were conducted in the open greenhouse at the Experimental Farm, Arab Al-Awamer Agricultural Station, Assiut, during the 2022 growing season to evaluate the efficiency of irradiated sesame seeds against Mp causing charcoal rot disease of sesame. The sowing date in both experiments was the 21st of April.
Formalin sterilized pots (35 cm in diameter) containing sterilized loam soil were infested with 10 ml Mp inoculum, irrigated, and left for a week before planting. Each pot was then seeded with five irradiated seeds exposed at 2 and 4 cm of the sesame cultivar Giza 32. Twelve pots (replicates) were used for each treatment, and pots sowed with non-irradiated seeds served as a control. Pots were irrigated when necessary and checked daily. After 28 days of planting, 20 plant samples (2 from each pot) of each treatment were randomly selected to calculate charcoal rot incidence based on root colonization by Mp in sesame plant samples by cutting one-centimeter-long root pieces after washing in tap water were surface sterilized with 1% sodium hypochlorite (SH) solution and transferred onto the PDA plates supplemented with 50 mg streptomycin sulfate L-1 medium at 5 pieces per plate and then incubated in the darkness at 28 °C for 7 days as mentioned before. The number of growing Mp colonies in each plate was then recorded, and the percent of charcoal rot incidence of sesame based on root colonization by Mp of each replicate was calculated using the following formula.
Charcoal rot incidence based on root colonization by Mp (%) = Number of pieces with fungal growth /Total number of pieces x 100
Moreover, the same selected plant samples were further used for sesame roots' biochemical and histological changes in response to infection with Mp concerning charcoal rot disease resistance. On the other hand, other left-potted plants were visually checked for the development of charcoal rot symptoms, and the incidence of charcoal rot disease was also determined after 90 days of planting using the following formula:
Charcoal rot (%) = Number of plants with charcoal rot symptoms/Total number of cultivated plants × 100
Field experiments
Field trials were carried out at the Experimental Farm of Arab-El-Awamer Research Station, Assiut, Egypt, to evaluate the efficiency of gamma-irradiated sesame seeds against Mp, causing charcoal rot disease of sesame under natural infestation with Mp during the 2022 and 2023 growing seasons. The sowing date in both experiments was the 1st of May. Irradiated sesame seeds were sown in rows in plots with 3.2 × 2.4 m, each having three rows and 60 cm apart between rows. Each row contained 15 hills spaced at 20 cm. Every hill was sown with five seeds, and non-irradiated sesame seeds were used as a control. A randomized complete block design of each trail with four replicates was adopted. Non-irradiated seeds served as a control. Sesame plants were then thinned to 2 plants per hill after 20 days from sowing. The cultural practices recommended for sesame production were adopted throughout the growth season. After three months, the growing sesame plants were checked for the development of charcoal rot symptoms, and the percentage of charcoal rot incidence in sesame was determined, as mentioned before. Ten days before harvesting, 10 plants were randomly selected from each plot to assess plant height (cm), number of capsules, and seed yield (g) per plant. Also, oil was extracted from the seeds by the cold extraction method. At room temperature, about 50 g of crumpled sesame seeds were shaken with petroleum ether (1:10 w/v) for 24 h. Then, the solvent was removed from the oil using a rotary evaporator. Lastly, the oil was placed in a glass vessel at ambient temperature to delete residual solvent. The oil was stored at 4 °C, and then seed oil content (%) was calculated according to Hassan et al. (2019).
Biochemical and histological changes in roots of sesame plants originated from gamma-irradiated and non-irradiated seeds in response to the infection with Mp causing charcoal rot disease:
A- Biochemical changes
A-1- Total protein content
The total protein contents of the randomly sampled sesame plant roots at 7, 14, 21, and 28 days old originating from gamma-irradiated and non-irradiated seeds were estimated following the method described by Bradford (1976) using crystalline bovine serum albumin (BSA) as a standard. The roots of plant samples from each treatment were collected at 28 days old, and then 1 g of each plant root was heated at 85 °C with 1 N NaOH. The hydrolyzed protein was then determined using Bio-Rad assay dye, and the developed color was measured at 595 nm. The total protein content in each tested sample was then calculated as mg g-1 fresh weight from the standard curve of BSA.
A-2- The activity of oxidative enzymes peroxidase (PO) and polyphenol oxidase (PPO)
Otherwise, the PO and PPO enzyme extraction was performed from the randomly sampled sesame plant roots at 7, 14, 21, and 28 days old, originating from gamma-irradiated and non-irradiated seeds according to the method described by Maxwell and Bateman (1967). Root tissue samples (1 g fresh weight) were ground in a sterile mortar with 10 ml of 0.1 M phosphate buffer (pH= 7) and strained through layers of disinfected muslin cloths. The root tissue extract of each sample was filtrated by centrifuging at 2500 g and 4 °C for 10 min, and the supernatant was then used as enzyme extract. A reaction mixture contained 0.5 ml of freshly dissolved 0.5% Catechol, 1 ml of 0.1 M phosphate buffer, 4.5 ml SDW, and 0.2 ml of enzyme extract. The activity of PO and PPO enzymes was determined by measuring the absorbance at 470 and 480 nm for PO and PPO, respectively, after 15 min. Then, the PO and PPO activity was expressed as absorbance g-1 fresh weight 15 min-1.
A-3- Total phenolic content
To ascertain the total phenolic contents, fresh roots (1 g) of each tested root sample were extracted in 50% methanol (12 v:v) for 90 minutes at 80 °C from sesame plant roots that were randomly sampled at 7, 14, 21, and 28 days old and originated from gamma-irradiated and non-irradiated seeds. Following a 15-minute centrifugation at 14000 g, the extract was utilized to quantify the amount of free and cell wall-bound phenolic using the Folin-Ciocaleus (FC) reagent, following the protocol outlined by Kofalvi & Nassuth (1995). After neutralizing the pellet with 0.5 ml of 2 N HCl and saponifying it for 24 hours at room temperature to liberate the bound phenolic, the pellet was centrifuged at 14000 g for 15 minutes. Using an FC assay, the supernatant was utilized to bind the phenolic determination. The 100 μL of methanol and NaOH extracts were diluted to 1.0 ml with distilled water, then combined with 2.5 ml of 20% Na2 CO3 and 0.5 ml of 2 N FC reagent. After the combination was let to stand at room temperature in the dark for 20 minutes, each sample's absorbance was measured using a spectrophotometer set to measure wavelengths of 725 nm. The Gallic acid (1 mg ml-1) was dissolved in distilled water to create a stock solution. Then, different concentrations between 1 and 10 μg ml-1 were made. After adding 1.5 ml of FC reagent and waiting five minutes for each concentration to be used, 4 ml of 20% Na2 CO3 solution was added, and the mixture was finished with 10 ml of distilled water. After 20 minutes of storage, the mixture's absorbance at 725 nm was determined. The total phenolic content (μg ml-1) of the samples was calculated by extrapolating a standard curve made with Gallic acid as the standard. To calculate mg of total phenolic g-1 of fresh weight, the absorbance values were converted.
A-4- Analysis of root lignin content
Using a blade mill (Polymix PX-A10), root samples were frozen after 28 days following the procedure outlined by Fagerstedt et al. (2015). At 103 °C, the dry solids content of the ground root samples was ascertained. Using a Soxhlet apparatus, three-gram samples of air-dried root powders were extracted for six hours using acetone (KCL, 1982). The residues were dried at 103 °C, allowed to cool in a desiccator, and then weighed following the solvents' evaporation. The Klason method was used to calculate the quantity of acid-insoluble lignin (Dence, 1992). Three centiliters of 72% sulfuric acid were applied to the 300 mg of extracted root powder samples while they were under vacuum for an hour. After diluting the mixtures with around 82 cm3 of water, they were autoclaved for an hour at 125 °C. Using a 52 × 47 mm glass fiber filter, the precipitates were gathered using suction filtration and subsequently cleaned with water. The filters containing the Klason lignin, which is acid-insoluble, were dried at 103 °C, chilled in a desiccator, and then weighed. The amount of lignin that was soluble in acid was then measured by diluting the filtrate with water to a volume of 250 cm3. Using sulfuric acid with the same concentration as a blank, the absorption of the acid solutions containing the dissolved lignin was measured at 203 nm. Using a spectrophotometer to measure absorbance, the total lignin content (Klason lignin plus acid-soluble lignin) of the unextracted roots was computed as follows:
Klason lignin (%) = p (100 − u) / m, in which p = precipitate (g), u = extractives (%), and m = calculated dry weight of the extracted sample (g). Based on the absorption of carbohydrates, the acid-soluble lignin content was adjusted using a lignin absorptivity of 128 L g−1 cm−1 (KCL, 1982). The measured lignin content of the roots sample, expressed as mg g-1 fresh weight, was calculated by taking the mean of the duplicate measurements of each tested sample.
B- Root histological features
For investigating root anatomical structures, random root samples from the randomly sampled sesame plant roots at 28 days old originating from gamma-irradiated and non-irradiated seeds were taken from the top 4–6 cm (2 cm length) along the taproot and stored in formalin-alcohol-glacial acetic acid (90:5:5, v/v/v) for at least 24 h, according to the method described by Dossa et al. (2017). Dehydration was done by double-staining with safranin and light green, and then the transverse sections were processed using a microtome following the descriptions of Pandey & Chadha (1996). Images of the root sections were acquired with a Lecia microscope and photographed by its camera at (×20-40) magnifications.
Statistical analysis
Analysis of variance (ANOVA) was carried out using the Mstatc program. The least significant difference (L.S.D.) at P≤0.05 was applied to distinguish differences among treatments (Gomez & Gomez, 1984).