2.2. Pathogens and inoculation
FOL and FORL were isolated from protected tomato cultivation fields in the West Mediterranean region of Turkey based on consistent tomato production, specifically fields having been cultivated since minimum 4 years and with a defined distance between locations (Fig. 1, Table 2). Necrotic tissue fragments were surface-sterilized (2% NaOCl) and plated on potato dextrose agar (PDA). Conidia were produced in potato dextrose broth (PDB) under standard conditions [42]. All tomato FOL and FORL isolates rendered colonies with conidia and mycelia with morphological characteristics typical of F. oxysporum [42]. The characteristic morphological and molecular properties of fungal isolates were compared with a reference FORL culture obtained from Dr. J. Scott (University of Florida, USA). The culture characteristics and micromorphology were investigated by using PDA and carnation leaf agar [26]. Symptoms include typical one-sided wilting and dark brown vascular discoloration. Upper stem tissues and wilted seedlings were disinfested by immersion in a 1.0% aqueous solution of sodium hypochlorite for 2 min, rinsed in sterile water, and placed on PDA medium. After 72 h, hyphal growth was recovered and re-subcultured on PDA and incubated at 25 °C in 12-h light/dark cycles. Identification was based on colony morphology and conidial characteristics. White cottony mycelium, reddish coloration of the medium, ovoid two-celled macroconidia, and large macroconidia, all characteristic of F. oxysporum, were observed [33]. Each pathogen sample showing characteristics of F. oxysporum was inoculated to tomato plants through wounded roots by submerging for 10 min in a conidial suspension (7 × 107 conidia/ml in sterile H2O), while control plants were only dipped in sterile tap water. Disease symptoms were observed during the post inoculation period of 20 days. Each isolate was inoculated to tomato seedlings with 5 replicates. Afterwards, pathogens were re-isolated from the discoloured stem vascular tissue and inoculated to tomatoes in order to complete Koch's postulates. Re-isolation was done from the discoloured stem vascular tissue. Necrotic tissue fragments were surface-sterilized (2% NaOCl) and plated on PDA.
2.2. โรคและ inoculationFOL and FORL were isolated from protected tomato cultivation fields in the West Mediterranean region of Turkey based on consistent tomato production, specifically fields having been cultivated since minimum 4 years and with a defined distance between locations (Fig. 1, Table 2). Necrotic tissue fragments were surface-sterilized (2% NaOCl) and plated on potato dextrose agar (PDA). Conidia were produced in potato dextrose broth (PDB) under standard conditions [42]. All tomato FOL and FORL isolates rendered colonies with conidia and mycelia with morphological characteristics typical of F. oxysporum [42]. The characteristic morphological and molecular properties of fungal isolates were compared with a reference FORL culture obtained from Dr. J. Scott (University of Florida, USA). The culture characteristics and micromorphology were investigated by using PDA and carnation leaf agar [26]. Symptoms include typical one-sided wilting and dark brown vascular discoloration. Upper stem tissues and wilted seedlings were disinfested by immersion in a 1.0% aqueous solution of sodium hypochlorite for 2 min, rinsed in sterile water, and placed on PDA medium. After 72 h, hyphal growth was recovered and re-subcultured on PDA and incubated at 25 °C in 12-h light/dark cycles. Identification was based on colony morphology and conidial characteristics. White cottony mycelium, reddish coloration of the medium, ovoid two-celled macroconidia, and large macroconidia, all characteristic of F. oxysporum, were observed [33]. Each pathogen sample showing characteristics of F. oxysporum was inoculated to tomato plants through wounded roots by submerging for 10 min in a conidial suspension (7 × 107 conidia/ml in sterile H2O), while control plants were only dipped in sterile tap water. Disease symptoms were observed during the post inoculation period of 20 days. Each isolate was inoculated to tomato seedlings with 5 replicates. Afterwards, pathogens were re-isolated from the discoloured stem vascular tissue and inoculated to tomatoes in order to complete Koch's postulates. Re-isolation was done from the discoloured stem vascular tissue. Necrotic tissue fragments were surface-sterilized (2% NaOCl) and plated on PDA.
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