We brought together insights from the initial screening and factorial experiments for a final test of the effects of six disinfection treatments on the bacterial and fungal loads on seedlings after 24 h and on seed germination success. Disinfection treatments were bleach (10% commercial CloroxTM, 0.6% sodium hypochlorite), hypochlorous acid (HCA; freshly produced Force of NatureTM Multi-purpose cleaner, ~800 ppm chlorine), vinegar (diluted HeinzTM (Sharpsburg, PA, USA) white vinegar, 3% acetic acid), H2O2 (3%), 55 °C water; Sonication in 23 °C (room temperature) water, and a control treatment of sterile deionized water (23 °C). Note that H2O2 was used at 3% rather than the higher concentrations tested earlier because concentrations of food-grade H2O2 at higher concentrations are widely restricted for consumer purchase. Each treatment was replicated 5 times. The entire experiment was conducted twice with treatments lasting 5 min and twice for treatments lasting 15 min.
To conduct each experiment, 200 broccoli seeds were placed in sterile test tubes with a slip-on cap (35 tubes total). The disinfecting agent was poured into the tubes to cover the seeds. At the conclusion of the treatment time, the agent was decanted from the tubes, and seeds were rinsed 3 times with sterile deionized water. Sterile deionized water was then added to each tube to cover seeds and allowed to soak for 4 h before draining. At 24 h from the initial treatment, 9 mL of sterile deionized water was added to each tube. Tubes were vortexed for 10 s, then two 100-fold dilutions of the solution were prepared. This created three 100-fold dilutions: 10−2, 10−4, and 10−6; aliquots (100 µL) of each of the three dilutions were individually transferred to 100-mm Petri dishes containing 0.1TSA and spread evenly with a sterile bent glass rod. The same was done for the 10−2 on MEA medium for quantification of fungi. Seeds from each tube were then placed into a Petri dish lined with sterile filter paper; percent germination was recorded 5 days after initial treatments. Bacterial and fungal densities were expressed as log10(CFU per 200 seeds + 10); the 10 was added to allow for the inclusion of replicates that recovered zero bacteria at the 10−2 dilution and was chosen as 10-fold below the expected minimum level of detection). Percent seed germination was measured after 5 days.
Differences among treatment means were tested using analysis of variance, treating each experimental repetition as a block (function aov in R). Soaking times of 5 min and 15 min were analyzed separately. Least squares means and 95% confidence intervals for the mean of each treatment were calculated using emmeans function (emmeans package in R). Post-hoc comparison of means was performed using Tukey’s HSD multiple comparisons test (TukeyHSD in R) with 95% confidence.
To conduct each experiment, 200 broccoli seeds were placed in sterile test tubes with a slip-on cap (35 tubes total). The disinfecting agent was poured into the tubes to cover the seeds. At the conclusion of the treatment time, the agent was decanted from the tubes, and seeds were rinsed 3 times with sterile deionized water. Sterile deionized water was then added to each tube to cover seeds and allowed to soak for 4 h before draining. At 24 h from the initial treatment, 9 mL of sterile deionized water was added to each tube. Tubes were vortexed for 10 s, then two 100-fold dilutions of the solution were prepared. This created three 100-fold dilutions: 10−2, 10−4, and 10−6; aliquots (100 µL) of each of the three dilutions were individually transferred to 100-mm Petri dishes containing 0.1TSA and spread evenly with a sterile bent glass rod. The same was done for the 10−2 on MEA medium for quantification of fungi. Seeds from each tube were then placed into a Petri dish lined with sterile filter paper; percent germination was recorded 5 days after initial treatments. Bacterial and fungal densities were expressed as log10(CFU per 200 seeds + 10); the 10 was added to allow for the inclusion of replicates that recovered zero bacteria at the 10−2 dilution and was chosen as 10-fold below the expected minimum level of detection). Percent seed germination was measured after 5 days.
Differences among treatment means were tested using analysis of variance, treating each experimental repetition as a block (function aov in R). Soaking times of 5 min and 15 min were analyzed separately. Least squares means and 95% confidence intervals for the mean of each treatment were calculated using emmeans function (emmeans package in R). Post-hoc comparison of means was performed using Tukey’s HSD multiple comparisons test (TukeyHSD in R) with 95% confidence.
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