Cabbage seed samples
The 4 batches of cabbage seeds (MB1414 batch 7669, MB1414 batch 54, MB1415 batch 717 and MB1415 batch 720) used in this study were provided by the company ANSEME (Cesena, Italy).
Phytosanitary evaluation of seeds
To evaluate the phytosanitary conditions of the seed batches a modified protocol from ISTA (2019) was used. A total of 1000 seeds (4 g) from each batch were suspended in 25 ml of sterile PBS solution (0.05 M phosphate) plus Tween™ 20 (0.02% v/v) (Merck, Readington, New Jersey, USA), preliminary chilled to 2–4 °C, in a 100 ml conical flask. The suspension was shaken for 2.5 h at room temperature on a rotary shaker at 100–125 rpm. Two tenfold serial dilutions were obtained and 100 µl of each solution and of the undiluted seed extract were plated on the semi-selective medium mFS, and distributed over the surface with a sterile bent rod. The plates were incubated at 28–30 °C upside down and examined after 4 days. A negative and a positive control were included in each test. The negative control was obtained preparing dilutions from a sample of the extraction medium (PBS plus Tween™ 20), without seeds, and plating on the media used for the samples. The positive control was obtained preparing a suspension of a strain of Xcc in sterile PBS plus Tween™ 20. The suspension concentration was adjusted to 102 to 104 CFU/ml and 100 µl of it was plated on the selective media and spread over the surface with a sterile bent rod.
To confirm the identification, representative colonies were plated on YDC medium and incubated at 28–30 °C for 24–48 h.
Three representative bacterial colonies, isolated from lot 720, were tested with specific real-time PCR protocol to confirm the identification as Xcc according to the ISTA protocol (2019). The DNA was extracted from the colonies (Xcc1, Xcc2, Xcc3) obtaining a suspension of each colony in 1 ml of sterile Ringer's solution, then centrifuged at 8000 rpm for 5 min at room temperature. The supernatant was removed, and the pellet suspended again in 500 µl of NaOH (0.5 M) and incubated for 10 min at 100 °C.
The DNA was tested using the specific primers and probe designed by Köhl et al. (2011) and the universal primers and probes designed by Wu et al. (2008) to validate the reaction. The reaction was set up in a final volume of 25 µl containing 0.5 µl of each primer and probe, 5 µl of GoTaq® Probe qPCR Master Mix (Promega, Madison, USA), 11.5 µl of nuclease free water and 5 µl of DNA. The cycle was carried out in a StepOne Plus ™ Real-Time PCR System (Applied Biosystems) thermal cycler with an incubation at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The results were visualized through the “StepOne Software” program, connected to the thermal cycler.
A total of 400 seeds from each sample were incubated on top of paper, soaked with a 0.05% sodium hypochlorite solution, at 20 °C for 5 days, under a 12 h near-ultraviolet light (NUV) 12 h dark cycle. The same protocol was adopted to test the germinability of the seeds after disinfection treatments.
Disinfection of cabbage seeds under controlled conditions
The seed lot no.720 was used to evaluate the different methods applied because of its high level of contamination with Xcc. A total of 1400 seeds, for each treatment tested, were closed in two layers of sterile gauze and submerged in the disinfectant solutions, following the experimental scheme shown in Table 1. At the end of the treatment the seeds were let dry under the flow of a laminar hood overnight. Four hundred seeds were used for the germination test, while the other 1000 seeds were used to evaluate the effectiveness of the treatment in reducing their level of contamination, with the method described above for phytosanitary evaluation of seeds. Hot water (50 °C) was tested for its effectiveness in containing Xcc by immersion of the seeds for 20 and 30 min. Seeds were disinfected with: (1) a solution of hydrogen peroxide, H2O2 (3%); (2) with a 0.5% sodium hypochlorite (NaClO) water solution; (3) a commercial product formulated for sanitization of different surfaces containing a mixture of peracetic acid (CH3CO3H, 5%), hydrogen peroxide (H2O2, 20%) and acetic acid (CH3CO2H, 10%) (JetFive, Certis Europe B.V., Italy) from now PAAH, at the final concentration of 0.8%, tested at room temperature and at 50 °C; and at the concentration of 1.6% tested at room temperature; 4) electrolyzed water, obtained by applying an electrical charge to a mixture of tap water and salt causing the release of hypochlorous acid (HClO) which acts as disinfectant (Anolyte—Envirolyte, Italy). Each treatment was performed for 30 min, except treatment with hydrogen peroxide (3%), that was tested also for 15 min.
Seed disinfection procedure at the company facilities
The seeds belonging to two batches (717 and 7669) were tested to assess the efficacy of the treatment with hydrogen peroxide (3%). Different amounts of seeds (50 g, 100 g, 200 g, 300 g, 500 g, 1000 g) were closed in cotton bags and submerged in the solution for 30 min. At the end of the treatment the seeds were rinsed under tap water for 3 min, centrifugated at 150 rpm for 2 min at room temperature and then dried in the oven at 37 °C for 1 or 2 h. Seeds were tested to assess the viability and the efficacy of the treatment by following the protocols described above.
The data were subjected to the analysis of variance (ANOVA), following a Log10 transformation of the quantity of Xcc recorded, for data normalization. The Tukey’s test was used to explore differences between multiple group means (P ≤ 0.05). Data were finally back‐transformed to the original. Statistical analysis were performed with the Statistical Package for Social Science (SPSS, IBM, Chicago, IL, USA) version 27.0.