Pmaxx

Each sample was divided into three portions: one portion was heat-inactivated at 85°C for 10 min, while the other two portions were kept at room temperature. PMAxx (Biotium, Inc., Hayward, CA, USA) was dissolved in deionized water to obtain a stock solution of 1 mM. Then, 10 μL of the PMAxx solution was added to 190 μL aliquots of both the non-heated and heat-treated samples, resulting in a final concentration of 50 μM. Additionally, 10 μL of deionized water was added to an untreated aliquot as a control template for standard qPCR. These three aliquots were incubated in the dark at 37°C for 15 min with occasional mixing to allow reagent penetration. Subsequently, the samples were irradiated with a PMA-Lite™ LED photoactivator (E90002, Biotium) at room temperature for 15 min. The experiment was repeated in triplicate, and DNA extraction of the samples was performed. Only when the CT values of the heat-treated-PMAxx treated aliquots were significantly higher than those of the PMAxx treated aliquots, the presence of ASFV viral particles was considered to be existed.

PMAxx^™ (Biotium Inc., CA, United States) was mixed with each mock to the final concentration of 75μM, 100μM, and 150μM. Afterward, the samples were placed on ice for 30min, in the dark with intermittent mixing. Subsequently, samples were exposed to blue LED light at 464nm at 30-s intervals for a total of 2min (Young et al., 2017 (link)). Therefore, samples were centrifuged at 10,000×g for 5min (Young et al., 2017 (link)). The supernatant was discarded, and the remaining DNA was extracted using GenElute^™ Bacterial Genomic DNA Kits (Sigma-Aldrich) according to the manufacturer’s instructions.

PMAxx (Biotium, Hayward, CA, United States), an improved version of the PMA dye for the selective detection of live bacteria by qPCR, was used as follows. PMAxx was diluted in sterile water to obtain a 2 mM stock solution and stored at -20°C in the dark until used. Separate flasks of PWW (50 ml) were inoculated with a cocktail of L. monocytogenes containing live (exponential phase), heat-treated, and chlorine-treated cells to approximately 10⁴ cfu/ml. From each flask, 10 ml of PWW was centrifuged at 4,000 rpm for 10 min at 4°C. The supernatant was removed, and the cell pellets resuspended in PBS at a final volume of 1,000 μl supplemented with PMAxx to obtain a final dye concentration of 50, 75, and 100 μM. After PMAxx addition, the samples were incubated at 200 rpm in the dark at room temperature or 40°C for 10–60 min. Stained samples were subsequently exposed to blue-light PMA-Lite LED photolysis (Interchim, Montluçon, France) for 15 min. In parallel, 10 ml of inoculated PWW was taken from each flask to determine the level of total bacteria by qPCR. Bacteria cells were concentrated by centrifugation (4,000 rpm, 4°C, 10 min). The supernatant was discarded, and untreated and PMAxx-treated pellets were kept at -20°C until DNA genomic extraction.

To use the strain specific primers and probe for viable count determination, the assay needs to be coupled with a viability dye treatment (Gobert et al., 2018 (link)). To find the optimal concentration of viability dye that can effectively inactivate DNA from dead cells, heat-killed and non-heated cells from one mono-strain reference sample were treated with the viability dye PMAxx (40069, Biotium Inc., Hayward, CA, United States) at final concentrations of 0, 25, 50, 100, and 150 μM as previously described (Shehata and Newmaster, 2021 (link)). Once a potential optimal PMAxx concentration was determined, it was confirmed using five mono-strain reference samples. To liberate DNA following PMAxx treatments, bead beating in BeadBug™ prefilled tubes (Z763764, Sigma-Aldrich, St. Louis, MO, United States) was performed for 5 min at 3,000 rpm (Hansen et al., 2018 (link)). The liberated DNA was used in real-time PCR.

The PMA treatment was designed based on Fittipaldi et al.^{30 (link)} and works analyzing plant material²⁷ or more complex samples^{26 (link)}. Unless otherwise stated, 0.4–0.5 mL plant macerates were treated with PMA (Biotium Inc., CA, USA) at a final concentration of 100 μM, in 2 mL tubes and incubated in the dark for 5 min with shaking (150 rpm). Tubes were then placed horizontally on ice, and exposed to light from two halogen bulbs (500 W each) at a distance of 20 cm from the light source, as described elsewhere^{26 (link),27} . In other works, the usual times for PMA photolysis range from 2 to 20 min^{30 (link)}. In our case, light exposure periods above 5 min were enough to provide consistent results among sample replicates. Hence, a standard 10-min PMA photo-activation was applied in all the experiments. For natural canker analysis, a commercial, improved version of PMA, known as PMAxx (Biotium Inc., CA, USA), was also used the same as described for PMA. After the PMA (or PMAxx) treatment, samples were centrifuged at 13,000 rpm for 10 min, the supernatant discarded and pellets stored at −80 °C until use.

A cryopreserved cell suspension of A. aerophoba (Aa18) was thawed and divided into four 150 μL aliquots. Two aliquots served as total prokaryotic community controls (Cryostock samples) and were stored at 4°C for a few hours until DNA extraction as described below. To assess the viable prokaryotic community after cryopreservation (Figure 1), two aliquots were treated with a propidium monoazide dye (PMAxx, Biotium Hayward, CA, United States) following manufacturer’s instructions (here referred to as PMA samples). PMA permanently modifies DNA of membrane-impaired, dead cells and thus only DNA from viable cells with intact membranes is amenable to PCR amplification and sequencing (Nocker et al., 2007 (link); Emerson et al., 2017 (link)). After photo-activation of the dye (using the PMA-Lite LED Photolysis Device), cells were pelleted for subsequent DNA extraction.

Reagents used to develop the assays and perform viability experiments were purchased from different companies. In summary, the PMAxx (40,069, 20 mM in H₂O) was purchased from Biotium (United States), TritonX-100 from Sigma-Aldrich (United States), Virkon™ S from DuPont (United States), and Disinfectant Basi containing 4.0–4.99% (w/v) chlorine from Yiheng (Dezhou, China). Primers and probes were synthesized by Sangon Biotech (Shanghai, China). All other chemical reagents used in the experiments were purchased from Sinopharm (Shanghai, China) except otherwise stated. Double distilled water was used in all experiments.