Establishment and preliminary application of the PMA-PCR, PMA-LAMP, and PMA-qPCR assays for the detection of viable <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> cells in Kiwifruit

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is the most devastating disease affecting kiwifruit production worldwide, posing a major threat to industry sustainability. This study aimed to establish a rapid and reliable molecular detection system capable of differentiating viable from dead Psa cells by combining propidium monoazide (PMA) with conventional PCR, loop-mediated isothermal amplification (LAMP), and quantitative PCR (qPCR). We subsequently applied this methodology to analyze viable Psa in commercially available kiwifruit pollen. The results demonstrated that under optimized conditions (130 μg/mL PMA concentration, 5 minutes dark incubation and 20 minutes light exposure time), PMA effectively inhibited DNA amplification from dead cells while preserving detection sensitivity for viable cells. The optimized PMA-PCR, PMA-LAMP, and PMA-qPCR assays consistently achieved detection thresholds of 10² CFU/mL for viable Psa cells in pure culture, while yielding negative results for heat-inactivated Psa cells at concentrations up to 10⁸ CFU/mL. Moreover, this approach was successfully extended to the detection of viable Psa cells in commercial kiwifruit pollen samples. In conclusion, the developed assays enable accurate discrimination between viable and dead Psa cells, providing a promising tool for the early detection of live Psa in kiwifruit pollen and contributing to effective disease management and prevention.