Use of immuno-flow cytometry and real-time PCR disclose the epidemiological behaviour of Erwinia amylovora populations during the winter in Portuguese pear orchards

The persistence and progression of Erwinia amylovora in naturally infected orchards is still not fully understood and the majority of studies have been carried out under controlled or artificial infections. A major hurdle stems from the inability to characterize the viability of the bacterium in its natural state. Most standard tests, including real-time qPCR, fail to differentiate live from dead cells, while culture dependent methods lack sensitivity. Even in orchards with recurring infections, the detection of E. amylovora is erratic. Immuno-flow cytometry (IFCM) is considered a high throughput tool in clinics and food industry but has been rarely used as diagnostic test for plant pathogens. Two pear orchards with fire blight history, located in the central part of Portugal, were selected for epidemiological monitoring between February and June, from 2019 to 2022. A total of 440 samples were tested using EPPO standard diagnostic protocols. To address the epidemiological behaviour of E. amylovora, we devised an IFCM test using specific monoclonal antibodies and viability dyes, validated in a subset of 149 samples, testing positive in routine diagnostic tests. Our IFCM test was able to differentiate live and dead E. amylovora in naturally infected samples with concentrations as low as 3.54 × 10⁴ cell·mL⁻¹, confirming positive results for 91% these samples, while 19.4% were considered positive by isolation, 38% using conventional PCR and 95% for real-time qPCR. Temporal analysis showed the highest number of positive samples regardless of the method employed in April, May and June, while viability values measured with IFCM were the highest in April, coinciding with the exit of dormancy and early flowering of pear trees. Integrative analysis of E. amylovora viability and population dynamics over the growing season allowed identification of distinct outbreak risks associated with these Portuguese pear orchards.