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Abstract Legionellae are opportunistic bacteria that cause various conditions after exposure to contaminated aerosols, ranging from a serious type of pneumonia to a mild case of an influenza-like illness. Despite the risks of exposure, little is known about the occurrence of Legionella in natural environments and, even though studies have shown that there is a potential risk of transmission via inhalation, it does not have to be detected in groundwater that is used for irrigation. The culture methods traditionally used to detect Legionella have several limits that can be partly solved by applying molecular techniques. Samples from 177 wells in Apulia, Southern Italy, were collected twice, in winter and in summer, and analyzed. When compared with the guidelines, 145 (81.9%) of the sampled wells were suitable for irrigation use. The culture-based method highlighted the presence of different species and serogroups of Legionella in 31 (21.2%) of the 145 wells that were shown to be suitable for irrigation use. A greater number of wells returned positive results for Legionella in summer than in winter (p = 0.023), and the median concentrations were mostly higher in summer (500 CFU/L) than in winter (300 CFU/L). The median temperature in the Legionella positive well waters was significantly higher than that in the negative ones, both in winter and in summer (p < 0.001). Using molecular techniques, Legionella non-pneumophila was found in 37 of the 114 wells earlier detected as suitable for irrigation use but negative for Legionella by the culture-based methods. The distribution of Legionella differ significantly in porous aquifers compared to the karst-fissured ones both with quantitative polymerase chain reaction (qPCR) (p = 0.0004) and viable cells by propidium monoazide (PMA-qPCR) (p = 0.0000). Legionella concentrations were weakly correlated with temperature of water both with qPCR (ρ = 0.47, p = 0.0033) and PMA-qPCR (ρ = 0.41, p = 0.0126). Our data suggest that water that aerosolizes when sprinkled on plants represents a potential source of Legionellosis, with a higher risk from exposure in summer. On a practical level, this finding is important for workers (farmers and gardeners) who are in contact with waters used for irrigation. Highlights • Legionella was not related to the suitability of wells for irrigation according to Italian law. • Molecular methods showed non-cultivable Legionella in wells suitable for irrigation. • Legionella was similarly distributed in the various aquifers examined. • Sprinkler irrigation is a potential source of Legionellosis. [ABSTRACT FROM AUTHOR]