Identification and molecular characterization of oomycete isolates from trout farms in Croatia, and their upstream and downstream water environments

Oomycetes from the genus Saprolegnia are opportunistic pathogens that cause significant losses in salmonid aquaculture. Despite this, studies reporting dominant Saprolegnia species in different fish farming facilities, as well as analyses of their spreading to natural environments, are still scarce. In this study, we have for the first time identified oomycete species present in four different trout farms in Croatia. We have collected 220 oomycete isolates, both from affected tissue (46 in total: adult trout - 28, eggs - 13, and alevins - 5) and from water (174 in total: in the fish farm – 78, upstream – 50, and downstream - 46). We have used Bayesian inference to reconstruct phylogenetic relationship among the internal transcribed spacer (ITS) sequences of the collected isolates and referent strains, and determined that the isolates belonged to three different oomycete genera: Saprolegnia (64% of isolates), Pythium (35%), and Leptolegnia (1%). Saprolegnia isolates were classified into four species: S. parasitica with 53 isolates, S. australis - 52, S. delica – 25, and S. ferax – 11. Pythium and Leptolegnia isolates couldn't be identified to the species level and probably belong to so far undescribed species since their sequences didn't group with previously described species. Next, isolates from the affected tissue were mostly S. parasitica (32), while S. australis, S. delica, and S. ferax were less common (≤ 4 isolates per species). Furthermore, we used hempseed baits to capture oomycetes from water and positioned them inside the fish farms, as well as upstream (between 55 and 155 m) and downstream (between 95 and 140 m) of the fish farms. According to correspondence analysis, Saprolegnia species showed a strong association with fish farms and downstream locations, while upstream locations were associated with Pythium species, highlighting a possible role of trout farms as a source of spreading Saprolegnia species into the environment.