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Oomycete pathogens in freshwaters, such as Saprolegnia parasitica and Aphanomyces astaci, are responsible for fish/crayfish population declines in the wild and disease outbreaks in aquaculture. Although the formation of infectious zoospores in the laboratory can be triggered by washing their mycelium with natural water samples, the physico-chemical properties of the water that might promote sporulation are still unexplored. We washed the mycelia of A. astaci and S. parasitica with a range of natural water samples and observed differences in sporulation efficiency. The results of Partial Least Squares Regression (PLS-R) multivariate analysis showed that SAC (spectral absorption coefficient measured at 254 nm), DOC (dissolved organic carbon), ammonium-N and fluoride had the strongest positive effect on sporulation of S. parasitica, while sporulation of A. astaci was not significantly correlated with any of the analyzed parameters. In agreement with this, the addition of environmentally relevant concentrations of humic acid, an important contributor to SAC and DOC, to the water induced sporulation of S. parasitica but not of A. astaci. Overall, our results point to the differences in ecological requirements of these pathogens, but also present a starting point for optimizing laboratory protocols for the induction of sporulation.

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*Original abstract online at https://doi.org/10.3390/microorganisms10030520


Furthermore, the PLS-R modelling results suggest that increased fluoride and ammonium concentration could also act as a sporulation trigger, at least for S. parasitica. The range of fluoride concentrations in freshwater is between 0.01 and 0.3 mg/L [57], which is in accordance with the average fluoride concentration of 0.1 mg/L in our dataset. The induction of sporulation by environmentally relevant fluoride concentrations could be explained as a response to unfavorable environmental conditions, as in the case of the humic substances mentioned above. High fluoride concentrations have been shown to have negative effects on microbial physiology [58,59,60,61], but there are no data yet on the toxicity of fluoride to oomycetes. Furthermore, in some of our samples, ammonium-N concentrations were above the threshold of 0.3 mg/L set by national legislation [45].
Environmentally relevant ammonium concentrations (0.05 and 0.5 mg/L) were related to an increased susceptibility of rainbow trout (Oncorhynchus mykiss) to saprolegniosis [62,63]. This was explained by the host stress response and specific impairments of the defense mechanisms against saprolegniosis, but, considering our results, might also partly be due to the ammonium-induced sporulation increase and thereby the increased virulence of the pathogen. However, it was shown that in the presence of high ammonium-N concentrations (8 and 16 mg/L, not found in our dataset) the relative abundance of oomycetes in freshwater habitat can decrease [64]. Similarly, our analysis of S. parasitica load in water samples by droplet digital PCR (ddPCR) showed a negative correlation with ammonium and fluoride concentration (unpublished results). Additional experiments, using a series of increasing ammonium concentrations in the water, should be performed to determine the ammonium concentration range that promotes sporulation, and to compare the effects of ammonium towards mycelium and zoospores of pathogenic oomycetes.
Altogether, our results suggest that some substances might suppress the mycelial growth of the pathogen in a certain range of environmentally relevant concentrations and at the same time promote sporulation, thus facilitating the spread of the pathogens into more favorable environment. Thus, the effects of these compounds, such as ammonium and fluoride, on the sporulation intensity and virulence of freshwater oomycete pathogens should be tested, as it was tested here with HA. Furthermore, one of the limitations of this study is that we used only a single isolate from each species. This is unlikely to be representative at the species level, as significant within-species differences in mycelial growth rate, sporulation temperature, zoospore motility, and other parameters were observed for both A. astaci [65] and S. parasitica [66,67,68]. Therefore, a wider range of isolates/genotypes should be included in in vitro sporulation intensity tests in the future.
The knowledge obtained would enable, from an ecological standpoint, the prediction of the water conditions that might promote the pathogen spreading in natural environments and aquaculture facilities, and thereby aid in the development of preventive measures. On the other hand, the research community working on pathogens from the order Saprolegniales would greatly benefit if the composition of water used for sporulation would be standardized. A range of artificial water samples, with defined composition, could be designed and tested to provide optimal and reproducible oomycete sporulation in laboratory conditions.

Supplementary Materials

The following are available at https://www.mdpi.com/article/10.3390/microorganisms10030520/s1, Table S1: Water samples collected at different locations in Croatia; Table S2: Physico-chemical parameters of the collected water samples and the average number of zoospores obtained; Table S3: Correlation matrix describing the relationship between sporulation efficiency of A. astaci and S. parasitica (response variable) and physico-chemical parameters of water (explanatory variables).