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Propolis inhibits life stages of aquaculturally important oomycete pathogens Aphanomyces astaci and Saprolegnia parasitica

Introduction Saprolegnia parasitica and Aphanomyces astaci (Oomycetes) are pathogens with negative impact in freshwater aquaculture. Saprolegnia parasitica causes saprolegniosis, a disease affecting mostly salmonid fishes, while A. astaci causes crayfish plague. Chemicals harmful to humans and the environment are being used globally in aquaculture facilities to prevent the spread of these pathogens. Thus, the development of new, ecologically acceptable methods for their control is urgently needed. The aim of this study was to examine whether propolis, known for its antimicrobial properties and stimulatory effect on the host immune system, can inhibit the life stages of A. astaci and S. parasitica in vitro. Materials and methods Two propolis formulations were used: P1 (pure propolis in ethanol), and P2 (propolis in ethanol with the addition of sage and pepper mint). Their chemical composition was determined by ultra- performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) and gas chromatography/mass spectrometry (GC-MS). Two main life stages of the oomycete pathogens, namely mycelium and zoospores, were treated with propolis formulations and their main components, pinocembrin and chrysin. The effect of these compounds on mycelial growth was assessed using the disk diffusion assay and radial growth inhibition test, followed by determination of EC50 values. To test the inhibition of zoospore germination, sporulation was induced by washing the mycelium grown in liquid PG1 with natural water. Test compounds were added to the resulting zoospore suspension in a range of concentrations, and their effect was assessed by comparing the percentage of germinated zoospores with the results of control experiments. Malachite green, with known toxicity towards A. astaci and S. parasitica, was used as a positive control. Results Both propolis samples were rich in volatile and polyphenol compounds. As shown by UPLC-MS/MS analysis, chrysin was most abundant in both propolis samples (up to app. 50 µg/mL), followed by pinocembrin in P2 (4 µg/mL). Significantly higher number of different volatile components was found by GC-MS in P2 than in P1 propolis sample, probably because of addition of sage and pepper mint. In case of S. parasitica, both propolis formulations moderately inhibited the mycelial growth: EC50 (P1) = 206.2 µg/mL, EC50 (P2) = 206.6 µg/mL for P2, when compared with a known inhibitory compound malachite green (EC50 = 0.1 µg/mL). In contrast, EC50 values for P1, P2, and malachite green for mycelial growth inhibition of A. astaci were up to 36 times lower (5.6, 8.6, and 0.02 µg/mL, respectively). Considering zoospore germination, the minimal concentration that caused complete inhibition of S. parasitica germination (minimum inhibitory concentration, MIC) was 62.0 and 39.0 µg/mL for P1 and P2 propolis samples, respectively, while for malachite green it was 0.08 µg/mL. Similar results were obtained for Aphanomyces astaci zoospores: MIC was 31.3 and 39.0 µg/mL for P1 and P2, respectively, while malachite green inhibited zoospore germination at a concentration of 0.04 µg/mL. The inhibitory potential of main propolis components, chrysin and pinocembrin, was also tested but these compounds didn´t show significant anti-oomycete activity when applied in concentrations determined in propolis samples. This suggests that the observed anti-oomycete activity of propolis formulations was probably due to synergistic activity of a number of minor bioactive components. Conclusions Our results demonstrate the inhibitory activity of propolis towards life stages of pathogenic oomycetes S. parasitica and A. astaci. Interestingly, S. parasitica mycelium was relatively resistant to propolis in comparison to mycelium of A. astaci, but zoospores of both species (as main infection agents) were highly susceptible. Future in vivo testing is needed to explore the suggested host- protective effects of propolis during the infection process and to demonstrate its applicability as a feed additive in the fish farms.

propolis, Aphanomyces astaci, Saprolegnia parasitica, antyoomicete activity, bioactive compounds

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