In Vitro Activity of Essential Oils against Saprolegnia parasitica.

Saprolegnia spp. water molds severely impact fish health in aquaculture, fish farms and hobby fish tanks colonizing mature and immature stages of fishes, as well as eggs. Considering that there are no drugs licensed for treating and/or control the organism, efficient and environmental low-impact methods to control these oomycetes in aquaculture are needed. The aim of the present report was to evaluate the in vitro sensitivity of Saprolegnia parasitica to essential oils (EOs) from Citrus aurantium L., Citrus bergamia Risso et Poiteau, Citrus limon Burm. f., Citrus paradisi Macfad, Citrussinensis Osbeck, Cinnamomum zeylanicum Blume, Cymbopogon flexuosum (Nees ex Steud.) Watson, Foeniculum vulgare Mill., Illicium verum Hook.f., Litsea cubeba (Lour.) Pers., Origanum majorana L., Origanum vulgare L., Pelargonium graveolens L'Hér., Syzygium aromaticum Merr. & L.M.Perry, and Thymus vulgaris L., by microdilution test. The most effective EOs assayed were T. vulgaris and O. vulgare, followed by C. flexuosum, L. cubeba and C. bergamia. These EOs could be of interest for controlling Saprolegnia infections. Nevertheless, further safety studies are necessary to evaluate if these products could be dispersed in tank waters, or if their use should be limited to aquaculture supplies.

In vitro activity of chemicals and commercial products against Saprolegnia parasitica and Saprolegnia delica strains.

Oomycetes of the genus Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Following the ban of malachite green in food fish production, the demand for new treatments pushes towards the selection of more safe and environment-friendly products. In the present work, in vitro activity of ten chemicals and three commercial products was tested on different strains of Saprolegnia, using malachite green as reference compound. The compounds were screened in agar and in water to assess the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), respectively. Two strains of Saprolegnia parasitica and one isolate of Saprolegnia delica were tested in triplicate per each concentration. Among tested chemicals, benzoic acid showed the lowest MIC (100 ppm) followed by acetic acid, iodoacetic acid and copper sulphate (250 ppm). Sodium percarbonate was not effective at any tested concentration. Among commercial products, Virkon™ S was effective in inhibiting the growth of the mycelium (MIC = MLC = 1,000 ppm). Actidrox® and Detarox® AP showed MIC = 5,000 and 1,000 ppm, respectively, while MLCs were 10-fold lower than MICs, possibly due to a higher activity of these products in water. Similarly, a higher effectiveness in water was observed also for iodoacetic acid.

Elucidating the Diversity of Aquatic Microdochium and Trichoderma Species and Their Activity against the Fish Pathogen Saprolegnia diclina.

Animals and plants are increasingly threatened by emerging fungal and oomycete diseases. Amongst oomycetes, Saprolegnia species cause population declines in aquatic animals, especially fish and amphibians, resulting in significant perturbation in biodiversity, ecological balance and food security. Due to the prohibition of several chemical control agents, novel sustainable measures are required to control Saprolegnia infections in aquaculture. Previously, fungal community analysis by terminal restriction fragment length polymorphism (T-RFLP) revealed that the Ascomycota, specifically the genus Microdochium, was an abundant fungal phylum associated with salmon eggs from a commercial fish farm. Here, phylogenetic analyses showed that most fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride species. Phylogenetic and quantitative PCR analyses showed both a quantitative and qualitative difference in Trichoderma population between diseased and healthy salmon eggs, which was not the case for the Microdochium population. In vitro antagonistic activity of the fungi against Saprolegnia diclina was isolate-dependent; for most Trichoderma isolates, the typical mycoparasitic coiling around and/or formation of papilla-like structures on S. diclina hyphae were observed. These results suggest that among the fungal community associated with salmon eggs, Trichoderma species may play a role in Saprolegnia suppression in aquaculture.

Inhibition of dioscin on Saprolegnia in vitro.

As one of the most serious pathogens in the freshwater aquatic environment, Saprolegnia can induce a high mortality rate during the fish egg incubation period. This study investigated the anti-Saprolegnia activity of a total of 108 plants on Saprolegnia parasitica in vitro and Dioscorea collettii was selected for further studies. By loading on an open silica gel column and eluting with petroleum ether-ethyl acetate-methanol, dioscin (C45H72O16) was isolated from D. collettii. Saprolegnia parasitica growth was inhibited significantly when dioscin concentration was more than 2.0 mg L(-1). When compared with formalin and hydrogen peroxide, dioscin showed a higher inhibitory effect. As potential inhibition mechanisms, dioscin could cause the S. parasitica mycelium morphologic damage, dense folds, or disheveled protuberances observed by field emission scanning electron microscopy and the influx of Propidium iodide. The structural changes in the treated mycelium were indicative of an efficient anti-Saprolegnia activity of dioscin. The oxidative stress results showed that dioscin also accumulated reactive oxygen species excessively and increased total antioxidant and superoxide dismutase activity. These situations could render S. parasitica more vulnerable to oxidative damage. Additionally, when dioscin concentration was less than 2.0 mg L(-1), the survival rate of embryos was more than 70%. Therefore, the use of dioscin could be a viable way of preventing and controlling saprolegniasis.

Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin.

The mucosal surfaces of wild and farmed aquatic vertebrates face the threat of many aquatic pathogens, including fungi. These surfaces are colonized by diverse symbiotic bacterial communities that may contribute to fight infection. Whereas the gut microbiome of teleosts has been extensively studied using pyrosequencing, this tool has rarely been employed to study the compositions of the bacterial communities present on other teleost mucosal surfaces. Here we provide a topographical map of the mucosal microbiome of an aquatic vertebrate, the rainbow trout (Oncorhynchus mykiss). Using 16S rRNA pyrosequencing, we revealed novel bacterial diversity at each of the five body sites sampled and showed that body site is a strong predictor of community composition. The skin exhibited the highest diversity, followed by the olfactory organ, gills, and gut. Flectobacillus was highly represented within skin and gill communities. Principal coordinate analysis and plots revealed clustering of external sites apart from internal sites. A highly diverse community was present within the epithelium, as demonstrated by confocal microscopy and pyrosequencing. Using in vitro assays, we demonstrated that two Arthrobacter sp. skin isolates, a Psychrobacter sp. strain, and a combined skin aerobic bacterial sample inhibit the growth of Saprolegnia australis and Mucor hiemalis, two important aquatic fungal pathogens. These results underscore the importance of symbiotic bacterial communities of fish and their potential role for the control of aquatic fungal diseases.

Evaluation of the antifungal activity of Zataria multiflora, Geranium herbarium, and Eucalyptus camaldolensis essential oils on Saprolegnia parasitica-infected rainbow trout (Oncorhynchus mykiss) eggs.

The purpose of the present study was to evaluate and assess the capability of Zataria multiflora, Geranium herbarium, and Eucalyptus camaldolensis essential oils in treating Saprolegnia parasitica-infected rainbow (Oncorhynchus mykiss) trout eggs. A total of 150 infected eggs were collected and plated on glucose-pepton agar at 24°C for 2 weeks. The antifungal assay of essential oils against S. parasitica was determined by a macrodilution broth technique. The eggs were treated with essential oils at concentrations of 1, 5, 10, 25, 50, and 100 ppm daily with three repetitions until the eyed eggs stage. Of 150 eggs examined, S. parasitica (54.3%), Saprolegnia spp. (45%), and Fusarium solani (0.7%) were isolated. The minimum inhibitory concentrations of Z. multiflora, E. camaldolensis, and G. herbarium essential oils against S. parasitica were 0.9, 2.3, and 4.8 ppm, respectively. Zataria multiflora and E. camaldolensis at concentrations of 25, 50, and 100 ppm, and G. herbarium at concentration of 100 ppm had significant differences in comparison with negative control (p<0.05). The results revealed that malachite green, followed by Z. multiflora, E. camaldolensis, and G. herbarium treated eggs had remained the most number of final eyed eggs after treatment. The highest final larvae rates belonged to malachite green, E. camaldolensis, Z. multiflora, and G. herbarium, respectively. The most hatching rates were recorded with malachite green (22%), and then Z. multiflora (11%), E. camaldolensis (7%), G. herbarium (3%), and negative control (1%). Zataria multiflora and E. camaldolensis were more effective than G. herbarium for the treatment of S. parasitica-infected rainbow trout eggs in aquaculture environment.

Microwell enumeration of viable Saprolegniaceae in water samples.

Existing methods for enumeration of viable Saprolegniaceae propagules in water are laborious, time consuming and prevent examination of large numbers of samples or samples with high spore loads. In the present study a microwell plate (MWP) assay for estimation of Saprolegniaceae in water samples, modified from Hagen (1992), was evaluated. The ability of the assay to recover Saprolegniaceae was assessed by applying it to spore suspensions with four predetermined concentrations, 500-10,000 spores per liter of samples tested. The method also was used to analyze a set of field samples and compare it to a standard filtration method to ascertain its practicability. The MWP assay underestimated the number of spores in the test suspensions with predetermined concentrations. The accuracy of the assay varied with spore concentration, giving the lowest recovery (62.5%) at low spore numbers and the highest (86%) at intermediate concentrations (1000-5000 spores/L) for both isolates and growth media. The findings indicate that spores aggregate with increasing concentration. When applied to field samples the assay clearly distinguished among samples with presumptive differences in spore load and yielded significantly higher counts than the filtration method. The results justify the MWP method foruse in estimation of Saprolegniaceae in water bodies particularly relevant for monitoring of spore load in aquaculture as well as in ecological studies.

Reduction in vegetative growth of the water mold Saprolegnia parasitica (Coker) by humic substance of different qualities.

Humic substances (HS) account for 50-80% of the dissolved organic matter in non-eutrophicated freshwater ecosystems. HS are not inert, but are taken up by and may interact with aquatic organisms. However, at present no information is available on the interaction of HS with fungi, for instance, the fish-pathogenous species Saprolegnia parasitica. To fill this gap, we tested effects of HS on S. parasitica growth in-vitro using 25-500mgL(-1) carbon of HS on GY-agar. We investigated 20 HS including natural organic matter (NOM) samples, two lignite derived HS, and one synthetic HS. The aim was (1) to find out, if there are inhibiting effects and (2) if potential effects can be explained by humic matter properties by structure activity relationships. The growth of S. parasitica was related to the growth on HS-free agar controls. Characterization of HS and NOMs included elemental analysis, high-pressure size exclusion chromatography (HPSEC), UV/VIS, FTIR-, and EPR-spectroscopy in order to obtain information on elemental and structural composition including various metals, molecular weights of the HS fraction, aromaticity, free organic radicals, and functional groups. NOMs with high moieties of high-molecular carbohydrates supported the growth of S. parasitica, all other HS and NOMs reduced it. However, no inhibition of the development of the sporangia and primary zoospores was found. Therefore, the impact of the HS on S. parasitica has to be classified as fungistatic, rather than fungicidal. Synthetic and lignite-derived HS were among the most efficient HS sources. Growth inhibition was correlated (p<0.05) with the molecular weights of the HS-fraction, sUVa, COOH groups, C and H. Our results suggest that especially HS with higher molecular weights and aromaticity which contain a high number of organic radicals are the most efficient in reducing fungal growth. Furthermore, highly functionalized HS seem to be important for the observed effect. The development of internal oxidative stress could be a mechanism explaining the observed growth inhibition of S. parasitica.

Saprolegnia bulbosa sp. nov. isolated from an Argentine stream: taxonomy and comparison with related species.

Saprolegnia bulbosa sp. nov. was isolated from floating and decaying twigs and leaves in El Gato stream, Partido de La Plata, Buenos Aires Province, Argentina. The distinctive characteristics of S. bulbosa are the product of smooth oogonia and predominantly contorted monoclinous, androgynous and diclinous antheridia. The oogonial stalks are usually bent, curved or once coiled; oospores are subcentric, (1) 2-15 (45) per oogonium and are variable in size. Taxonomical description of this new species, its comparison with related oomycetes of the genus and the nucleotide sequences of the internal transcribed region (spacers ITS1, ITS2 and the gene 5.8S) of its rRNA gene are given here.

Morphological and biochemical alterations of oomycete fish pathogen Saprolegnia parasitica as affected by salinity, ascorbic acid and their synergistic action.

Vegetative growth of Saprolegnia parasitica decreased by increasing the concentration of NaCl and ascorbic acid. Under these conditions, the morphological features of the vegetative hyphae were distinguishable from those used as controls. NaCl and ascorbic acid in combination improved the tolerance of S. parasitica to high levels of salinity. Sporangial formation, release and proliferation were very sensitive to even lower levels of salinity. For instance, at 0.03 M NaCl sporangia formation was rarely observed. Ascorbic acid alone had a little effect on sporangial formation and release, but when combine with NaCl the developmental processes were improved. Reduction of numbers and plasmolysis of oogonia were found at various NaCl concentrations, whereas ascorbic acid stimulated the formation of these reproductive organs at low concentrations. The synergistic effect of NaCl and ascorbic acid improved and overcomed the symptoms of oogonial plasmolysis. Protease activity of S. parasitica was significantly reduced at all NaCl concentrations, whilst ascorbic acid significantly increased and inhibited it at low concentrations and at moderate and high concentrations, respectively. The combination of these compounds reduced protease activity at all tested concentrations with significant difference at the highest concentration. The total free amino-acids content of S. parasitica mycelia was significantly reduced at all the NaCl concentrations, whereas ascorbic acid significantly increased it at low but inhibited it at higher concentrations. The combination of NaCl and ascorbic acid significantly increased the accumulation of free amino-acids at low and moderate concentrations, but decreased them at high concentrations. Total protein content was reduced at all tested concentrations of NaCl and ascorbic acid had also similar effect. However, the combined effect of NaCl and ascorbic acid significantly enhanced and reduced total protein content at low and high concentrations, respectively. Treatments with NaCl induced proline accumulation in S. parasitica, which paralleled the salt concentration.

Time series analysis demonstrates the absence of pulsatile hyphal growth.

Hyphal tip growth has been previously reported as pulsatile, defined as regularly alternating fast and slow rates of extension. The growth of pollen tubes, and hyphae of Neurospora crassa and Saprolegnia ferax were analysed using high spatial and temporal resolution. By using long (100-500 s) records of growth rate, sampled every second, it was possible to apply rigorous statistical analysis of the time series. As previously demonstrated, pollen tubes can show pulsatile growth, detectable with this system. In contrast, hyphal growth rates do not show any evidence of pulsatile growth; instead, growth rates appear to fluctuate randomly. It is concluded that pulsatile growth is not a common feature of hyphal tip growth.