Search Results (33)

Chestnut blight, caused by Cryphonectria parasitica (Murrill) M.E. Bar, is a destructive fungal disease threatening chestnut cultivation and production. In response to the limitations of chemical control, biological control using antagonistic microbes has gained increasing attention. A rhizosphere-derived bacterium, strain D39, was isolated from healthy chestnut trees and identified as Bacillus amyloliquefaciens based on morphological characteristics and the phylogenetic analysis of 16S rRNA and gyrA genes. The antifungal activity of strain D39 against C. parasitica was evaluated using dual-culture and double-layer Oxford cup assays. The strain exhibited broad-spectrum and stable antagonistic effects and harbored five key genes associated with antimicrobial compound biosynthesis (srfAA, ituC, fenD, bmyB, and bacA), as confirmed by PCR. A 145 kDa extracellular protein with strong antifungal activity was extracted and purified by ammonium sulfate precipitation, DEAE ion-exchange chromatography, and Sephadex G-75 gel filtration. LC-MS analysis identified the protein as a serine peptidase belonging to the S8 family, and its structure was predicted using multiple bioinformatic tools. In pot experiments, treatment with the strain D39 significantly reduced disease severity, achieving control efficiencies of 66.07% and 70.89% at 10 and 20 days post-inoculation, respectively. These results demonstrate that B. amyloliquefaciens D39 has strong potential as a biocontrol agent against chestnut blight, offering an effective and environmentally friendly alternative for disease management. Full article

Potassium (K) fertilization is crucial for plant resistance to pathogens, but the underlying mechanisms remain unclear. Here, we investigate the molecular mechanism by which the addition of K promotes resistance in Nicotiana benthamiana to Phytophthora parasitica. We found that N. benthamiana with high K content (HK, 52.3 g/kg) produced more ethylene in response to P. parasitica infection, compared to N. benthamiana with low-K content (LK, 22.4 g/kg). An exogenous ethylene application effectively increased resistance in LK N. benthamiana to the level under HK status, demonstrating the involvement of ethylene in the HK-associated resistance in N. benthamiana. Further, transcriptome analysis showed that NbSAMS1, encoding ethylene biosynthesis, was induced to upregulate P. parasitica about five times higher in HK than in LK N. benthamiana. NbSAMS1 overexpression enhanced resistance in LK plants, whereas NbSAMS1 silencing reduced resistance in HK plants, confirming its importance in conferring resistance. Furthermore, we identified a calcium-binding protein, NbCBP1, which interacted with NbSAMS1, promoting its expression in HK N. benthamiana. Silencing NbCBP1 compromised resistance in HK N. benthamiana, whereas its overexpression improved resistance in LK N. benthamiana. Notably, NbCBP1 protected NbSAMS1 from degradation by the 26S proteasome, thereby sustaining ethylene accumulation in HK N. benthamiana in response to P. parasitica infection. Thus, our research elucidated some mechanisms of the NbCBP1-NbSAMS1 module associated with disease resistance in HK N. benthamiana. Full article

We have investigated the changes in the microbial communities on the surface of trout eggs and the skin of adult trout in relation to the presence of Saprolegnia parasitica. This pathogen causes saprolegniosis, a disease responsible for significant losses in salmonid farms and hatcheries. It is known from other disease systems that the host-associated microbiome plays a crucial role in the defence against pathogens, but if the pathogen predominates, this can lead to dysbiosis. However, analyses of the effects of S. parasitica on the diversity, composition, and function of microbial communities on fish skin and eggs are scarce. Thus, we have collected skin swabs from injured and healthy trout (N = 12), which differed in S. parasitica load, from three different fish farms in Croatia (Kostanjevac, Radovan, and Solin), while trout egg samples (N = 12) were infected with S. parasitica in the laboratory. Illumina sequencing of the V4 region of the 16S rRNA marker gene showed that infection with S. parasitica reduced the microbial diversity on the surface of the eggs, as evidenced by decreased Pielou’s evenness and Shannon’s indices. We further determined whether the bacterial genera with a relative abundance of >5.0% in the egg/skin samples were present at significantly different abundances in relation to the presence of S. parasitica. The results have shown that some genera, such as Pseudomonas and Flavobacterium, decreased significantly in the presence of the pathogen on the egg surface. On the other hand, some bacterial taxa, such as Acinetobacter and Janthinobacterium, as well as Aeromonas, were more abundant on the diseased eggs and the injured trout skin, respectively. Finally, beta diversity analyses (weighted UniFrac, unweighted UniFrac, Bray–Curtis) have shown that the sampling location (i.e., fish farm), along with S. parasitica infection status, also has a significant influence on the microbial communities’ composition on the trout skin and eggs, demonstrating the strong influence of the environment on the shaping of the host surface microbiome. Overall, we have shown that the presence of S. parasitica was associated with changes in the diversity and structure of the trout skin/egg microbiome. The results obtained could support the development of new strategies for the management of saprolegniosis in aquaculture. Full article

In this study, using Illumina sequencing, we sequenced first the complete mitochondrial genome (mitogenome) of two deep-sea eels, Histiobranchus bathybius and Simenchelys parasitica, collected from the East Mariana Basin in the Western Pacific Ocean. The complete length of the H. bathybius and S. parasitica mitogenomes were 16,696 and 16,687 bp, respectively, each containing 37 genes (13 protein-coding genes, 22 tRNA genes, and 2 ribosomal RNA genes). To enhance the accuracy of the identification of H. bathybius and S. parasitica, we performed a phylogenetic analysis of multiple deep-sea eels based on the mitochondrial DNA gene (cytochrome c oxidase subunit I [COI]) using the maximum likelihood method. Our phylogenetic tree analysis confirmed that the specimens collected in this study are congeneric species of H. bathybius and S. parasitica reported in previous studies. Based on these results, we report the first complete mitogenomes of H. bathybius and S. parasitica and a new record for the two species in the East Mariana Basin. Full article

Saprolegnia sp. is a pathogenic oomycete responsible for severe economic losses in aquaculture. To date, there is no treatment for its control that is effective and does not pose a threat to the environment and human health. In this research, two dihydrochalcones 1 and 2, and three chalcones 3–5, isolated from the resinous plant Adesmia balsamica, as well as their synthesized oxyalkylated derivatives 6–29 already reported and a new synthesized series of oxyalkylchalcones 30–35, were evaluated for their anti-saprolegnia activity and structure–activity relationship as potential control and treatment agents for strains of Saprolegnia parasitica and S. australis. Among the molecules tested, natural 2′,4′-dihydroxychalcone (3) and new oxyalkylchalcone 34 were the most potent antisaprolegnia agents against both strains, even with better results than the commercial control bronopol. On the other hand, the structure–activity relationship study indicates that the contributions of steric and electrostatic fields are important to enhance the activity of the compounds, thus the presence of bulky substituents favors the activity. Full article

This study is the first report of parasite and fungal disease separated from domestically reared Atlantic salmon in Korea; the characteristics of the pathogens were identified, and histopathological analysis was conducted. Fungal and parasitic diseases were detected in Atlantic salmon and were isolated as Saprolegnia parasitica and Neoparamoeba perurans based on morphological and genetic analysis. External symptoms observed in Atlantic salmon infected with S. parasitica include fin ulcers and hemorrhage, abdominal hemorrhage, and necrosis of the gills and gill covers. The histopathological analysis results showed necrosis, hemorrhaging, and inflammatory cell infiltration in the abdominal muscles, while only inflammatory cell infiltration was observed in the gill covers. The clinical symptoms observed in Atlantic salmon infected with N. perurans included excessive mucus secretion in the gills, a dense amoebic presence on the gill filaments, respiratory distress, and opening of the mouth and gill covers after death. Through histopathological analysis, we observed lesions in epithelial cells, characterized by the proliferation of epithelial cells and the fusion of secondary lamellae. Numerous lamellae were observed to be attached or fused with each other. To ensure the successful establishment of the Atlantic salmon aquaculture industry in Korea, it is essential to swiftly quarantine infected fish based on the morphological characteristics of S. parasitica and N. perurans revealed in this study, along with the external symptoms of Atlantic salmon infected with these pathogens. Developing disease control strategies based on the findings of this research is imperative. Full article

The natural pesticide phenazine-1-carboxylic acid (PCA) is known to lack phloem mobility, whereas Metalaxyl is a representative phloem systemic fungicide. In order to endow PCA with phloem mobility and also enhance its antifungal activity, thirty-two phenazine-1-carboxylic acid-N-phenylalanine esters conjugates were designed and synthesized by conjugating PCA with the active structure N-acylalanine methyl ester of Metalaxyl. All target compounds were characterized by ¹H NMR, ¹³C NMR and HRMS. The antifungal evaluation results revealed that several target compounds exhibited moderate to potent antifungal activities against Sclerotinia sclerotiorum, Bipolaris sorokiniana, Phytophthora parasitica, Phytophthora citrophthora. In particular, compound F7 displayed excellent antifungal activity against S. sclerotiorum with an EC₅₀ value of 6.57 µg/mL, which was superior to that of Metalaxyl. Phloem mobility study in castor bean system indicated good phloem mobility for the target compounds F1–F16. Particularly, compound F2 exhibited excellent phloem mobility; the content of compound F2 in the phloem sap of castor bean was 19.12 μmol/L, which was six times higher than Metalaxyl (3.56 μmol/L). The phloem mobility tests under different pH culture solutions verified the phloem translocation of compounds related to the “ion trap” effect. The distribution of the compound F2 in tobacco plants further suggested its ambimobility in the phloem, exhibiting directional accumulation towards the apical growth point and the root. These results provide valuable insights for developing phloem mobility fungicides mediated by exogenous compounds. Full article

Saprolegniosis is a major destructive disease in freshwater aquaculture. The destructive economic impact of saprolegniosis on freshwater aquaculture necessitates further study on the range of Saprolegnia species within Atlantic salmon fish farms. This study undertook a thorough analysis of a total of 412 oomycete and fungal isolates that were successfully cultured and sequenced from 14 aquaculture sites in Scotland across a two-year sampling period. An ITS phylogenetic analysis of all isolates was performed according to whether they were isolated from fish or water samples and during enzootic or epizootic periods. Several genera of oomycetes were isolated from sampling sites, including Achlya, Leptolegnia, Phytophthora, and Pythium, but by far the most prevalent was Saprolegnia, accounting for 66% of all oomycetes isolated. An analysis of the ITS region of Saprolegnia parasitica showed five distinct phylotypes (S2–S6); S1 was not isolated from any site. Phylotype S2 was the most common and most widely distributed phylotype, being found at 12 of the 14 sampling sites. S2 was overwhelmingly sampled from fish (93.5%) and made up 91.1% of all S. parasitica phylotypes sampled during epizootics, as well as 67.2% of all Saprolegnia. This study indicates that a single phylotype may be responsible for Saprolegnia outbreaks in Atlantic salmon fish farms, and that water sampling and spore counts alone may be insufficient to predict Saprolegnia outbreaks in freshwater aquaculture. Full article

Saprolegnia parasitica Coker, 1923 is a primary fish pathogen and one of the most common water molds in freshwater ecosystems. In our study, nineteen strains of S. parasitica were isolated, identified, and characterized using morphological and genetic markers. On the basis of the abundance of zoosporangia, gemmae, the formation of gemma chains, and the induction of zoospore release, three morphotypes were differentiated. A species-level molecular identification of isolates was performed using the ITS 1 and 2 regions. A total of six genotypes were distinguished based on partial DNA sequences of the genes RNA polymerase II subunit B (RPB2) and serine hydroxymethyltransferase (SHMT). In five settings of in vitro culture conditions differing in the mineral content and the temperature of water and in the presence of a host or bait, we found that the addition of fish skin extract boosted the formation of asexual reproductive and persistent vegetative structures in cultures, whereas an unfavorable environment did not support the formation of these structures in vitro. Full article

Secondary metabolites—organic compounds that are often bioactive—produced by endophytes, among others, provide a selective advantage by increasing the organism’s survivability. Secondary metabolites mediate the symbiotic relationship between endophytes and their host, potentially providing the host with tolerance to, and protection against biotic and abiotic stressors. Secondary metabolites can be secreted as a dissolved substance or emitted as a volatile. In a previous study, we isolated bioactive endophytes from several macroalgae and tested them in vitro for their ability to inhibit major disease-causing pathogens of aquatic animals in the aquaculture industry. One endophyte (isolate Abp5, K. flava) inhibited and killed, in vitro, the pathogen Saprolegnia parasitica, an oomycete that causes saprolegniasis—a disease affecting a wide range of aquatic animals. Here, using analytical chemistry tools, we found that Abp5 produces the volatile organic compound (VOC) 8-nonenoic acid. Once we confirmed the production of this compound by the endophyte, we tested the compound’s ability to treat S. parasitica in in vitro and in vivo bioassays. In the latter, we found that 5 mg/L of the compound improves the survival of larvae challenged with S. parasitica by 54.5%. Our isolation and characterization of the VOC emitted by the endophytic K. flava establish the groundwork for future studies of endophytic biocontrol agents from macroalgae. Use of this compound could enable managing oomycete agricultural pathogens in general, and S. parasitica in particular, a major causal agent in aquaculture diseases. Full article

Previous studies have demonstrated that the strains Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 are probiotics against vibriosis or lactococosis in sea bass or rainbow trout. In this study, the utility of these bacterial strains in the control of saprolegniosis was evaluated. For this purpose, both in vitro inhibition studies and competition for binding sites against Saprolegnia parasitica and in vivo tests with experimentally infected rainbow trout were carried out. In the in vitro tests, the three isolates showed inhibitory activity upon mycelium growth and cyst germination and reduced the adhesion of cysts to cutaneous mucus; however, this effect depended on the number of bacteria used and the incubation time. In the in vivo test, the bacteria were administered orally at 10⁸ CFU g⁻¹ in the feed or at 10⁶ CFU ml⁻¹ in the tank water for 14 days. None of the three bacteria showed protection against S. parasitica infection either through water or feed, and the cumulative mortality reached 100% within 14 days post infection. The obtained results show that the use of an effective probiotic against a certain disease in a host may not be effective against another pathogen or in another host and that the results obtained in vitro may not always predict the effects when used in vivo. Full article

Rhamnolipids have significant emulsifying activity and the potential to become a component of pesticide emulsifier. Rhamnolipids are usually composed of two main components: mono-rhamnolipids (Rha-C₁₀-C₁₀) and di-rhamnolipids (Rha₂-C₁₀-C₁₀). The proportion of di-rhamnolipids in the products ranged between 15% and 90%, affected by the production strains and fermentation process. In this paper, three kinds of rhamnolipid products containing di-rhamnolipids proportions, of 25.45, 46.46 and 89.52%, were used to test their emulsifying ability toward three conventional solvents used in pesticide (S-200, xylene, cyclohexanone) and antifungal activities against five strains of plant pathogenic fungi (Phytophthora capsici, Phytophthora parasitica var.nicotianae, Colletotrichum destructivum, Colletotrichum sublineolum, Fusarium oxysporum). The results indicated that although the CMC of the three rhamnolipids were significantly different, their emulsification properties had no remarkable differences, at a concentration of 10 g/L. However, their antifungal activities were significantly different: the more di-rhamnolipids, the stronger the antifungal activity. This work helps to promote the application of rhamnolipids as pesticides adjuvants. Full article

Saprolegnia parasitica induces heavy mortality in aquaculture. The detection of S. parasitica is often time consuming and uncertain, making it difficult to manage the disease. We validated a previously published real-time quantitative PCR (qPCR) assay to confirm the presence of S. parasitica in fish and in water using environmental DNA (eDNA) quantification. Analytical sensitivity and specificity of the assay was assessed in silico, in vitro and the qPCR assay was compared with microbiological cultivation methods to detect and quantify S. parasitica in water samples from a controlled fish exposure experiment and from fish farms. Furthermore, we compared the use of an agar cultivation method and the qPCR assay to detect S. parasitica directly from mucus samples taken from the fish surface. The analytical sensitivity and specificity of the qPCR assay were high. The qPCR assay detected 100% of S. parasitica-positive water samples. In a field study, the qPCR assay and a microwell plate (MWP) enumeration method correlated significantly. Furthermore, the qPCR assay could be used to confirm the presence of S. parasitica in skin mucus. Thus, the qPCR assay could complement diagnostic methods in specifically detecting saprolegniosis in fish and used as a surveillance method for S. parasitica pathogen in aquaculture environments. Full article

Saprolegniasis, which is caused by Saprolegnia parasitica, leads to considerable economic losses. Recently, we showed that metalaxyl, bronopol and copper sulfate are good antimicrobial agents for aquaculture. In the current study, the efficacies of metalaxyl, bronopol and copper sulfate are evaluated by in vitro antimicrobial experiments, and the mechanism of action of these three antimicrobials on S. parasitica is explored using transcriptome technology. Finally, the potential target genes of antimicrobials on S. parasitica are identified by protein–protein interaction network analysis. Copper sulfate had the best inhibitory effect on S. parasitica, followed by bronopol. A total of 1771, 723 and 2118 DEGs upregulated and 1416, 319 and 2161 DEGs downregulated S. parasitica after three drug treatments (metalaxyl, bronopol and copper sulfate), separately. Additionally, KEGG pathway analysis also determined that there were 17, 19 and 13 significantly enriched metabolic pathways. PPI network analysis screened out three important proteins, and their corresponding genes were SPRG_08456, SPRG_03679 and SPRG_10775. Our results indicate that three antimicrobials inhibit S. parasitica growth by affecting multiple biological functions, including protein synthesis, oxidative stress, lipid metabolism and energy metabolism. Additionally, the screened key genes can be used as potential target genes of chemical antimicrobial drugs for S. parasitica. Full article

The purpose of the study was to investigate the antifungal activity of essential oils derived from the Zingiberaceae family, such as Zingiber officinale Roscoe, Alpinia officinarum. Hance, and Curcuma longa Linn., against Phytophthora parasitica Dastur, (the pathogen that causes root and stem rot diseases). In vitro antifungal activity was measured using poisoned food technologies at a final concentration of 1000 mg/L in a completely randomized design with triplications. The essential oil of A. officinarum demonstrated the highest antifungal activity against P. parasitica, according to findings (p < 0.05). Gas chromatography-mass spectrometry was also used to analyze the oil. Twenty constituents representing 99.1% of the total content were identified. Eucalyptol was the most abundant component in A. officinarum rhizome oil (52%). The oil’s half-inhibitory concentration (IC₅₀) of 432.89 mg/L had higher antifungal activity than eucalyptol (>1000 mg/L). The results suggested that A. officinarum oil should be expanded further for a new generation of fungicides as an environmentally acceptable agent and to reduce the use of chemicals in crop protection. Full article