Kocuria flava, a Bacterial Endophyte of the Marine Macroalga Bryopsis plumosa, Emits 8-Nonenoic Acid Which Inhibits the Aquaculture Pathogen Saprolegnia parasitica

Abstract

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.

Keywords: 8-nonenoic acid; aquaculture; disease management; endophyte; macroalga; secondary metabolite.

Figure 1

In vitro bioactivity assays of K. flava isolate ABp5 against the aquaculture pathogen Saprolegnia parasitica. ABp5 was grown in the middle of an NPDA plate for a week (A + B, right plates) before a plug of solid medium harboring live S. parasitica mycelia was added to the plates. Control plates were inoculated with S. parasitica in the absence of ABp5 (A + B, left plates). (A) A two-compartment petri plate used for the bioactive volatile emission assay. The divider prevents any direct contact of ABp5 and its secreted secondary metabolites with the pathogen, allowing only volatile movement in the headspace between the compartments. (B) One-compartment petri plate. In this assay, ABp5 can inhibit the pathogen through both direct and indirect contact with secondary metabolites (secreted or emitted through the agar or the headspace, respectively). In both panels A and B, S. parasitica was fully inhibited by ABp5. (C) Pathogen viability assay. S. parasitica from both assays (direct and headspace exposure) were transferred to a plate with new medium plate in the absence of ABp5. There was no growth of pathogens from either assay (shown for the headspace experiment only); a plug from the control plate was viable and grew as expected (right side of the plate).

Figure 2

8-nonenoic acid inhibitory activity against Saprolegnia parasitica in agar plates exam. Inhibition was recorded after seven days. S. parasitica plugs, 100 μg of 8-nonenoic acid (left disk) and ethanol (right disk) as a control. Inhibition is expressed as avoidance of growth towards the paper disk loaded with 8-nonenoic acid while the Saprolegnia mycelium grows with no disturbance towards the ethanol loaded discs.

Figure 3

Identification of 8-nonenoic acid. (A) Comparative analysis of GC–MS retention times (RT) of ABp5 and authentic standard. 8-Nonenoic acid standard peak (light orange) with RT of ~14 min post-injection and the ABp5 peaks (red), released from the SPME, showing one peak at ~14 min post-injection with similar features (shape and slope) as the authentic standard. (B) Fractionation comparing the peak identified as 8-nonenoic acid secreted by ABp5 (red-line columns above axis) and the peak produced by the purchased authentic standard (blue-line columns below axis). The fractionations given by the GC–MS are identical for both.

Figure 4

Bioactivity of 8-nonenoic acid against Saprolegnia parasitica. Different concentrations of 8-nonenoic acid were added to solid (PDA) and liquid (DDW and PDB) media. Results present the percentage of maximal (control) growth of S. parasitica at the different concentrations. The results were subjected to ANOVA followed by Tukey–Kramer multiple comparison test; different letters above points on curve indicate a significant difference between solid and liquid media at p ≤ 0.05.

Figure 5

Effect of 8-nonenoic acid on Saprolegnia parasitica pathogenicity on tilapia eggs. Percent survival of tilapia eggs 5 days post-inoculation with S. parasitica in the presence or absence of 8-nonenoic acid is presented as a column graph. Control: eggs with ethanol only; 8-nonenoic acid 5 mg/L: eggs with 8-nonenoic acid only at a concentration of 5 mg/L; S. parasitica + 8-None 5 mg/L: eggs inoculated with S. parasitica and 5 mg/L 8-nonenoic acid; S. parasitica: eggs with S. parasitica only. Results were subjected to ANOVA followed by Tukey–Kramer multiple comparison test; different letters above the bars indicate a significant difference between treatments at p ≤ 0.05.

Figure 6

Effect of 8-nonenoic acid on tilapia eggs/larvae inoculated with Saprolegnia parasitica, evaluated 5 days post-inoculation. (A) Eggs with ethanol only: all of the larvae developed and hatched normally. (B) Eggs with 5 mg/L 8-nonenoic acid: 98% of the larvae developed and hatched normally. (C) Eggs with 5 mg/L 8-nonenoic acid inoculated with S. parasitica: 77% developed and hatched normally, dead eggs did not develop fungal-like growth. (D) Eggs inoculated with S. parasitica: 22.5% survived, infected eggs developed fungal-like growth.