## 3 Discussion  
ABp5 was previously isolated [18] as an endophyte from the seaweed _B. plumosa_. ABp5 demonstrated inhibitory activity against the fish pathogen _S. parasitic_ in in vitro assays. We therefore decided to isolate and identify the secondary active molecules that it secretes. Because of the endophyte's activity in antibiosis petri-plate assays, we initially assumed that the active compounds are secreted into the growth medium. However, our attempts to additionally extract active crude from liquid growth medium were unsuccessful. Yet, when we re-cultured ABp5 from this liquid growth medium on solid agar medium, it was once again found to be active against _S. parasitic_. Therefore, we assumed that the active metabolites are not secreted by the bacteria but are emitted as active volatile compounds. To test this assumption, we conducted an assay designed to evaluate the activity of volatile  
Figure 5: Effect of 8-nonenoic acid on _Saprolegnia parasitic_ pathogenicity on tilapia eggs. Percent survival of tilapia eggs 5 days post-inoculation with _S. parasitic_ 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. parasitic_ + 8-None 5 mg/L: eggs inoculated with _S. parasitic_ and 5 mg/L 8-nonenoic acid; _S. parasitic_: eggs with _S. parasitic_ 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 \(p\leq 0.05\). Figure 6: Effect of 8-nonenoic acid on tilapia eggs/larvae inoculated with _Saprolegnia parasitic_, 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. parasitic_: 77% developed and hatched normally, dead eggs did not develop fungal-like growth. (**D**) Eggs inoculated with _S. parasitic_: 22.5% survived, infected eggs developed fungal-like growth. metabolites by using a two-compartment petri plate. Our results demonstrated inhibition of _S. parasitica_ by the volatiles of ABp5 in this assay. To identify the VOCs emitted by ABp5, we used SPME. The fiber was incubated with the growing endophyte prior to GC-MS analysis. The GC-MS analysis suggested 10 low-molecular-weight (151-256) molecules from different families of secondary metabolites (Table 1). We purchased nine of them; the volatile compound oxime-, methoxyphenyl- was not available for purchase, but a review of the literature showed that this volatile is known for its anti-viral, anti-bacterial and anti-fungal bioactivity [27; 28]. The other nine metabolites were tested in in vitro assays for their inhibitory activity against the aquaculture pathogen _S. parasitica_. Of these, only 8-nonenoic acid exhibited meaningful biological activity against _S. parasitica_. 8-Nonenoic acid is a medium-chain fatty acid with the molecular formula C\({}_{9}\)H\({}_{16}\)O\({}_{2}\). It is mainly a starting or intermediate material for reactions found in nature [29; 30] or in the industry [31; 32]. However, it is found as an end product in substances secreted by different animals as a defense mechanism or for behavioral purposes [33; 34]. It is also found in extracts of different plants and is considered to have antimicrobial properties [35; 36]. Although identification of the active bacterial compound from the NIST library was 8-nonenoic acid, we confirmed its identity by two more methods: biological and chemical. The biological method consisted of an antibiosis activity test of the standard against _S. parasitica_ (Figure 2). The chemical method consisted of exposing SPME fiber to the 8-nonenoic acid standard and the endophyte under the same conditions and analyzing it by GC-MS.