Screening antifungal activity of macroalgae from SE Spain highlights the invader Rugulopteryx okamurae

This study demonstrates that macroalgae from southeastern Spain, particularly the invasive species *Rugulopteryx okamurae*, possess significant antioxidant and antifungal activities against *Fusarium oxysporum* TR4, supporting the potential valorization of invasive algal biomass as a source of bioactive compounds.

The Gist

Imagine the Mediterranean Sea as a bustling, crowded city. Recently, a few "uninvited guests" (invasive algae) have moved in, taking up too much space and pushing out the locals. One of the most notorious troublemakers is a brown seaweed called Rugulopteryx okamurae. Usually, scientists and environmentalists see these invaders as a problem to be cleaned up and thrown away.

But this study asks a different question: What if we stop seeing them as trash and start seeing them as treasure?

Here is the story of how the researchers turned a sea problem into a potential solution, explained simply.

1. The "Sea Garden" Cleanup Crew

The researchers went to the southeastern coast of Spain and collected five different types of seaweed. Some were native residents, but three were the "invaders" (Rugulopteryx okamurae, Batophora sp., and Codium fragile).

Think of these seaweeds as different types of plants in a garden. Some are green, some are brown, and some are red. The scientists wanted to know: If we squeeze the juice out of these plants, what kind of "superpowers" do they have?

2. The Extraction: Making "Magic Elixirs"

To get the good stuff out, the scientists didn't just boil the seaweed. They used two different "solvents" (liquids) to wash the seaweed, kind of like using different detergents to clean clothes:

• Methanol: A polar liquid (like water) that grabs onto water-loving molecules.
• Ethyl Acetate: A less polar liquid (more like oil) that grabs onto fat-loving molecules.

They turned the dried seaweed into a powder, soaked it in these liquids, and collected the resulting "elixirs."

3. The Two Superpowers Tested

The team tested these elixirs for two specific superpowers:

Superpower A: The "Rust Remover" (Antioxidant Activity)

Inside our bodies (and in plants), harmful molecules called "free radicals" act like rust on a car or rust on a metal fence. They cause damage and aging. Antioxidants are like a rust-proof coating that stops this damage.

• The Test: They used a chemical that turns purple. If the seaweed extract could turn it back to yellow, it meant it was fighting the "rust."
• The Result: The brown seaweeds, especially the invader Rugulopteryx okamurae and the green Batophora sp., were the champions. Their methanol extracts were so good at stopping the "rust" that they blocked more than 40% of the damage. It's like finding a superhero in the group of troublemakers.

Superpower B: The "Fungus Fighter" (Antifungal Activity)

There is a scary fungus called Fusarium that attacks banana crops, causing them to wilt and die. Farmers have almost no way to stop it. The scientists wanted to see if seaweed could be a natural shield against this fungus.

• The Test: They put the seaweed extracts on a plate with the fungus and watched to see if the fungus stopped growing.
• The Result: The methanol extract from the invader Rugulopteryx okamurae was the biggest winner. It slowed down the fungus significantly. The red seaweed (Palisada tenerrima) also did a decent job. It's as if the invader seaweed grew a shield that the fungus couldn't break through.

4. The "Pareto" Puzzle: Finding the Perfect Balance

The researchers had a lot of data. Some seaweeds were great at stopping rust but bad at fighting fungus. Others were the opposite. They used a special math trick (called Pareto analysis) to find the "Goldilocks" extract—the one that wasn't necessarily the best at just one thing, but was excellent at everything at once.

The Winner: The methanol extract from the invader Rugulopteryx okamurae.

• It had a high yield (easy to get a lot of it).
• It was a great rust remover.
• It was a strong fungus fighter.
• It was packed with "good stuff" like phenols and flavonoids (nature's vitamins).

5. What's Inside the Magic Potion?

Using high-tech microscopes (LC-MS/MS), the scientists peeked inside the winning extract. They found a cocktail of interesting chemicals:

• Oxylipins: Molecules that help plants heal and fight stress.
• Terpenes: Fragrant compounds often found in essential oils.
• Fucoxanthin: A pigment that gives brown algae their color and is famous for being healthy.

The Big Takeaway

This paper tells a story of turning a problem into a solution.

Instead of just trying to kill the invasive seaweed that is clogging up the Mediterranean, the researchers suggest we should harvest it. By turning this "weed" into a natural medicine or agricultural shield, we can:

1. Clean up the ocean (by removing the invader).
2. Help farmers save their banana crops from fungus.
3. Create natural antioxidants for health products.

In short: The "bad guy" of the sea might just be the hero we need to save our crops and our health, provided we know how to squeeze the right juice out of it.

Technical Summary

1. Problem Statement

The Mediterranean Sea faces dual challenges:

1. Ecological: Invasive macroalgae species (specifically Rugulopteryx okamurae, Codium fragile, and Batophora sp.) are proliferating, disrupting local ecosystems and biodiversity.
2. Agricultural/Health: There is a critical lack of effective treatments for Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), the pathogen causing Fusarium wilt in banana crops. Additionally, there is a growing demand for natural antioxidants to combat oxidative stress-related diseases.

Objective: The study aims to evaluate the potential of macroalgae from the southeastern coast of Spain (both native and invasive) as sources of bioactive compounds. Specifically, it seeks to determine if these algae possess antioxidant and antifungal properties and to assess whether the biomass of invasive species can be valorized for biotechnological applications.

2. Methodology

Sample Collection and Identification

• Species Studied: Five macroalgae were collected: Rugulopteryx okamurae (invasive), Dictyota fasciola (native), Batophora sp. (invasive), Codium fragile (invasive), and Palisada tenerrima (native).
• Identification: Morphological identification was confirmed via specialized keys. Molecular identification was performed using DNA extraction (CTAB protocol) and PCR amplification of specific genes (psbA for brown algae, rbcL for green, COI for red/brown), followed by Sanger sequencing and BLASTn analysis.

Extraction and Preparation

• Solvents: Methanol (MeOH, polar) and Ethyl Acetate (EtOAc, semi-polar).
• Process: Freeze-dried algae were ground and macerated at a 1:10 (w/v) ratio. Extraction involved heating at 40°C for 2 hours followed by stirring at 4°C for 48 hours.
• Yield Calculation: Extracts were evaporated and yields were calculated as a percentage of dry weight.

Chemical Characterization

• Total Phenolic Content (TPC): Quantified using the Folin-Ciocalteu method (expressed as µg Gallic Acid Equivalents/mg).
• Total Flavonoid Content (TFC): Measured using the aluminum chloride method (expressed as µg Quercetin Equivalents/mg).
• Metabolite Profiling: LC–MS/MS (HPLC coupled to Q-TOF) was used to identify specific metabolites, particularly in the most promising extracts.

Bioactivity Assays

• Antioxidant Activity: Assessed via the DPPH radical scavenging assay at concentrations of 0.25–1 mg/mL. Ascorbic acid served as the positive control.
• Antifungal Activity: Tested against Fusarium oxysporum f. sp. cubense TR4 using an agar well diffusion assay. Extracts (10 mg and 20 mg per well) were applied, and radial fungal growth was monitored for 10 days. Nystatin was used as a positive control.

Statistical and Multi-Objective Analysis

• Statistics: ANOVA (two-way and three-way) and Generalized Linear Models (GLM) were used to analyze differences in yield, chemical content, and bioactivity.
• Pareto Optimization: A multi-objective Pareto analysis was applied to identify extracts that offered the best simultaneous balance of six criteria: antifungal inhibition (at two doses), DPPH scavenging, TPC, TFC, and extraction yield.

3. Key Results

Extraction Yields and Chemical Composition

• Yields: Methanolic extracts generally yielded higher amounts (up to ~12.5% for D. fasciola and R. okamurae) compared to ethyl acetate extracts.
• Phenolics & Flavonoids: Methanol consistently extracted higher levels of phenolics and flavonoids.
  • R. okamurae (MeOH) showed the highest phenolic content (62.3 µg GAE/mg).
  • D. fasciola (MeOH) showed the highest flavonoid content (44.6 µg QE/mg).
  • Batophora sp. and R. okamurae generally outperformed other species in antioxidant potential.

Antioxidant Activity

• All extracts exhibited DPPH scavenging activity.
• Top Performers: Batophora sp. and R. okamurae (methanolic extracts) showed the highest activity, inhibiting >44% of DPPH radicals at 1 mg/mL.
• Solvent Effect: Methanolic extracts generally outperformed ethyl acetate extracts, except for Batophora sp., where the ethyl acetate extract showed superior activity.

Antifungal Activity

• Most Potent: The methanolic extract of R. okamurae demonstrated the strongest antifungal activity against Foc TR4, achieving 30.5% inhibition at 20 mg/well. This was statistically significant and comparable to or better than the commercial control (nystatin) at certain time points.
• Secondary Performer: P. tenerrima (methanolic) showed notable inhibition (~20%).
• Other Species: D. fasciola and C. fragile showed variable or lower effects. Batophora sp. showed mild/inconsistent inhibition.
• Dose-Response: Higher doses (20 mg/well) generally resulted in greater growth reduction, particularly evident from day 6 onwards.

Metabolite Profiling (LC–MS/MS)

• R. okamurae (MeOH): Revealed a diverse profile including phenolic derivatives, oxylipins (e.g., 12-OPDA), terpenoid-like compounds (bisabolene-type sesquiterpenoids), and carotenoids (fucoxanthin).
• R. okamurae (EtOAc): Showed a similar but less complex profile, with lower diversity of polar metabolites, correlating with lower bioactivity.

Multi-Criteria Optimization (Pareto Analysis)

• The analysis identified three Pareto-optimal extracts: R. okamurae (MeOH), D. fasciola (MeOH), and Batophora sp. (MeOH).
• Winner: R. okamurae (MeOH) was the globally optimal candidate, offering the best compromise between high antifungal inhibition, strong antioxidant capacity, high phenolic content, and high extraction yield.

4. Significance and Contributions

1. Valorization of Invasive Species: The study provides strong evidence that the invasive alga Rugulopteryx okamurae is not just an ecological pest but a valuable resource. Its biomass can be converted into high-value bioactive extracts, offering a sustainable strategy for managing its environmental impact.
2. Novel Antifungal Source: The research identifies a natural, plant-derived candidate (R. okamurae extract) capable of inhibiting Foc TR4, a pathogen for which no effective treatment currently exists. This opens new avenues for sustainable agriculture and banana crop protection.
3. Methodological Insight: The application of Pareto optimization effectively prioritized extracts based on a multidimensional balance of yield, chemistry, and bioactivity, moving beyond single-parameter screening.
4. Chemical Insights: The identification of oxylipins, sesquiterpenoids, and fucoxanthin in R. okamurae suggests specific mechanisms for its bioactivity, providing a roadmap for future isolation and structural characterization of the active compounds.
5. Regional Potential: It highlights the southeastern Spanish coast as a rich reservoir of bioactive macroalgae, supporting the development of a local blue biotechnology sector.

Conclusion

The study concludes that macroalgae from SE Spain, particularly the invasive Rugulopteryx okamurae, are potent sources of antioxidants and antifungals. The methanolic extract of R. okamurae stands out as a promising candidate for agricultural and biomedical applications, supporting a circular economy approach where invasive biomass is transformed into functional products.