Algal extract as a key protective agent: Jania rubens–enriched PVA/CMC coatings for postharvest onion disease control

Abstract

Background

Onion (Allium cepa L.) is one of the major vegetable crops that are damaged and lost by pathogenic fungal infection during storage due to a lack of proper storage conditions. This study investigates the antifungal potential and mechanism of action of Jania rubens extract against two onion pathogens, Aspergillus niger and Talaromyces calidominioluteus.

Results

The acetone extract (AE) of Jania rubens demonstrated notable inhibitory effects against the tested fungi, exhibiting potential antifungal activity relative to the fungicide fluconazole. Gas Chromatography-Mass Spectrometry (GC–MS) profiling revealed a rich composition of bioactive compounds, including phytol, thymol, estragole, and fatty acids, known for their antimicrobial properties. In practical application on onion bulbs, dipping in polyvinyl alcohol/carboxymethyl cellulose (PVA/CMC) incorporated with Jania rubens extract significantly improved the physiological status (fresh and dry weight), provided a stronger and more sustained reduction in oxidative stress markers (H₂O₂ and MDA), and a prolonged decrease in fungal colony-forming units over two months compared to infected onion and PVA/CMC coating. At the morphological level, Transmission Electron Microscopy (TEM) confirmed the direct antifungal action, showing severe ultrastructural damage in fungal hyphae, including cell wall degradation, plasma membrane detachment, and cytoplasmic disorganization. Additionally, molecular analysis provided further clarity, revealing the extract's ability to downregulate key fungal genes: FKS1, CHS2, and CHS (Cell Wall Biosynthesis), ERG11 (Ergosterol Biosynthesis), SOD5 (Antioxidant Defense).

Conclusion

Collectively, these findings confirm that Jania rubens extract exerts a potent antifungal effect by simultaneously targeting the fungal cell wall, membrane, and antioxidant pathways. The extract represents a promising, eco-friendly alternative for the effective postharvest management and preservation of stored onions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08563-1.

Background

Onion (Allium cepa) is a vital vegetable crop cultivated globally for its culinary, medicinal, and economic importance [1]. Allium plants usually produce bulbs, which are of economic importance because of their edible fleshy scale leaves, unique flavor, and nutritional value. Allium species are also often used in traditional medicine due to their anticarcinogenic, antibiotic, antithrombotic, and cardioprotective properties [2]. Therefore, the long-term storage of onions is necessary to ensure a steady supply throughout the year. One of the major causes of postharvest losses in onions is fungal infection during long-term storage [3]. Additionally, post-harvest fungal infections pose a serious threat to onion storage, leading to substantial economic losses and reduced market quality, with losses ranging from 15% to 30% [4, 5]. Among the most prevalent fungal pathogens affecting stored onions are Aspergillus niger and Talaromyces calidominioluteus. A.niger is responsible for black mold, characterized by black spores on the outer scales of onions, leading to tissue degradation and shortened shelf life [6]. On the other hand, infected onion bulbs by T.calidominioluteus, a close relative of T. Minioluteus, often exhibit postharvest stem rot and tissue decay, leading to softening and discoloration of bulb tissues. A related species, T. minioluteus, was previously isolated from decayed onion bulbs in Serbia, and its pathogenicity was confirmed [7]. T. Calidominioluteus, alongside T. adpressus, P. gladioli, P. Polonicum, as one of the main isolates from naturally diseased lily bulb tissues during storage [8].

Traditional methods of fungal control include low-temperature storage; for instance, maintaining onions below 4 °C can reduce the frequency of infections from storage pathogens to 10–20%. However, low-temperature storage requires a higher cost because the harvest time of onions is in the summer [9, 10]. Generally, many farmers are using the curing treatment after harvest before storage, and then pesticides or sulfur treatment during storage [11]. Excessive use of chemical fungicides has led to environmental pollution, potential human health hazards, and the emergence of resistant fungal strains [12]. Furthermore, consumer preferences are shifting toward organic and chemical-free food products, necessitating the development of alternative, eco-friendly preservation methods [13]. Therefore, new technologies that can economically and effectively extend the shelf life of onions are needed.

In recent years, natural antimicrobial agents have gained increasing attention for their potential use in food preservation. One promising approach involves the use of bio-based coatings. These coatings, often made from biopolymers, offer a barrier against moisture loss and gas exchange, which helps to reduce microbial spoilage and extend shelf life [14, 15]. Biobased coatings derived from marine organisms, particularly macroalgae and seagrasses, are rich in bioactive compounds with antimicrobial properties [16]. Macroalgae, especially red algae (Rhodophyta), and seagrasses produce diverse antifungal compounds including terpenoids, flavonoids, and phenols effective against plant pathogens [17–20]. This bioactivity is exemplified by species such as Jania rubens, a red macroalga belonging to the Corallinaceae family, has a unique structure and is common in diverse marine habitats. Studies show that its extract demonstrates significant antimicrobial effects [21]. Further research supports that extracts contain secondary metabolites active against microorganisms[22]. Unlike synthetic fungicides, these biocoatings are biodegradable, non-toxic, and environmentally sustainable, making them ideal candidates for food preservation applications [23, 24]. While the use of various algae-based coatings for food preservation is well-documented, the specific integration of Jania rubens extracts into a PVA/CMC (Polyvinyl Alcohol/Carboxymethyl Cellulose) matrix remains a specialized and targeted approach for onion protection. This study aims to develop a Jania rubens–based biocoating to protect stored onions from A. niger and T. calidominioluteus. It focuses on formulating the biocoating and evaluating its effects on fungal inhibition, shelf life, and overall storage quality. This work supports efforts to reduce post-harvest losses and promote environmentally friendly storage practices.

Materials and methods

Preparation of Jania rubens extract

Jania rubens was collected during November of 2024 from the El Qoseir coast along the Red Sea, Egypt (26°10′28″ N, 34°14′26″ E). The algal samples were formally identified by Mahmoud M. Abu-Faddan, Assistant researcher at Taxonomy and biodiversity of aquatic biota lab., National Institute of Oceanography and Fisheries (NIOF), Egypt. The collected samples were transferred to the Faculty of Science, Tanta University. Herbarium specimens were deposited in National Institute of Oceanography and Fisheries (NIOF) with accession number NIOF-EG-JANI-2024-009. To remove adhering sand and debris, the samples were thoroughly washed several times, air-dried, and then finely ground using a mechanical grinder. For preparation of Jania rubens extract, around 4 g of the dried algal powder was separately soaked in 40 mL of methanol (ME), acetone (AE) or methanol:acetone (1:1,, v/v) (MAE) on a heat magnetic stirrer at 70 °C for 5 hours. The resulting mixtures (0.1 g/mL) were filtered, and the filtrate was evaporated to dryness at room temperature to obtain the crude extract.

Isolation of onion rot fungi

Aspergillus niger AUMC 17180 and Talaromyces calidominioluteus AUMC 17181 were isolated from bulb onions that had been stored at home and exhibited clear symptoms of rot. The outer dry scales were removed, and the bulbs were washed under running water to eliminate soil and debris. The onion bulbs were then surface-sterilized in 70% ethanol for 60 seconds, rinsed three times with sterile distilled water, and blot-dried using sterile cotton. Rotten portions of the bulbs were aseptically excised with a sterile scalpel and inoculated onto potato dextrose agar (PDA) petri dishes, which were incubated at 28 °C for 7 days.

Molecular identification of fungal strains

Fungal isolates were maintained on Potato sucrose agar (PSA) plates, sent to mycological centre, faculty of science, Assiut university, Egypt for the taxonomic identification. Genomic DNA was subsequently extracted by SolGent Co. (Daejeon, South Korea) using the Solg™ Genomic DNA Prep Kit. The internal transcribed spacer (ITS) region was amplified via PCR using universal primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) [25] in an ABI 9700 thermal cycler with Solgent EF-Taq polymerase. Each 25 µL reaction mixture consisted of 2.5 µL 10× buffer, 0.5 µL 10 mM dNTPs, 1.0 µL of each 10 pmol/µL primer, 0.25 µL (2.5 U) EF-Taq, 1.0 µL template DNA, and 18.75 µL nuclease-free water. Thermal cycling parameters included an initial denaturation at 95 °C for 15 min, followed by 30 cycles of 95 °C for 20 s, 50 °C for 40 s, and 72 °C for 1 min, with a final 5-min extension at 72 °C [26]. Following amplification, PCR products were purified with the SolGent PCR Purification Kit-Ultra and verified via 1% (w/v) agarose gel electrophoresis against a 100 bp ladder. Target bands were excised and sequenced bidirectionally using BigDye ddNTP terminator chemistry (SolGent Co.). Finally, raw chromatograms were assembled into contigs using CLC Main Workbench (QIAGEN), queried against the GenBank database via NCBI BLASTn, and aligned with reference sequences [27]. The evolutionary relationships among the sequences were analyzed using the Neighbor-Joining (NJ) method in MEGA7 software, and the consistency of the tree topology was evaluated by bootstrap analysis with 1000 replicates.

Antimicrobial assay

The antimicrobial activity of Jania rubens extracts was evaluated using the agar well diffusion technique [28]. Freshly prepared fungal spore suspensions were obtained from 7-day-old cultures grown on potato dextrose agar (PDA) at 28± 2 °C. Spores were harvested by flooding plates with sterile 0.85% saline, gently scraping with a sterile L-shaped spreader, and filtering through four layers of sterile cheesecloth to obtain primarily single spores. Spore concentration was adjusted to 1 × 10⁶ spores/mL using a hemocytometer under phase-contrast microscopy. For the assay, 100 µl of the microbial suspension was evenly spread over the surface of PDA plates. Using a sterile cork borer, wells of 6–8 mm in diameter were aseptically created in the agar. Each well was then filled with 100 µl of the tested extracts at 500 µg/mL, Fluconazole positive control (10 µg/mL), solvent negative control) methanol and acetone). The plates were incubated at 28 °C for 72 hours for fungal growth. The diameters of the inhibition zones surrounding the wells were measured in millimeters [29].

Minimum inhibitory concentrations (MICs)- broth dilution method

For filamentous fungi, mature culture slants were flooded with sterile distilled water, and the surface mycelia were gently scraped using a sterile wooden applicator stick to obtain a conidial suspension. The suspension was left to stand for approximately 5 minutes to allow the sedimentation of larger particles. The supernatant was then adjusted spectrophotometrically to the optical density recommended for each fungal species, yielding an inoculum concentration of 1 × 10⁶ conidia/mL. Different concentrations of algal extract were prepared as follow: (1) 250 µg/mL, (2) 500 µg/mL, (3) 1000 µg/mL, (4) 1500 µg/mL and (5) 5000 µg/mL in acetone, zero concentration was considered as a negative control. A prepared pure spore suspension of the selected fungi (0.5 ml of about 106 conidia/mL) was inoculated into in sterile test tubes, containing 9 ml of PS broth medium and 0.5 ml of each concentration, finally incubated at 27 °C for 3 days, then optical density of growth was measured spectrophotometrically at 620 nm for blank (growth medium) and each incubated mixture, results were tabulated and represented graphically, and MIC was recorded for each tested extract [30].

Phytochemical analysis of Jania rubens extract

For qualitative analysis, a specific amount of Jania rubens was soaked in 90% acetone to extract its bioactive components, followed by filtration to obtain the acetonic extract, which was subsequently analyzed using gas chromatography–mass spectrometry (GC–MS).along with phytochemical screening [31]. An aliquot of 1 µL from the acetonic extract was injected into a GC–MS system (Perkin Elmer, model Clarus 580/560S) equipped with a capillary column (Elite-5MS, 30 m × 0.25 mm ID × 0.25 µm film thickness) through an AS3000 autosampler operating in split mode. The GC oven temperature was initially set at 60 °C, then increased at a rate of 5°C/min to 210 °C with a 6-minute hold, followed by a further rise at 10°C/min up to 280°C. Helium served as the carrier gas in constant pressure mode with a flow rate of 1 mL/min. Mass spectrometry conditions included an ion source temperature of 200 °C, electron energy of 70 eV, mass scan range of 50–620 m/z in full scan mode. The chemical constituents of Jania rubens were identified primarily by matching mass spectra against the NIST and Pfleger mass spectral libraries for compound identification.

Preparation of algal extract-containing CMC/PVA coating

The CMC solution was prepared by dissolving 1 g of carboxymethyl cellulose (CMC) in 100 mL of deionized water under continuous stirring at 50 ± 5 °C until a clear and homogeneous solution was obtained. Separately, the PVA aqueous solution was prepared by dissolving 5 g of poly(vinyl alcohol) (PVA) in 100 mL of deionized water at 75 ± 5 °C with constant stirring until complete dissolution. Both solutions were then allowed to cool to room temperature. The two polymer solutions were subsequently mixed at a CMC:PVA ratio of 2:1 (v/v), corresponding to a CMC solution:PVA solution ratio of 2:1, under continuous stirring. The resulting polymer blend was stirred for 60 min to ensure complete homogenization. Thereafter, the algal extract was incorporated into the polymer blend at a final concentration of 1.0% (w/v) and stirred for an additional 30 min to obtain a uniform coating formulation. The final coating solution was allowed to stand to remove entrapped air bubbles prior to application [32]. Red onion bulbs (Allium cepa L., cv. Giza 6 Mohassan) were harvested during growing season 2025 from farmers located in Kom Hamada, Beheira, Egypt (30.76° N, 30.69° E). The samples were formally identified by Esraa E. Ammar, lecturer at Tanta University Herbarium (TANE), Egypt. The cleaned onion bulbs (washed with saline solution) were immediately dipped in to the coating solution then air-dried at room temperature for 30 minutes until a stable film was formed. The coated samples were compared with uncoated bulbs (negative control) and bulbs coated with CMC/PVA alone. All onion bulb samples were stored for two consecutive months under controlled conditions. The stored bulbs were infected with Aspergillus niger and T. calidominioluteus inoculum solution, administered at two-week intervals (one application every two weeks). Afreshly prepared fungal spore suspension was obtained from 7-day-old cultures grown on PDA and adjusted to a concentration of 1 × 10⁶ spores/mL using a hemocytometer. For inoculation, 1 mL of the spore suspension was evenly applied to the surface of each bulb using a sterile hand sprayer to ensure uniform coverage. Control bulbs were sprayed with an equal volume of sterile distilled water. After inoculation, bulbs were incubated under storage conditions at 28 ± 2 °C and relative humidity of 70–80%, and monitored regularly for symptom development. These applications were intended to ensure continuous exposure of the bulbs to the required concentration of the pathogenic fungi throughout the storage period, allowing for the assessment of fungal effects under extended storage conditions. From each sampling point, five replicates of bulb tissues were collected for physiological and genetic analyses. The fresh and dry weight of outer fleshy scales (cube 2*2 cm) derived from the bulbs was determined at 30, 60 days, and some oxidative stress markers (MDA and H₂O₂) were also quantified to evaluate the physiological responses of the bulbs to pathogenic stress during storage.

Oxidative stress markers

Malondialdehyde (MDA) content was determined spectrophotometrically by the thiobarbituric acid (TBA) method as recommended by Heath and Packer [33]. To calculate MDA content, the absorbance was estimated at 532 and 600 nm.

Following the protocol established by Junglee et al. [34], hydrogen peroxide (H₂O₂) content was determined in fresh fleshy scale tissue of onion bulbs. Tissue samples (0.5 g) were homogenized in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 12,000 × g for 15 min at 4°C. The supernatant (1 mL) was mixed with 1 mL of 100 mM potassium phosphate buffer (pH 7.0) and 2 mL of 1 M potassium iodide (KI). The absorbance was measured at 390 nm using a spectrophotometer.

Ultrastructure study

Small fragments of fresh specimens were fixed in 4F1G fixative prepared in phosphate buffer solution (pH 7.2) for 3 hours at 4 °C. The fixed samples were then post-fixed in 2% osmium tetroxide (OsO₄) in the same buffer at 4 °C for 2 hours. Subsequently, the specimens were washed in buffer and dehydrated at 4 °C through a graded acetone series. The dehydrated samples were then embedded in resin, polymerized by heating in an oven at 60 °C for 2 days, and sectioned into ultrathin slices (~90 nm thickness) using an ultramicrotome. The ultrathin sections were mounted on copper grids and stained with uranyl acetate for 5 minutes, followed by lead citrate for 2 minutes before examination under the JEOL JSM-1400 PLUS Transmission Electron Microscope, located at the Electron Microscopy Unit, Faculty of Science, Alexandria University (El-Shatby Campus), Egypt.

Evaluation of gene expression by quantitative reverse transcription PCR (qRT-PCR)

A 5-mm mycelial disc, taken from the growing margin of a 7-day-old colony, was inoculated into a 250 ml flask containing 50 ml of Potato Dextrose Broth (PDB). To the treatment group, 1 ml of algal extract (1500 µg/mL) was added. A control group, without algal extract, was inoculated simultaneously. All flasks were incubated statically at 28 ± 2°C.After 48 hours of incubation, mycelia were harvested by filtration, flash-frozen in liquid nitrogen, and ground to a fine powder. RNA was extracted utilizing the Qiagen RNase Mini Kit. Total RNA was extracted using the Qiagen RNeasy Mini Kit (Qiagen, Germany) following the manufacturer's protocol. cDNA was synthesized in a 20 μl reaction volume using M-MLV Reverse Transcriptase (Promega, USA), 5x RT buffer, 10 mM dNTPs, 50 ng/μl oligo(dT)18 primer, 40 U/μl RiboLock RNase inhibitor, and 200 U/μl M-MLV RT enzyme. The thermal cycling conditions were: 5 min at 65 °C (primer annealing/DNA denaturation), 60 min at 42 °C (reverse transcription), and 10 min at 95 °C (enzyme inactivation). Quantitative real-time PCR was conducted in triplicate using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, USA) in a 25 μl reaction volume containing 1 μl cDNA (1:10 dilution), 12.5 μl 2x Master Mix, 0.5 μM each primer (CHS, SOD5, FKS1, ERG11, CHS2; in Table S1), and nuclease-free water. Thermal cycling on the Rotor-Gene Q (Qiagen, Germany) consisted of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s (combined annealing/extension). Relative gene expression was quantified using the 2^(-ΔΔCt) method with ACTIN as the reference gene [35].

Statistical analysis

The findings were reported as the means of three replicates, and the standard error (SEs) was computed. One-way ANOVA was employed for statistical analysis to identify significant differences among treatments. Analyses were conducted using XLSTAT software (version 2014.5.03), with a significant threshold was set at Tukey’s test with p < 0.05.

Results

Molecular identification of fungal strains

ITS-based phylogenetic analysis identified the two fungal isolates as Aspergillus niger (AUMC 17180) and Talaromyces calidominioluteus (AUMC 17181). Strain AUMC 17180 shared 99.67–99.83% identity and 99–100% coverage with A. niger reference sequences, while strain AUMC 17181 showed 99.49–99.83% identity and 95–100% coverage with T. calidominioluteus sequences, each clustering with their respective type strains (ATCC16888 & CBS147313) (fig. 1)

Fig. 1.

ITS-based phylogenetic analysis of two fungal isolates as Aspergillus niger (AUMC 17180) and Talaromyces calidominioluteus (AUMC 17181)

Antifungal activities of Jania rubens

Results of our present work show that the three prepared J. rubens (ME, AE or MAE) extracts exhibited antifungal activity against T. calidominioluteus and A. niger, with varying inhibition zone diameters as represented in Table 1 and Fig. 2. Among the tested extracts, the AE showed the highest antifungal effect against both fungal species, recording inhibition zones of 31.3 ± 4.3 mmfor T. calidominioluteus and 34.4 ± 2.1 mmfor A. niger. These values were notably higher than those obtained by the reference fungicide, fluconazole, which showed inhibition zones of 14.9 ± 1.7 mmand 16.5 ± 1.7 mm, respectively. The MAE demonstrated moderate activity, with inhibition zones of 24.6 ± 2.3 mmagainst T. calidominioluteus and 25.96 ± 1.6 mmagainst A. niger. The ME also showed noticeable antifungal activity, producing inhibition zones of 21.6 ± 1.6 mmand 20.9 ± 0.07 mmagainst T. calidominioluteus and A. niger, respectively. In contrast, the negative controls (solvents alone) exhibited no inhibitory effect (0 mm), confirming that the antifungal activity was exclusively due to the bioactive compounds in the different J. rubens extracts. It is worth noting that A. niger exhibited slightly greater sensitivity to the J. rubens extracts than T. calidominioluteus, with the acetone extract producing the largest inhibition zone (34.4 ± 2.1 mm). These results indicate that J. rubens, especially its acetonic extract, has strong antifungal activity and may represent a viable natural alternative to synthetic antifungal agents (fluconazole).

Table 1.

Antifungal activity of J. rubens (ME, AE or MAE) extracts against T. calidominioluteus and A. Niger

Inhibition zone (mm)
Treatments		T. calidominioluteus	A. niger
ME		21.6 ± 1.6 bc	20.9 ± 0.07 b
AE		31.3 ± 4.3 a	34.4 ± 2.1 b
MAE		24.6 ± 2.3 ab	25.96 ± 1.6 a
+ve control (Fluconazole)		14.9 ± 1.7 c	16.5 ± 1.7 c
-ve control	Methanol Acetone	0 0	0 0

Different letters within a column indicate significant differences at p<0.05 according to Tukey’s test

Fig. 2.

Antifungal activity of J. rubens extracts (1; AE, 2; ME, 3; MAE, 4; +ve control) against (A); T. calidominioluteus and (B); A. niger

Minimum inhibitory concentrations (MICs)

MIC assay revealed that the algal extract inhibited both A. niger and T. calidominioluteus at 1500 µg/mL as shown in Table 2. Tubes at concentrations below this level displayed visible turbidity, while those at or above the MIC remained completely clear.

Table 2.

MIC of J. rubens extract against A. niger and T. Calidominioluteus

Fungal species	0 µg/mL	250 µg/mL	500 µg/mL	1000 µg/mL	1500 µg/mL	2000 µg/mL
A. niger	0.28 ± 0.07a	0.26 ± 0.02ab	0.22 ± 0.01ab	0.18 ± 0.01bc	0.13 ± 0.01c	0.13 ± 0.01c
T. calidominioluteus	0.82 ± 0.12a	0.28 ± 0.01b	0.23 ± 0.01bc	0.18 ± 0.01bc	0.14 ± 0.01c	0.14 ± 0.01c

Different letters within a column indicate significant differences at p<0.05 according to Tukey’s test

Qualitative GC‒MS analysis

The GC–MS analysis (Figure 3 and Table 3) identified the major phytochemical constituents of the J. rubens extract. The detected compounds were ranked in descending order according to their relative abundance (peak area %). The most abundant compounds included 1,3-Dioxolane, 2-pentadecyl-, thymol, oleic acid, palmitic acid, and linoleic acid, followed by phytol, estragole, and hexadecanoic acid, ethyl ester. Notably, several of the identified compounds, including thymol, phytol, estragole, eicosane, 1,3-Dioxolane, 2-pentadecyl-, and multiple fatty acid such as, oleic acid, palmitic acid, linoleic acid, have been previously reported to possess antimicrobial properties, which may contribute to the observed antifungal activity of the extract.

Fig. 3.

GC–MS chromatogram of J. rubens acetone extract

Table 3.

Phytochemical constituents identifed by GC–MS of J. rubens acetone extract

No	Area%	Compounds	Height	Molecular Formula
1	5.334	1,3-Dioxolane, 2-pentadecyl-	25,065,750	C18H36O2
2	3.600	Thymol	55,528,860	C10H14O
3	2.909	Oleic acid	81,677,048	C₁₉H₃₆O2
4	2.856	Palmitic acid	104,171,672	C17H34O2
5	2.052	linoleic acid	63,050,064	C₁₉H₃₄O2
6	1.586	Eicosane	19.845.362	C20H42
7	1.506	Phytol	46.862.920	C20H40O2
8	1.295	Estragole	24,047,194	C10H12O
9	1.249	Hexadecane, 2,6,10,14-tetramethyl	19,845,362	C20H40
10	1.187	Pentadecane	46,862,920	C15H32
11	0.967	Hexadecanoic acid, ethyl ester	19,755,474	C18H36O2
12	0.912	16-Methylheptadecanoic acid	23,219,590	C₁₉H₃₈O2
13	0.716	Isopropyl 5,11-dihydroxy-3,7,11-trimethyl-2-dodecenoate	10,769,312	C18H34O4
14	0.432	Dodecane, 2,6,10-trimethyl	17,460,546	C₁₅H₃₂

Effect of J. rubens extract on fresh weight and dry weight of stored onion bulbs

The data represented in Fig. 4 showed that the application of a PVA/CMC/algal extract coating significantly reduced the post-harvest weight degradation in stored onion bulbs by conferring resistance against the fungal infection. The fresh weight of stored onion bulbs had weight loss by 58% in A. niger-infected onion bulbs and 62% in T. calidominioluteus-infected onion bulbs compared to stored onion bulbs coated with PVA/CMC/algal extract during storage for one month. However, the fresh weight of stored onion bulbs coated with PVA/CMC had weight loss by 33% in A. niger-infected onion bulbs and 24% in T. calidominioluteus-infected onion bulbs compared to stored onion bulbs coated with PVA/CMC/algal extract during storage for one month. In the same manner, the dry weight of stored onion bulbs had weight loss by 61% in A. niger-infected onion bulbs and 52% in T. calidominioluteus-infected onion bulbs compared to stored onion bulbs coated with PVA/CMC/algal extract during storage for one month. However, the dry weight of stored onion bulbs coated with PVA/CMC had weight loss by 52% in A. niger-infected onion bulbs and 26% in T. calidominioluteus-infected onion bulbs compared to stored onion bulbs coated with PVA/CMC/algal extract during storage for one month. Overall, the PVA/CMC/algal extract coating significantly mitigated storage weight loss compared to both the infected control and the coating-only control, demonstrating its effectiveness in preserving onion bulb biomass.

Fig. 4.

Effect of Jania rubens extract on (A); fresh weight after one month, (B); fresh weight after two months, (C); dry weight after one month, (D); and dry weight after two months of stored onion bulbs. Different letters within a column indicate significant differences at p<0.05 according to Tukey’s test

Effect of J. rubens extract on oxidative stress markers of stored onion bulbs

The data represented in Fig. 5 showed that, storage for one month, the content of hydrogen peroxide (H₂O₂) in stored onion bulbs coated with PVA/CMC/algal extract was 0.4 and 2.2 n.mol/g.f.wt with A. niger-infected onion bulbs and T. Calidominioluteus-infected onion bulbs, respectively. This value increased up to 1.55 and 16.29 n.mol/g.f.wt in bulbs infected with A. niger and T. calidominioluteus, corresponding to increases of 287% and 640%, respectively. Similarly, malondialdehyde (MDA) levels in stored onion bulbs coated with PVA/CMC/algal extract was 290.6 and 520.8 n.mol/g.f.wt with A. niger-infected onion bulbs and T. Calidominioluteus-infected onion bulbs, respectively. This value increased up to 797.47 and 1720.5 n.mol/g.f.wt in bulbs infected with A. niger and T. calidominioluteus, corresponding to increases of 174% and 230%, respectively. However, in stored onion bulbs coated with PVA/CMC alone, H₂O₂ concentrations reached 1.06 n.mol/g.f.wt in A. niger-infected onion bulbs and 4.4 n.mol/g.f.wt in T. calidominioluteus-infected onion bulbs, which increased by 165% in A. niger-infected onion bulbs and 100% in T. calidominioluteus-infected onion bulbs, respectively compared with those observed in PVA/CMC/algal extract–coated bulbs. Likewise, MDA contents increased to 576.6 n.mol/g.f.wt in A. niger-infected onion bulbs and 874.2 n.mol/g.f.wt in T. calidominioluteus-infected onion bulbs, which increased by 98% in A. niger-infected onion bulbs and 68% in T. calidominioluteus-infected onion bulbs, respectively compared with those observed in PVA/CMC/algal extract–coated bulbs. Overall, H₂O₂ and MDA accumulated progressively after two months of storage (fig. 6).

Fig. 5.

Effect of Jania rubens extract on (A); H₂O₂ after one month, (B); H₂O₂ after two months, (C); MDA after one month, and (D); MDA after two months of stored onion bulbs. Different letters within a column indicate significant differences at p<0.05 according to Tukey’s test

Fig. 6.

Visual appearance of onion bulbs with PVA/CMC/Jania rubens extract, PVA/CMC, and control onions infected with T. calidominioluteus and A. niger respectively, after one months (A, B) and two months (C, D) of treatments

Assessment of fungal growth on onion bulbs

The antifungal activity of PVA/CMC and PVA/CMC/algal extract coating were evaluated based on their effect on the colony-forming ability of A. niger and T. calidominioluteus after one and two months of incubation (Table 4). The results revealed a notable reduction in the number of A. niger and T. calidominioluteus colonies on stored onion bulbs in presence of PVA/CMC/algal extract coating compared to PVA/CMC coated or Uncoated onion bulbs. After the one month, A. niger showed a marked decrease in colony count from 2.7×10³ in the untreated culture to 0.88×10³ with PVA/CMC coating and further to 0.48×10³ with PVA/CMC/algal extract coating. Similarly, T. Calidominioluteus exhibited a reduction from 2.52×10³ to 1.05×10³ with PVA/CMC coating and then to 0.9×10³ with PVA/CMC/algal extract coating. After two months, A. niger colony numbers increased in the control (5.38×10³) and PVA/CMC coating samples (1.22×10³), but remained low in the PVA/CMC/algal extract coating samples (0.95×10³). A similar pattern was observed for T. calidominioluteus, where the colony count reached 7.51×10³, 2.11×10³ with PVA/CMC coating, and only 0.92×10³ with PVA/CMC/algal extract coating. These findings clearly demonstrate that while PVA/CMC had a moderate inhibitory effect, J. rubens extract exhibited a much stronger and longer-lasting antifungal action.

Table 4.

Effect of PVA/CMC and PVA/CMC/Jania rubens extract on the colony-forming ability of A.niger and T.calidominioluteus after one and two months of incubation

Months	Treat/colony number
	A. niger			T. calidominioluteus
	Control	PVA/CMC	PVA/CMC/algal extract	Control	PVA/CMC	PVA/CMC/algal extract
1 st month	2.7×103 ±568.7a	0.88×103 ±26.5b	0.48×103 ±28.8c	2.52×103 ±46.2a	1.05×103 ±50b	0.8×103 ±10.2 c
2nd month	5.38×103 ±255.4a	1.22×103 ±46.2b	0.95×103 ±86.6c	7.51×103 ±144.3a	2.11×103 ±28.8b	0.92×103 ±30.4c

Different letters within a column indicate significant differences at p<0.05 according to Tukey’s test

Transmission electron microscopy (TEM) micrographs of stored onion bulbs treated with A. niger and T. calidominioluteus

Transmission electron micrographs of fungal hyphae of A. niger and T. calidominioluteus isolated from stored onion bulbs coated with PVA/CMC/algal extract revealed pronounced ultrastructural damage of fungal hyphae (Fig. 7). The fungal cell wall exhibits surface irregularities and partial degradation, accompanied by clear detachment of the plasma membrane from the wall. The cytoplasm shows reduced electron density and marked disorganization of internal contents, indicating early signs of cellular damage and stress. In contrast, The TEM micrographs of hyphae of A. niger and T. calidominioluteus isolated from infected onion bulbs show a well-organized tubular morphology of fungal hyphae. The fungal hyphae appeared with intact, thick, and continuous cell wall and the plasma membrane is closely appressed to the cell wall. The cytoplasm is dense and homogeneous, with no evident ultrastructural abnormalities.

Fig. 7.

Transmission electron micrographs of stored onion bulbs : A A. niger-infected onion bulbs, B A. niger-infected onion bulbs coated with PVA/CMC/algal extract, C T. calidominioluteus-infected onion bulbs, D T. calidominioluteus-infected onion bulbs coated with PVA/CMC/algal extract

Effect of Jania rubens extract on the expression of genes in A.niger and T.calidominioluteus

The relative expression levels of CHS, CHS2, SOD5, FKS1 and ERG11 genes were significantly downregulated following the application of the PVA/CMC/algal extract coating. In A. niger-infected bulbs, expression levels decreased to 0.48, 0.38, 0.13, 0.57, and 0.17 fold relative to the infected control for CHS, CHS2, SOD5, FKS1, and ERG11, respectively. Similarly, for T. calidominioluteus, the coating resulted in a reduction of expression to 0.25, 0.54, 0.67, 0.71, and 0.74 fold for CHS, CHS2, SOD5, FKS1, and ERG11, respectively, compared to the untreated infected bulbs (Fig. 8).

Fig. 8.

Effect of Jania rubens extract on the relative gene expression of of CHS, CHS2, SOD5, FKS1 and ERG11 of A. niger-infected and T. calidominioluteus-infected onion bulbs. Different letters within a column indicate significant differences at p<0.05 according to Tukey’s test

Discussion

The harvest of onions often coincides with the rainy season, leading to inadequate drying and subsequent fungal contamination during storage [9, 36]. The present study demonstrated the antifungal potential of Jania rubens extracts against two fungal pathogens, A.niger and T.calidominioluteus, isolated from infected onion bulbs. In vitro assays revealed that the acetone extract (AE) exhibited the strongest inhibitory effect, producing inhibition zones against T.calidominioluteus and A. niger. The antifungal potential of the acetone extract could be attributed to its ability to dissolve a broad range of moderately polar and non-polar bioactive compounds such as terpenoids, phenolics, and fatty acids, which are commonly reported in red macroalgae [37]. Specifically, the GC–MS identification of phytochemicals provides a chemical basis for these findings, as these are well-documented for their antimicrobial and antifungal potential. Notably, phytol and thymol were detected; both are recognized for their ability to disrupt fungal membrane integrity. While phytol is known to induce oxidative stress via reactive oxygen species (ROS) production, thymol further contributes by inhibiting ergosterol biosynthesis [38–42]. Similarly, estragole, an aromatic ether, has been reported to exert antifungal and antioxidant activities, which could synergize with other compounds [43]. Fatty acid such as palmitic, oleic, and linoleic acid were also identified. These compounds are known to integrate into lipid bilayers, causing membrane destabilization and leakage of cellular contents, thus enhancing antifungal effects [44, 45]. Moreover, Eicosane, a lipophilic hydrocarbon from the extract, disrupts fungal membrane integrity by integrating into the lipid bilayer. This alters fluidity, causes leakage, and inhibits spore germination [46, 47]. 1,3-Dioxolane, 2-pentadecyl may contribute to the antimicrobial activity of the extract, as dioxolane derivatives exhibit antimicrobial effects and long alkyl chains can disrupt microbial membranes [48, 49].

Following GC–MS identification of bioactive compounds in Jania rubens extract, its antifungal efficacy was tested on infected onion bulbs to evaluate its ability to suppress fungal infection and improve bulb health.The results clearly showed that PVA/CMC/Jania rubens extract was more effective than PVA/CMC coating in enhancing the physiological status of infected onion bulbs. In terms of growth parameters, bulbs treated with PVA/CMC/Jania rubens exhibited greater improvements in both fresh and dry weight compared to PVA/CMC coating during one month and second month. In contrast, the effect of PVA/CMC coating was less pronounced and declined with time, particularly in T.calidominioluteus-infected bulbs. This observation is supported by earlier studies which reported that Jania rubens possesses potent antimicrobial activity due to its diverse secondary metabolites [21] and that it can act as a natural preservative enhancing postharvest stability [37]. In addition, bulbs treated with PVA/CMC/Jania rubens showed stronger reductions in H₂O₂ and MDA compared to PVA/CMC coating, both at one month and two months of storage. This reflects the strong antioxidant properties of Jania rubens, which have been attributed to its high content of phenolic compounds and other bioactive metabolites [37, 50]. By contrast, PVA/CMC provided only partial reductions in oxidative markers, which is consistent with previous reports describing cellulose and chitosan coatings as passive barriers that primarily reduce water loss but lack significant biochemical activity [51]. Collectively, these results suggest that Jania rubens extract acts through a dual mechanism, direct antifungal activity, and indirect enhancement of host antioxidant defense. This dual action makes it a promising eco-friendly alternative to conventional PVA/CMC coatings for the postharvest management of onion bulbs.

To elucidate the antifungal mechanism of Jania rubens extract, TEM analysis was performed on A.niger and T.calidominioluteus cells following treatment.. Untreated hyphae showed normal intact cell walls, closely adhered membranes, and dense cytoplasm. In contrast, treated fungi exhibited severe ultrastructural damage, including cell wall degradation, membrane detachment, cytoplasmic disorganization, and collapsed hyphae. These findings indicate that Jania rubens extract exerts direct antifungal activity by disrupting fungal cell wall and membrane integrity, causing cytoplasmic leakage and cell death, consistent with its known antimicrobial potential [21, 37, 50].

The TEM-detected damage was further supported by molecular evidence clarifying the extract’s mode of action. Key genes involved in cell wall biosynthesis and stress tolerance were significantly downregulated, consistent with membrane- and wall-active metabolites identified by GC–MS. In A. niger and T. calidominioluteus, the expression of FKS1 and CHS genes was markedly suppressed following treatment. These two enzymes are responsible for the synthesis of β-glucan and chitin, the major structural polysaccharides that maintain cell wall strength and elasticity. Their downregulation implies that the algal extract disrupts the biosynthetic machinery of the fungal cell wall, likely as a secondary response to membrane stress induced by palmitic, and oleic acid, which are known to destabilize fungal membranes and impair coordinated cell wall assembly. Similar inhibition of CHS activity was reported in Candida albicans when treated with antifungal agents that destabilize the chitin synthase complex [52]. Furthermore, CHS2 plays a crucial role in the formation of the primary septum during cell division and in maintaining proper cell wall remodeling. In filamentous fungi, CHS2 is particularly essential for hyphal growth and septation, processes that are vital for colony expansion and reproduction [53–55]. The observed suppression of CHS2 expression suggests an additional layer of antifungal interference, as inhibition of this gene can lead to incomplete septum formation, abnormal hyphal morphology, and eventual cell lysis, in agreement with the severe ultrastructural damage observed under TEM [53].

In parallel, the significant inhibition of ERG11, a cytochrome P450 enzyme essential for ergosterol biosynthesis, suggests that Jania rubens extract directly targets membrane integrity. This effect is strongly supported by the detection of thymol and estragole in the GC–MS analysis, both of which are reported to interfere with ergosterol biosynthesis and membrane stability through cytochrome P450–related mechanisms. Ergosterol depletion disrupts membrane permeability and fluidity, thereby increasing susceptibility to cell lysis. Recent genomic studies confirmed that ERG11 downregulation directly correlates with loss of membrane stability and increased antifungal sensitivity[56]. Moreover, the significant reduction in SOD5 expression highlights the extract’s ability to impair fungal antioxidant defense systems. This observation can be mechanistically linked to the presence of thymol, identified by GC–MS, which are known to induce intracellular ROS accumulation either through redox cycling or inhibition of antioxidant enzymes [57]. Suppression of SOD activity results in oxidative stress, protein oxidation, and mitochondrial dysfunction, ultimately contributing to fungal cell death. Comparable oxidative stress–mediated antifungal responses associated with reduced SOD transcription have been reported in fungi exposed to phenolic-rich natural extracts [58, 59].

The time-dependent decrease in fungal colonies over the two-month incubation period further supports the stability and persistence of the antifungal compounds in the Jania rubens extract. Studies have shown that extracts and bioactive compounds from Jania sp. exhibit sustained antifungal activity over extended periods, effectively inhibiting fungal growth, spore germination, and colony formation. For example, water extracts and polysaccharides from Jania sp. demonstrated antifungal effects against Botrytis cinerea and maintained inhibitory activity in vitro and on fruit infections over time, indicating their potential for long-lasting crop protection. Additionally, Jania rubens extracts incorporated into hydrogels showed significant antifungal effects against Candida tropicalis with a rapid initial reduction in colony-forming units and continued inhibition over time, confirming the bioactive compounds’ stability and prolonged activity [60, 61]. This supports the interpretation that the antifungal metabolites from Jania rubens remain active and effective for at least two months under typical experimental conditions. Moreover, the lower MIC values recorded for PVA/CMC/Jania rubens extract compared to PVA/CMC confirm its strong and concentration-dependent antifungal efficiency.

Conclusion

In conclusion, this study demonstrates the potential of Jania rubens acetone extract as a natural antifungal agent for the postharvest preservation of onions. Under the experimental conditions, the extract exhibited inhibitory activity against A. niger and T. calidominioluteus, likely mediated by its profile of bioactive compounds. In laboratory trials, the application of PVA/CMC/algal extract improved the physiological status and reduced oxidative stress in infected onion bulbs more effectively than the PVA/CMC coating alone, suggesting a dual mechanism of direct antifungal action and indirect host defense enhancement. This mechanism was further supported by TEM and molecular analysis, which indicated damage to fungal cell walls and the downregulation of key genes, CHS, SOD5, FKS1, ERG11, and CHS2, essential for structural integrity. While Jania rubens extract shows promise as an eco-friendly antifungal, its classification as a definitive alternative to synthetic fungicides requires further validation. Future research should focus on direct comparative trials with commercial synthetic fungicides, and a comprehensive cost-benefit analysis.

Authors’ contributions

Conceptualization, D.E.E., M.A.E. and N.A.E; methodology, D.E.E., M.A.E. and N.A.E; software, D.E.E.; validation, D.E.E., M.A.E. and N.A.E; formal analysis, D.E.E.; investigation and visualization, D.E.E., M.A.E. and N.A.E; writing—original draft preparation, D.E.E., M.A.E., N.E.E., and N.A.E; writing—review and editing, D.E.E., M.A.E. N.A.E; F.A.S. and N.E.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R318), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Data availability

All data supporting the findings of this study are already presented in this published manuscript.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.