Biostimulant activity of brown seaweed species from Strangford Lough: compositional analyses of polysaccharides and bioassay of extracts using mung bean (Vigno mungo L.) and pak choi (Brassica rapa chinensis L.)

The aims of this study were to characterise the composition of seaweed species and to evaluate the efficacy of aqueous extracts as plant biostimulants. Five species (Ascophyllum nodosum, Fucus serratus, Fucus vesiculosus, Laminaria hyperborea and Sargassum muticum) of seaweed were harvested from Strangford Lough, Northern Ireland for the evaluation of polysaccharides, indole-3-acetic acid (IAA), carbon, nitrogen, lipid, ash and mineral contents. The compositional analyses of the five species and their freeze-dried extracts were also carried out using thermogravimetric analysis, scanning electron microscopy and X-ray microanalysis. The concentration of IAA in the acid extracts of the five species ranged between 2.74 and 46.8?ng?g^?1. The carbon, nitrogen, lipid and ash contents ranged between 25.0 and 38.6, 1.37%, and 3.16, 0.83%, and 3.98 and 18.10 and 47.68%, respectively. L. hyperborea and S. muticum contained the highest amounts of minerals. The biostimulant activities of acidic (pH?3.0), neutral (pH?6.5) and alkaline (pH?9.0) extracts were determined by mung bean bioassay. The alkaline extracts from F. vesiculosus and A. nodosum stimulated significantly (P?<?0.001) higher dry matter (DM, %) yield of the mung bean plants. The majority of the acidic extracts significantly (P?<?0.001) enhanced root formation on the mung bean stem cuttings compared to alkaline or neutral extracts. The acidic extracts of the five species, water control and a commercial product were evaluated as foliar feeds for pak choi plants using a hydroponic production system. The interaction of species, e.g. A. nodosum and F. vesiculosus and the two treatment dilutions on DM yield increases of pak choi were significant (P?<?0.05).     

Climate change impact on seaweed meadow distribution in the North Atlantic rocky intertidal

The North-Atlantic has warmed faster than all other ocean basins and climate change scenarios predict sea surface temperature isotherms to shift up to 600 km northwards by the end of the 21st century. The pole-ward shift has already begun for many temperate seaweed species that are important intertidal foundation species. We asked the question: Where will climate change have the greatest impact on three foundational, macroalgal species that occur along North-Atlantic shores: Fucus serratus, Fucus vesiculosus, and Ascophyllum nodosum? To predict distributional changes of these key species under three IPCC (Intergovernmental Panel on Climate Change) climate change scenarios (A2, A1B, and B1) over the coming two centuries, we generated Ecological Niche Models with the program MAXENT. Model predictions suggest that these three species will shift northwards as an assemblage or “unit” and that phytogeographic changes will be most pronounced in the southern Arctic and the southern temperate provinces. Our models predict that Arctic shores in Canada, Greenland, and Spitsbergen will become suitable for all three species by 2100. Shores south of 45° North will become unsuitable for at least two of the three focal species on both the Northwest- and Northeast-Atlantic coasts by 2200. If these foundational species are unable to adapt to the rising temperatures, they will lose their centers of genetic diversity and their loss will trigger an unpredictable shift in the North-Atlantic intertidal ecosystem.     

Floating seaweed and the influences of temperature, grazing and clump size on raft longevity — A microcosm study

Laboratory experiments were conducted to evaluate the longevity, and consequently also the rafting capacity of the brown seaweeds Fucus vesiculosus and Ascophyllum nodosum. The seaweed degradation process and the activity of the grazer Idotea baltica were strongly influenced by temperature: only at 5 °C, the seaweed growth exceeded the weight loss. At higher temperatures, seaweed fragments sank quickly (within 100 days at temperatures higher than 15 °C). This process was significantly accelerated in the presence of I. baltica, resulting in a decrease of raft longevity of 60-70%. At a constant temperature of 15 °C and in the absence of grazers, fragments of A. nodosum floated longer (mean 45 weeks) than fragments of F. vesiculosus (mean 15 weeks). The results indicate that floating seaweeds have the potential to stay afloat for a long time, but that their longevity is temperature-dependent and can be strongly reduced by grazing activity of associated herbivores.     

Improved methods of analysis for betaines in <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> and its commercial seaweed extracts

Beneficial effects of seaweeds and their extracts on crop performance have been attributed to a variety of compounds, including the betaines which are quaternary ammonium betaines. Methods of analysis of betaines published thus far suffer from low sensitivity, lack of baseline separation of individual betaines and from interference from other sample constituents. A rapid cleanup protocol and a sensitive LC-MS/MS method of analysis were developed to afford baseline separation of four betaines in the brown alga Ascophyllum nodosum and its commercial seaweed extract. Using this method, the presence of glycine betaine, δ-aminovaleric acid betaine, γ-aminobutyric acid betaine and laminine in A. nodosum, and commercial extracts derived from A. nodosum, were confirmed and quantified. The major betaine present was γ-aminobutyric acid betaine accounting for 0.008–0.014% of the dry weight of the seaweed and 0.014–0.027% of the dry weight of the commercial extracts. Seasonal variation in betaine content was observed. Differences in the total betaine content were observed between A. nodosum of the yellow (0.011–0.017% dry weight) and the olive green (0.017–0.021% dry weight) coloured morphologies.     

Geographic variation in feeding preference of a generalist herbivore: the importance of seaweed chemical defenses

The ecological impacts of generalist herbivores depend on feeding preferences, which can vary across and within herbivore species. Among mesoherbivores, geographic variation in host use can occur because host plants have a more restricted geographic distribution than does the herbivore, or there is local evolution in host preference, or both. We tested the role of local evolution using the marine amphipod Ampithoe longimana by rearing multiple amphipod populations from three regions (subtropical Florida, warm-temperate North Carolina and cold-temperate New England) and assaying their feeding preferences toward ten seaweeds that occur in some but not all regions. Six of the ten seaweeds produce anti-herbivore secondary metabolites, and we detected geographic variation in feeding preference toward five (Dictyota menstrualis, Dictyota ciliolata, Fucus distichus, Chondrus crispus and Padina gymnospora, but not Caulerpa sertularioides). Amphipod populations that co-occur with a chemically-rich seaweed tended to have stronger feeding preferences for that seaweed, relative to populations that do not co-occur with the seaweed. A direct test indicated that geographic variation in feeding preference toward one seaweed (D. ciliolata) is mediated by feeding tolerance for lipophilic secondary metabolites. Among the four seaweeds that produce no known secondary metabolites (Acanthophora, Ectocarpus, Gracilaria and Hincksia/Feldmannia spp.), we detected no geographic variation in feeding preference. Thus, populations are more likely to evolve greater feeding preferences for local hosts when those hosts produce secondary metabolites. Microevolution of feeding behaviors of generalist marine consumers likely depends on the availability and identity of local hosts and the strength of their chemical defenses.     

UPLC-MS profiling of low molecular weight phlorotannin polymers in Ascophyllum nodosum,Pelvetia canaliculata and Fucus spiralis

Phlorotannins are a group of complex polymers, found in particular brown macroalgae, composed solely of the monomer phloroglucinol (1,3,5-trihydroxybenzene). Their structural complexity arises from the number of possible linkage positions between each monomer unit. This study aimed to profile the phlorotannin metabolite composition and the complexity of isomerisation present in brown macroalgae Ascophyllum nodosum, Pelvetia canaliculata and Fucus spiralis using UPLC-MS utilising a tandem quadrupole mass spectrometer. Phlorotannin-enriched fractions from water and aqueous ethanol extracts were analysed by UPLC-MS performed in multiple reaction monitoring mode to detect molecular ions consistent with the molecular weights of phlorotannins. Ascophyllum nodosum and P. canaliculata appeared to contain predominantly larger phlorotannins (degree of polymerisation (DP) of 6–13 monomers) compared to F. spiralis (DP of 4–6 monomers). This is the first report observing the complex chromatographic separation and metabolomic profiling of low molecular weight phlorotannins consisting of more than ten monomers. Extracted ion chromatograms, for each of the MRM transitions, for each species were analysed to profile the level of isomerisation for specific molecular weights of phlorotannins between 3 and 16 monomers. The level of phlorotannin isomerisation within the extracts of the individual macroalgal species differed to some degree, resulting in substantially different numbers of phlorotannin isomers for particular molecular weights. A similar UPLC-MS/MS separation procedure, as outlined in this study, may be used in the future as a means of screening the metabolite profile of macroalgal extracts, therefore, allowing extract consistency to be monitored for standardisation purposes.     

Liquid seaweed extracts identified using <Superscript>1</Superscript>H NMR profiles

Aqueous extracts of Ascophyllum nodosum and several other brown seaweeds are manufactured commercially and widely distributed for use on agricultural crops. The increasingly regulated international trade in such products requires that they be standardized and defined to a degree not previously required. We examined commercially available extracts using quantitative ¹H NMR and principal components analysis (PCA) techniques. Extracts manufactured over a 4-year period using the same process exhibited characteristic profiles that, on PCA, clustered as a discrete group distinct from the other commercial products examined. In addition to recognizing extracts made from different seaweeds, analysis of the ¹H spectra in the 0.35–4.70 ppm region allowed us to distinguish amongst extracts produced from the same algal species by different manufacturers. This result established that the process used to make an extract is an important variable in defining its composition. A comparison of the ¹H NMR integrals for the regions 1.0–3.0 ppm and 3.0–4.38 ppm revealed small but significant changes in the A. nodosum spectra that we attribute to seasonal variation in gross composition of the harvested seaweed. Such changes are reflected in the PCA scores plots and contribute to the scatter observed within the data point cluster observed for Acadian soluble extracts when all data are pooled. Quantitative analysis using ¹H NMR (qNMR) with a certified external standard (caffeine) showed a linear relationship with extract concentration over at least an order of magnitude (2.5–33 mg/mL; R ² > 0.97) for both spectral regions integrated. We conclude that qNMR can be used to profile (or “fingerprint”) commercial seaweed extracts and to quantify the amount of extract present relative to a suitably chosen standard. Issued as NRCC no. 42,652.     

Characterization of microsatellite loci in the marine seaweed Ascophyllum nodosum (Phaeophyceae; Fucales)

Ascophyllum nodosum L. dominates rocky intertidal shores throughout the temperate North Atlantic. Six microsatellite loci were developed for A. nodosum using enriched libraries. The number of alleles ranged from 9 to 24 and heterozygosities from 0.2213 to 0.7785. Ascophyllum is monotypic. There was no cross‐reactivity observed with Fucus serratus, F. vesiculosus or F. evanescens.     

Antibacterial activity of marine macroalgae against fish pathogenic Vibrio species

In mariculture, diseases of microbial origin can cause significant economic losses worldwide; the evolution of microorganism resistance to antibiotics has resulted in a growing need for new antibacterial compounds that are effective in veterinary medicine and characterized by limited undesirable side effects. Increased attention has recently been turned to seaweeds as a promising source for metabolites with antimicrobial activity. Vibriosis is a common disease, caused by bacteria of the genus Vibrio, that can result in high mortality in aquaculture. The aim of this study was to identify seaweeds with antibacterial activity against some pathogenic Vibrio species, in order to identify a possible alternative to the commonly used antibiotics in aquaculture. Chloroform/methanol lipidic extracts of six seaweed species (Chaetomorpha linum, Cladophora rupestris, Gracilaria dura, Gracilaria gracilis, Gracilariopsis longissima, Ulva prolifera) were tested for their antibacterial activities against six fish pathogenic Vibrio species using the disc diffusion method. Different susceptibilities to lipidic algal extracts were observed. All six of the seaweed extracts tested demonstrated inhibition of Vibrio ordalii. The best was that from Gracilariopsis longissima, showing activity against Vibrio ordalii, Vibrio salmonicida, Vibrio alginolyticus and Vibrio vulnificus. The results confirmed the potential use of seaweed extracts as a source of antibacterial compounds or as a health-promoting feed for aquaculture.     

Isolation and cross‐species amplification of microsatellite loci from the fucoid seaweeds Fucus vesiculosus,F. serratus and Ascophyllum nodosum (Heterokontophyta,Fucaceae)

The Fucaceae is a family of brown seaweeds that dominate and frequently co‐occur on North Atlantic rocky shores. We developed nine polymorphic microsatellite markers for the fucoid seaweeds Fucus vesiculosus, F. serratus and Ascophyllum nodosum using a combined, enriched library. Six of these loci were polymorphic in at least two species, showing from two to eight alleles with heterozygosities ranging from 0.41 to 0.85. Loci were also tested on F. spiralis, revealing five polymorphic microsatellite loci in this species.     

Influence of day-night and tidal cycles on phenol content and antioxidant capacity in three temperate intertidal brown seaweeds

The daily variations of phenol contents and antioxidant capacities were surveyed in a 32-h field experiment in three temperate brown seaweeds belonging to Fucales - Pelvetia canaliculata, Ascophyllum nodosum and Bifurcaria bifurcata - living at different intertidal levels — high-, mid- and low-tide level, respectively. Phenolic compounds of brown seaweeds are secondary metabolites involved in many different protection mechanisms, as for example against grazer and pathogen attack as well as UV damage. This study was thus aimed at understanding the influence of both day/night and tidal cycles on the brown seaweed phenol pool with respect to their bathymetric level on the shore. These cycles affect the quantity and quality of light received by intertidal seaweeds (protection via the water layer during immersion), and the photoprotective role of phlorotannins was thus evaluated. Phenol levels and antioxidant capacities were monitored every hour during a tidal cycle and a half at the equinox spring tide, in March 2003. The three species contained rather high phenol levels, i.e. about 3, 6 and 4% DW in P. canaliculata, A. nodosum and B. bifurcata, respectively. Antioxidant capacities globally paralleled phenol contents in the three species under study. Moreover, the measured antioxidant capacities and the phenol contents of the extracts were significantly and positively correlated in the three species. Significant effect of neither cycles nor their interaction was observed in any species despite a trend to follow day/night alternation for P. canaliculata, and emersion/immersion cycle for A. nodosum. No trend was observed for the third species. However, significant correlations between phenol levels of P. canaliculata and A. nodosum and measured air-temperature were found. Our results suggested an effect of aerial, aquatic and both conditions on the phenolic pool of P. canaliculata, B. bifurcata and A. nodosum, respectively, and a fast evolution of the phenolic pool on a day time scale.     

Short-term effects of temperature upon the growth of intertidal fucales

The growth rate of five species of intertidal Fucales (Pelvetia canaliculata (L.) Dec. et Thur., Fucus spiralis L., Fucus vesiculosus L., Fucus serratus L., Ascophyllum nodosum (L.) Le Jolis) was measured at temperatures from 2.5 to 35 °C. An increase in temperature immediately causes a high growth rate, and during the first hour it increases linearly with temperature; at 35 °C it is 20 times the control at 7 °C. This acceleration of growth is based mainly on stored photosynthate. After the first few hours the growth rate decreases rapidly, particularly at the highest temperatures. After 2–3 weeks a temperature optimum below 17.5 °C is indicated. High temperatures, 30–35 °C, were lethal to all species, with a survival time corresponding to their vertical zonation in the natural habitat.     

Enhanced leaf chlorophyll levels in plants treated with seaweed extract

Application to the soil of an aqueous alkaline extract ofAscophyllum nodosum resulted in higher concentrations of chlorophyll in the leaves of treated plants in comparison to control plants treated with an equivalent volume of water. Positive results were obtained with all species tested (tomato, dwarf French bean, wheat, barley, maize). When the seaweed extract was applied as a foliar spray, similar effects on leaf chlorophyll contents were obtained, except in the case of dwarf French bean plants, for which no significant difference was recorded between test and control plants. When the betaines present in the seaweed extract were applied as a mixture in the same concentrations as those in the diluted seaweed extract (-aminobutyric acid betaine 0.96 mg L^–1, -aminovaleric acid betaine 0.43 mg L^–1, glycinebetaine 0.34 mg L^–1), very similar leaf chlorophyll levels were recorded for the seaweed extract and betaine treated plants. This suggests strongly that the enhanced leaf chlorophyll content of plants treated with seaweed extract is dependent on the betaines present.     

Rapid bioassays to evaluate the plant growth promoting activity of <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> (L.) Le Jol. using a model plant, <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh

Ascophyllum nodosum extract products are used commercially in the form of liquid concentrate and soluble powder. These formulations are manufactured from seaweeds that are harvested from natural habitats with inherent environmental variability. The seaweeds by themselves are at different stages of their development life-cycle. Owing to these differences, there could be variability in chemical composition that could in turn affect product consistency and performance. Here, we have tested the applicability of using Arabidopsis thaliana as a model to study the activity of two different extracts from A. nodosum. Three different bioassays: Arabidopsis root-tip elongation bioassay, Arabidopsis liquid growth bioassay and greenhouse growth bioassay were evaluated as growth assays. Our results indicate that both extracts promoted root and shoot growth in comparison to controls. Further, using Arabidopsis plants with a DR5:GUS reporter gene construct, we provide evidence that components of the commercial A. nodosum extracts modulates the concentration and localisation of auxins which could account, at least in part, for the enhanced plant growth. The results suggest that A. thaliana could be used effectively as a rapid means to test the bioactivity of seaweed extracts and fractions.     

Bioactive compounds in seaweed: functional food applications and legislation

Seaweed is more than the wrap that keeps rice together in sushi. Seaweed biomass is already used for a wide range of other products in food, including stabilising agents. Biorefineries with seaweed as feedstock are attracting worldwide interest and include low-volume, high value-added products and vice versa. Scientific research on bioactive compounds in seaweed usually takes place on just a few species and compounds. This paper reviews worldwide research on bioactive compounds, mainly of nine genera or species of seaweed, which are also available in European temperate Atlantic waters, i.e. Laminaria sp., Fucus sp., Ascophyllum nodosum, Chondrus crispus, Porphyra sp., Ulva sp., Sargassum sp., Gracilaria sp. and Palmaria palmata. In addition, Undaria pinnatifida is included in this review as this is globally one of the most commonly produced, investigated and available species. Fewer examples of other species abundant worldwide have also been included. This review will supply fundamental information for biorefineries in Atlantic Europe using seaweed as feedstock. Preliminary selection of one or several candidate seaweed species will be possible based on the summary tables and previous research described in this review. This applies either to the choice of high value-added bioactive products to be exploited in an available species or to the choice of seaweed species when a bioactive compound is desired. Data are presented in tables with species, effect and test organism (if present) with examples of uses to enhance comparisons. In addition, scientific experiments performed on seaweed used as animal feed are presented, and EU, US and Japanese legislation on functional foods is reviewed.     

Bioprospecting of brown seaweed (Ochrophyta) from the Yucatan Peninsula: cytotoxic,antiproliferative, and antiprotozoal activities

In the Yucatan Peninsula coast, a large diversity of seaweed species are found, and recent studies have reported the presence of metabolites with pharmaceutical importance. In this study, a biological screening of brown seaweed extracts from Dictyota ciliolata, Padina sanctae-crucis, Sargassum fluitans, and Turbinaria tricostata was carried out. Their cytotoxicity and antiproliferative activities were evaluated by the sulforhodamine B assay on human embryonic kidney (HEK 293), human breast cancer (MCF-7), human prostate cancer (LNCaP), and human hepatic cancer (Hep-G2) cell lines. Seaweed extracts were also tested for their anti-trichomonal (Trichomonas vaginalis) and anti-giardicidal (Giardia lamblia) properties. Fucan fractions were extracted using successive maceration with ethanol/water and freeze-dried. Organics extracts were obtained from ethanol residue from liquid–liquid fractionation. A total of four ethanol extracts, four fucan-rich fractions, four ethanolic extracts, and 12 organic fractions were obtained. Only the ethanolic extracts from Turbinaria tricostata and D. ciliolata were active against LNCaP (CC₅₀ of 24.4 and 29.3 μg mL^?1, respectively). Interestingly, the activity found in the extracts from D. ciliolata and Turbinaria tricostata was maintained when both extracts were subjected to a liquid–liquid fractionation with hexane on the LNCaP cell line (CC₅₀ of 24.4 and 25.2 μg mL^?1, respectively). The antiproliferative assays showed that both dichloromethane and ethanolic fractions from P. sanctae-crucis were active against MCF-7, with IC₅₀ of 26.1 and 29.8 μg mL^?1, respectively. These species have been selected for further bio-guided fractionation and isolation of active compounds.     

Temporal dynamics of inducible anti‐herbivory defenses in the brown seaweed Ascophyllum nodosum (Phaeophyceae)1

Anti‐herbivory defenses support persistence of seaweeds. Little is known, however, about temporal dynamics in the induction of grazer‐deterrent seaweed traits. In two induction experiments, consumption rates of the periwinkle Littorina obtusata (L.) on the brown seaweed Ascophyllum nodosum (L.) Le Jolis were measured in 3‐d intervals. Changes in palatability of directly grazed A. nodosum were tested every 3 d with feeding assays using fresh and reconstituted seaweed pieces. Likewise, assays with fresh A. nodosum assessed changes in seaweed palatability in response to water‐borne cues from nearby grazed conspecifics. Consumption rates of L. obtusata varied significantly during the 27‐d induction phase of each experiment. Direct grazing by L. obtusata lowered palatability of fresh and reconstituted A. nodosum pieces to conspecific grazers after 15 d as well as after 6 and 12 d, respectively. After 12, 18, and 24 d, fresh A. nodosum located downstream of L. obtusata‐grazed conspecifics was significantly less palatable than A. nodosum located downstream of ungrazed conspecifics. Changes in L. obtusata consumption rates and A. nodosum palatability during both induction experiments suggest temporal variation of grazer‐deterrent responses, which may complicate experimental detection of inducible anti‐herbivory defenses.     

The effects of priming treatments using seaweed suspensions on the water sensitivity of barley (Hordeum vulgare L.) caryopses

Two seaweed suspensions, one prepared from Ascophyllum nodosum (Linnaeus) Le Jolis (ANS), another from Laminaria hyperborea (Gunn.) Foslie (LHS), were evaluated for their effects on the water sensitivity of barley (Hordeum vulgare L.) seeds. Priming in either seaweed suspension did not reduce viability. Priming in ANS was beneficial to seeds germinated in elevated water levels. The suspension of A. nodosum reduced the water sensitivity of barley seeds better than either water or polyethylene glycol (PEG) priming treatments. ANS also reduced the microbial population on the seeds by 86%. The hygroscopic properties and the antibiotic effect of the suspension of A. nodosum resulted in greater oxygen availability to the embryo, enabling more seeds to germinate under oxygen deficient conditions.     

Effect of seaweed extracts and plant growth regulators on high-frequency in vitro mass propagation of Lycopersicon esculentum L (tomato) through double cotyledonary nodal explant

An efficient and reproducible two-step in vitro propagation system for tomato (Lycopersicon esculentum) was developed by using the combinations of seaweed biostimulant (Gracilaria edulis and Sargassum wightii) extracts and plant growth regulators (PGRs). Double cotyledonary nodal (DCN) explants of Co-3 cultivar were initially cultured on Murashige and Skoog (MS) and Gamborg’s medium (B5) containing thidiazuron (TDZ) and 6-benzylaminopurine (BA); the best responding cytokinin was tested in combinations with different auxins (NAA, IAA and IBA), and seaweed extracts (G. edulis and S. wightii) of about basal MS medium +10–70% was used for shoot proliferation. The best organogenic culture response was obtained on MS medium fortified with 1.5 mg L^?1 TDZ and 1.5 mg L^?1 IBA. Up to 24 shoots per explants were formed at an optimal duration of exposure to 35 days. Mini shoots of about 3–4 cm were transferred to medium supplemented with MS + iP, MS + zeatin, MS + G. edulis and MS + S. wightii at different concentrations. High frequency of shoot elongation was observed in the medium supplemented with 30% G. edulis (15.2 cm), and profuse rooting was observed in the medium supplemented with 50% S. wightii of about 16.1 cm. Shoot elongation and rooting were observed in the medium supplemented with seaweed extracts. The plantlets were transferred to the plant growth chamber (70% of relative humidity and 9 light cycles) and maintained in it for a week, and then they were transferred to a greenhouse condition. The plant growth chamber to green house transferred plantlets showed an increase in the survival rate from 70 to 85%. Thus a two-step regeneration protocol was developed in this study with a combination of seaweed extracts and PGRs, which provides a basis for the production of transgenics with high frequency and survivability of tomato plants.     

Priming of Nicotiana benthamiana antioxidant defences using brown seaweed extracts

Extracts from the brown seaweeds Cystoseira myriophylloides, Laminaria digitata and Fucus spiralis were evaluated as plant defence inducers against the wild fire disease of Nicotiana benthamiana. Seeds’ imbibition in aqueous seaweed extracts (SE) results in plants with reduced disease severity. In addition, bacterial populations were significantly reduced in these plants when compared to those germinated in sterile distilled water. They primed N. benthamiana for H₂O₂ accumulation and for enhanced activity of several antioxidant enzymes including catalase, ascorbate peroxidase, guaiacol peroxidase and polyphenol oxidase. These results revealed that soaking of seeds in SE before sowing allows N. benthamiana to reduce pathogen attack and can be easily applied in practice.