In the Mediterranean Sea, Cystoseira sensu lato (s.l.) (Phaeophyceae) form extensive forests, which are among the most productive habitats in the coastal zone (Fabbrizzi et al., 2020 ). In recent decades, Cystoseira s.l. canopies have declined significantly in many coastal regions and are now critically threatened (e.g. Mangialajo et al., 2008; Falace et al., 2010; Vergés et al., 2014; Thibaut et al., 2015; Blanfuné et al., 2016; Valdazo et al., 2017), due to multiple stressors such as overgrazing, eutrophication, habitat fragmentation and climate change (e.g Thibaut et al., 2005; Mangialajo et al., 2008; Thibaut et al., 2015; Blanfuné et al., 2016; Mancuso et al., 2018; Bevilacqua et al., 2019; Falace et al., 2021). There is little evidence of natural recovery of damaged populations, hampered by low zygote and embryo dispersal (Clayton, 1990; Buonomo et al., 2017), so proactive actions are needed to support the regeneration of such populations. Restoration is increasingly recognized as useful tool to trigger the recovery of degraded coastal habitats (Abelson et al., 2020), as stated by the UN Decade of Ecosystem Restoration (2021-2030; https://www.decadeonrestoration.org/). Several techniques have been advanced for the restoration of Cystoseira s.l.: transplantation of adult thalli (Falace et al., 2006; Susini et al., 2007), in situ deployment of receptacles (Verdura et al., 2018; Medrano et al., 2020) and outplantation of seedlings grown ex situ (Falace et al., 2006; Sales et al., 2011; Falace et al., 2018a; Verdura et al., 2018; De La Fuente et al., 2019; Orlando-Bonaca et al., 2021; Savonitto et al., 2021; Lardi et al., 2022; Orlando-Bonaca et al., 2022). The latter two techniques are preferable to avoid further depletion of donor populations (De La Fuente et al., 2019).

Optimizing growth conditions can maximize zygote settlement and survival of the most critical embryonic stages and reduce the cost of long-term maintenance (Savonitto et al., 2021; Clausing et al., 2022; Orlando-Bonaca et al., 2022). Nevertheless, overgrowth of bacteria, epiphytic diatoms and filamentous algae could strongly limit growth and development of zygotes (Orlando-Bonaca et al., 2021; Clausing et al., 2022; Lardi et al., 2022). To overcome these issues, commercially manufactured extracts of a variety of seaweeds have recently been used to reduce epiphyte attachment (Jiksing et al., 2022) and as biostimulants to increase survival, growth and stress tolerance of macroalgae, including brown algae (Hurtado and Critchley, 2018; Umanzor et al., 2019; Hurtado and Critchley, 2020; Umanzor et al., 2020a; Umanzor et al., 2020b; Ali et al., 2021; Han et al., 2022; Jiksing et al., 2022; Umanzor et al., 2022). Macroalgae-derived biostimulants are also used as a sustainable option to improve agronomic production, plant growth and health (Crouch and Van Staden, 1993; Battacharyya et al., 2015; Hurtado and Critchley, 2020; Samuels et al., 2022), thanks to their broad spectrum of constituents (i.e., macro- and microelements, amino acids, hormones, phenolic compounds and saccharides) (Khan et al., 2009; Ali et al., 2021; Sujeeth et al., 2022). Biostimulants are defined as substances and materials, excluding pesticides and nutrients, that can modify the physiological processes of plants improving their growth, development and/or responses to stress (du Jardin, 2012; Rouphael and Colla, 2020). In particular, they promote natural processes for efficient nutrient uptake and utilization, chlorophyll content and photosynthesis, stress resistance, root development and also trigger early flowering and seed germination (Blunden and Wildgoose, 1977; Crouch and Van Staden, 1993; Van Oosten et al., 2017; Ali et al., 2021; Shukla et al., 2021; Sujeeth et al., 2022).

The interactions between host and microbiome can also play a crucial role in development and welfare in the early life stage, as has been shown for corals and sponges (Taylor et al., 2007; Cleary et al., 2022; Peixoto et al., 2022; Thatcher et al., 2022). In the marine field, microbe-assisted crop improvement is still in its infancy compared to agriculture, where plants benefit from mixtures of diverse microbes to protect them from pests or pathogens, improve nutrient supply and directly promote growth through microbial antagonistic interactions (Berg et al., 2021). A growing body of literature (reviewed in Florez et al., 2017 and Duarte et al., 2018) on microbe-algae highlights the paramount importance of the tiny unseen majority in morphological development (Matsuo et al., 2003; Grueneberg et al., 2016), antimicrobial activity and control of microbial colonization via quorum sensing (Manefield et al., 1999; Holmström et al., 2002; Rao et al., 2005; Wang et al., 2009) and algal status under healthy and stressful conditions across the lifespan (Ivanova et al., 2002; Mancuso et al., 2016; Minich et al., 2018; Juhmani et al., 2020). The microbial biofilm forms an outer layer over the host epidermis that plays a functional role between the host and the environment, and is almost considered a new and functionally different “tissue” (Steinberg et al., 2011). The microbes living on the algal surface have an abundance of 10⁶ to 10⁹ bacteria per cm² and a high phylogenetic diversity (Martin et al., 2014), and differ from the surrounding seawater community (Bengtsson et al., 2012; Michelou et al., 2013; Florez et al., 2017). The algae provide a rich substrate for the microorganisms to thrive, and antagonistic relationships can strongly shape the community alongside algal physiology. As the macroalgal surface is highly dynamic, the functioning of the microbial interface intimately depends on the complex relationships between the host and all-associated organisms (Trevathan-Tackett et al., 2019; Dittami et al., 2021). Hence it is useful to view it as a network of interactions forming a functional synergistic unit called a holobiont (Zilber-Rosenberg and Rosenberg, 2008). The associated microbes play a crucial role in the biology, ecology, and well-being of their hosts, and therefore altering these associations can lead to a pathological state within the holobiont (Egan and Gardiner, 2016; Pitlik and Koren, 2017; Sullivan et al., 2018; Longford et al., 2019; van der Loos et al., 2019).

In the context of ex-situ restoration, it may therefore be relevant to investigate the microbial biofilm associated with the algae in mesocosm cultures to identify possibly probiotic communities with beneficial effects on the algal fitness once they are outplanted in the field. As the application of algal-derived biostimulants in ecological restoration also needs to be explored (Hurtado and Critchley, 2020; Jiksing et al., 2022), in the present study, we used a commercial extract derived from Macrocystis pyrifera (Linnaeus) C. Agardh (Laminariales, Ochrophyta) as an additive to culture media for the cultivation of Ericaria amentacea (C.Agardh) Molinari & Guiry (Fucales, Ochrophyta) seedlings.

Within this framework, the aims of this study were: (i) to investigate the effects of different culture media on the survival, growth and photosynthetic efficiency of E. amentacea seedlings both in culture and in the field, (ii) to identify the microbial biofilm associated with E. amentacea seedlings during an ex-situ experiment and (iii) to create conditions to promote potentially beneficial microbes by using a seaweed extract.

Our results show that the commercial seaweed extract AlgatronCifo^® and von Stoch medium (VS) influenced seedling survival and growth as well as microbial biofilm communities. Overall, the A2 culture survived best in the field. VS, VSA and A1 were the second- best treatments, A3 performed poorly, while C seedlings did not survive in the field.

Brown algae extract with biostimulant properties have recently been used to improve harvest in aquaculture (Hurtado and Critchley, 2018; Umanzor et al., 2019; Hurtado and Critchley, 2020; Umanzor et al., 2020a; Umanzor et al., 2020b; Han et al., 2022; Jiksing et al., 2022; Umanzor et al., 2022). The priming effect is likely due to their complex composition and the presence of compounds with biostimulatory activities like polysaccharides (Shukla et al., 2021) and secondary metabolites. Phenolic compounds (Stirk et al., 2020; Sujeeth et al., 2022) play a crucial role in cell wall formation and early development of brown algae (Schoenwaelder, 2002), help adapt to environmental stressors and defend against biological pressures such as grazers, pathogens and epiphytes (Stiger-Pouvreau et al., 2014; Generalić Mekinić et al., 2019). In plants, algal-derived extracts have been shown to induce changes in the microbial community and inhibit plant pathogens (see references in Ali et al., 2021; Shukla et al., 2021).

In our study, the culture media (i.e., VS) and the addition of Algatron had a significant effect on the structure and composition of the biofilm communities associated with the seedlings ( Figure 11 ). The biofilm communities shifted in distinct ways over time, with an overall decrease in the diversity of OTUs compared to adult thalli. This could be due to the more controlled and less variable culture environment compared to the intertidal zone where E. amentacea thrives, where desiccation, wave dynamics, temperature stress and ever-changing seawater conditions (i.e., nutrients, salinity, pH, pollution, etc.) strongly influence the algae and their biofilm.

In our experimental setting, we cannot unambiguously distinguish between the effect of the additive on algal physiology, which in turn modified the microbes (indirect effect of biostimulant and nutrients, via algal metabolism, Ren et al., 2022) and the direct effect of biostimulant/nutrients on microbial growth and community structure.

At the end of the mesocosm culture (T3), the treatments with VS (i.e., VS, VSA) and A1 and A2 showed comparable seedling coverage. However, A2 and A1 also showed optimal photophysiological fitness (F_v/F_m > 0.69). The VS and VSA treatments had the lowest F_v/F_m values (0.51 ± 0.02 and 0.46 ± 0.02, respectively), indicating stress to the photosynthetic apparatus (Celis-Plá et al., 2014; Smolina et al., 2016; Falace et al., 2018b; Savva et al., 2018; Verdura et al., 2021). After the field period, all treatments showed good photosynthetic efficiency, with A2 having the highest coverage, being 1.8, 1.9, 2.2 and 6.0 times higher than VS, VSA, A1 and A3, respectively.

We hypothesize that the higher survival is due to the interplay of nutrients, presence/absence of epiphytes and their antagonistic or beneficial relationships with the E. amentacea seedlings and finally priming by the algal extract.

While E. amentacea is mainly restricted to oligotrophic waters and is sensitive to eutrophication (Pinedo et al., 2007; Mangialajo et al., 2008; Thibaut et al., 2014), seedlings may have a higher nutrient uptake capacity than adult thalli, as observed in other Fucales (Thomas et al., 1985; Sánchez de Pedro et al., 2022). Although culture media were frequently renewed, C-culture seedlings likely suffered from nutrient limitation ( Supplementary Table 1 ), which may have affected many processes like photosynthetic capacity, embryonic development and growth (Duarte, 1992; Roleda and Hurd, 2019). Lower growth of E. amentacea seedlings in seawater compared to VS was also observed by Susini (2006). The lowest performance in terms of survival and growth of C seedlings was followed by total failure in the field. We found that the C biofilm differed the most from the other treatments (in the PCoA plot) despite the high alpha diversity. This treatment shared fewer OTUs with the adult thalli (13/64 scoring > 1% relative abundance) than the other treatments with nutrient addition, indicating the importance of microbial interactions and algal growth in co-shaping the microbial biofilm, as reported in Sargassum (Hervé et al., 2021) and Macrocystis (Michelou et al., 2013).

The VS and VSA treatments had high nutrient concentrations, with the highest nitrate ( NO 3   − ) and phosphate ( PO 4   3 − ) levels of all treatments ( Supplementary Table 1 ), which were 5.25 and 5.32 fold and 26.5 and 32.3 fold higher than the C-treatment, respectively. NO 3   − is one of the two main sources of nitrogen (N), which is essential for macroalgal growth as it is a major component of the photosynthetic apparatus, amino acids and cellular enzymes, and thus can be a critical limiting factor in the marine environment (Hurd et al., 2014). As well, phosphorus (P) is a vital nutrient for macroalgae, involved in algal photosynthesis and respiration, particularly energy transfer through the synthesis of adenosine triphosphate (ATP) and other energy-rich compounds (Lobban and Harrison, 1994). The nutrient-rich conditions, characterized by a lower N:P ratio in VS and VSA (10.29 and 65.26 respectively, Supplementary Table 1 ) which fall within the optimal range for seaweeds growth (i.e., from 10:1 to 80:1; Suthar et al., 2019), simultaneously promoted the highest growth of seedlings, but also, as expected, of epiphytes. Indeed, fast-growing opportunistic epiphytes (i.e., filamentous algae, diatoms, cyanobacteria) have a competitive advantage at high nutrient concentrations by maximizing rapid nutrient uptake and photosynthetic efficiency (Fujita, 1985; Carpenter, 1990; Duarte, 1995). Furthermore, when growing on the outer surface of seedlings, they compete for light (Pang et al., 2011) and act as a barrier to carbon uptake (Sand-Jensen, 1977), resulting in lower photosynthetic efficiency of their hosts. In algal cultures, epiphyte infestation has been shown to impair algal productivity (Lüning and Pang, 2003; Ward et al., 2020) and is one of the main barriers to ex situ culture of Cystoseira s.l. (e.g. Orlando-Bonaca et al., 2021; Clausing et al., 2022; Lardi et al., 2022). In addition, epiphytes can in some cases damage host tissues (Hayashi et al., 2010) and facilitate colonization by opportunistic bacteria (Vairappan et al., 2008; Jiksing et al., 2022) that cause disease.

Contrary to the treatments with VS (i.e., VS, VSA), in the Algatron treatment, the main source of N was ammonium ( NH 4   + ) ( Supplementary Table 1 ), which may be a more efficient form of N fertilizer for brown algal growth (Dā Costa Braga and Yoneshigue-Valentin, 1996; Smart et al., 2022). Among these treatments, A3 showed the lowest performance in terms of growth and survival. Seedlings cultivated in A3 treatment were exposed to the highest ammonium concentration ( NH 4   + ) ( Supplementary Table 1 ), which might have been the cause of the low cover despite the good Fv/Fm. In an indoor culture of Sargassum spp. seedlings, NH 4   + was shown to effectively promote their growth (Han et al., 2018) and increase photosynthesis (Hong et al., 2021), but a too high ammonium content (i.e., 900 μmol·L^-1) can negatively affect growth (Hong et al., 2021). It has also been noted that algal extracts can have both positive and negative effects depending on the concentration (Kapoore et al., 2021). A1 and A2 seedlings had on average the same F_v/F_m, with A2 seedlings showing higher growth and survival, indicating a more suitable concentration than A1. In treatments A1 and A2, NH 4   + was supplied at concentrations 4.5 and 2 times lower, respectively, than in A3 ( Supplementary Table 1 ), which promoted the growth of E. amentacea without favoring the development of epiphytes. Apart from high NH 4   + availability, other constituents of the algal extract Algatron may have induced changes in seedlings metabolism and signaling pathways specifically related to nutrient uptake and/or nutrient translocation improving nutrient use efficiency, as observed in plants (Jannin et al., 2013; Saa et al., 2015; Sujeeth et al., 2022). Nevertheless, in our experimental settings we cannot unambiguously assess the effect of the biostimulant versus the ammonium ( NH 4   + ) per se. The toxicity of A3 medium to the seedlings could be due to high ammonium concentration. On the other hand, the lower cover (%) of seedlings under A1 than in A2 media showed indeed an effect of biostimulant because nutrients (i.e., nitrate and ammonium) were not limiting in the A1 medium. Further studies are required to disentangle a possible biostimulant action from the NH 4   + effect on the seedling growth. Moreover, complementary studies using -omics techniques (i.e., metabolomics and/or transcriptomics) could identify the modes of actions and the metabolic pathways by which the complex extract might improve seedlings performance.

The lower occurrence of opportunistic epiphytes can also be attributed to the lower PO 4 3 − concentrations compared to VS and VSA, since fast-growing species have much higher P-demands than slower growing algae (Pedersen et al., 2010). According to the N:P ratio at which nutrients were supplied in Algatron treatments (A1 = 278.5; A2 = 324.58; A3 = 350.93, respectively), there would be an external P-limitation also for seedlings development, nevertheless the culture media were suitable for seedling growth, especially in A2, where the PO 4 3 − was 5.85 times higher than in the C treatment despite a relatively more balanced N:P ratio in the latest (i.e., 60.1) ( Supplementary Table 1 ). Indeed, to avoid misuse, the N:P ratio should be interpreted along with the absolute amounts of each nutrient (Dodds, 2003).

The good performance in terms of growth, survival and photophysiological fitness of the seedlings in A2 treatment might have played an important role in the success of this treatment in the field. In previous ex-situ outplantings, high detachment of E. amentacea seedlings was documented after transfer to the field (De La Fuente et al., 2019; Clausing et al., 2022). The high survival of A2 seedlings in the field might be related to the ability of the seedlings to anchor to the tiles due to the alginate-enriched algal extract, which can act as a metabolic stimulant and trigger favorable physiological responses (Briceño-Domínguez et al., 2014; Shukla et al., 2021). Although the synthetic pathways of alginates are not fully understood, it is thought that alginates are first synthesized as a polymer of mannuronic acid (a component of alginic acid) by alginate synthase and that some of the mannuronic acid is converted to guluronic acid (Michel et al., 2010). Alginates can influence the mechanical properties of cell walls in the rhizoids of developing zygotes of Fucales (Linardic, 2018; Yonamine et al., 2021) and provide for a stronger rhizoid system. We can assume that the A2 seedlings had stronger rhizoids that allowed them to adhere more firmly to the tiles. Considering that E. amentacea thrives in wave-exposed sites (Agardh, 1842; Boudouresque, 1971; Thibaut et al., 2014), it follows that well-developed rhizoids could promote better seedling survival.

The 20 most dominant families we found on the seedlings and adult thalli of E. amentacea were also identified as the most abundant on other brown algae (Florez et al., 2017 and reference), as Fucales and Laminariales. This suggests a great microbial plasticity that allows them to thrive with ecologically diverse algae. Within these families, there are many multitudes of microbial adaptive strategies for growth, nutrient uptake, and antagonistic interactions. From an algal perspective (Florez et al., 2017), studies have shown that Rhodobacteraceae, Flavobacteriaceae, Alteromonadaceae, Vibrionaceae, and Halomonadaceae can induce morphogenesis and degrade algal compounds. Rhodobacteraceae, Flavobacteriaceae, Alteromonadaceae, Vibrionaceae, Halomonadaceae Bacteriovoraceae, Saprospiraceae may be related to pathogenesis and antibacterial activity. Furthermore, microbially induced diseases have been identified in Laminaria, such as hole-rotten disease, red spot disease, spot-wounded fronds and swollen gametophytes and filamentous fading caused by Pseudomonas, Vibrio, Halomonas and Alteromonas strains (Egan et al., 2014). It follows that biofilm-associated microbes can become opportunistic pathogens when the host is stressed by temperature, UV and nutrient stress, eutrophication or other anthropogenic induced challenges (Egan et al., 2014; Duarte et al., 2018; van der Loos et al., 2019).

Overall, the adult thalli have structured seedlings biofilm community composition. Taxa that were dominant in the adult thalli decreased in the seedlings and vice versa. Taxa that were dominant in the seedlings were copiotrophs, particle- or biofilm-associated (Lauro et al., 2009; Heins and Harder, 2023) and high-efficient organic matter degraders (Cottrell and Kirchman, 2000). Given these features, we can surmise that these microbes were closely associated with algal growth. The seedling biofilm communities were characterized by metabolisms predicted by the FAPROTAX annotation, which primarily used chemical energy released by breaking chemical bonds to degrade organic carbon from the primary production of algae and alginates contained in the algal extract Algatron.

Index species analysis based on pre-infield biofilm communities revealed that the best performing treatment, A2, was uniquely characterized by 5 Gram-negative aerobic genera Postechiella, Roseovarius, Winogradskyella and Arenibacter. Many strains from these genera have been isolated from diverse marine systems, indicating the huge metabolic flexibility (Lee et al., 2012; Luo and Moran, 2014; Zhuang and Luo, 2020). Furthermore, these unique taxa have been reported as easy to cultivate and purify, in relation to the fact that not more than 1% of the microbes in every environment can be cultivated. This is an extremely important and desirable feature for planning future probiotic-focused bacterial strain cultivation efforts. Postechiella, Winogradskyella and Arenibacter (Flavobacteriaceae) have been shown to be able to degrade agar, DNA, starch and many diverse sugars beside proteins and lipids (Lee et al., 2012; Gutierrez et al., 2014; Kurilenko et al., 2019). The microbial degradative activities could be important for supplying the algae with nutrients from the carbohydrate, protein and lipid pools of the algal extract, which can then be used for primary production. Another possible function is the control of biofilm structure and dynamics by DNase and sugar and protein hydrolysis activities (Whitchurch et al., 2002; Wang et al., 2004). Arenibacter is also able to degrade polycyclic aromatic hydrocarbon compounds. This is interesting given that E. amentacea, like the other brown algae, produces polyphenols (Generalić Mekinić et al., 2019) and Algatron contains these compounds. Polyphenols are important for chemical defense against herbivory (i.e., non palatability) and microbes, and for protection against oxidative stress by absorbing harmful UV and excessive irradiation. Thus, for microbes being able to degrade a toxic substance as phenols could therefore be beneficial for more efficient microbial competition for space and nutrients. The Roseovarius (family Rhodobacteraceae) genome presents pathways for the production of thiamine and cobalamin (Luo and Moran, 2014). Thiamine and cobalamin are important micronutrients for algae (Croft et al., 2006), playing key roles in their central metabolism. Many genera belonging to the Rhodobacteraceae produce these vitamins and share them with primary producers within the virtuous cycle of organic matter production and remineralization (Bertrand and Allen, 2012; Luo and Moran, 2014).

A2, A1, VSA and VS shared the taxon Psychroserpens (Flavobacteriaceae, Ping et al., 2022), which is able to degrade alginate and casein, suggesting a role in degrading sugar-rich and phospho-protein rich compounds within the biofilm. Interestingly, some strains require vitamins for growth thus being a competitor for these micronutrients in the algal biofilm.

A few studies have found that secondary metabolites from macroalgal extracts have strong effects on microbial surface colonization, altering the bacterial biofilm formation and community composition under laboratory and field conditions (Sneed and Pohnert, 2011; Egan et al., 2013; Lachnit et al., 2013).

Further experiments should elucidate the direct or indirect effect of nutrients and biostimulants in promoting a probiotic microbial community by investigating the gene expression of algae and microbes along the diverse treatments and their degree of interdependence. It would be important to track O₂ evolution using optode technique in the key treatments to guide microbial sampling when changes in algal performance occur. Such an approach will allow coupling algal performance with microbial community response and fill the gap in the functional role of microbes in macroalgal holobiont health (Duarte et al., 2018).

Given the seedling success of A2, we propose treatment tailored probiotic consortia candidates characterized by the unique treatment-taxa (A2: Postechiella, Roseovarius, Winogradskyella and Arenibacter) and the reminder community. At this stage, we cannot disentangle the net contribution of the unique vs. shared vs. reminder community in relation to the overall seedling fitness. Within the holobiont concept, we can hypothesize that the nature and intensity of interactions might be important in defining probiotic consortia. Furthermore, the effect of algal extract or nutrients on the algae and/or biofilm could have important consequences for tuning the overall interaction network. Our study has shown that ex-situ restoration of macroalgae could benefit from both the use of commercial algal extracts in ex-situ cultures and the identification of probiotic consortia candidates that promote seedling growth and optimal protection against biotic and abiotic stressors.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BioProject, PRJNA922753.

AF, SK, FM designed the study. LA, FM, SK, AS, CB, FG, AF performed the laboratory measurements. AP, FM, SB, AS, SN performed the formal data analysis. FM, SK, AF wrote the original draft of the manuscript. All authors contributed to the article and approved the submitted version.