2.3.3. Accumulation of Lignin Content

Different trend of the lignin deposition in treated roots was exhibited following elicitation by FSF and BBF (Figure 5). The level of this metabolite increased slightly after 12 h then greatly increased (*p* < 0.05) after 48 h of FSF treatment. This response was expected since lignin is a phenols polymer whose maximum accumulation was obtained after 24 h with FSF (Figure 4). On the other hand, with BBF treatment (Figure 5), the lignin contents undergo a weak increase after 24 h and 48 h of elicitation while after 96 h a higher increase was noted, this could be explained by the possibility of the polymerization of phenolic compounds increased after 72 h of BBF treatment. The highest content of lignin was obtained at 48 h in response to FSF and after 96 h of BBF treatment, it is three times higher than that obtained in control roots.

The crude fucoidans from *F. spiralis* (FSF) and *B. bifurcata* (BBF) were extracted and structurally characterized; the results obtained show a significant proportion of L-fucose in FSF and BBF with a higher degree of sulfation (45 and 49% *w*/*w* respectively). A biological test showed that the crude fucoidans (FSF and BBF) exhibit an eliciting effect of the defence mechanisms in date palm roots. These mechanisms were initiated by the induction of PAL activity. FSF and BBF expressed a slightly differential effect on PAL activity, which could be due to the difference in structure between the two fucoidans. Indeed, the structural characterization revealed differences in sulfate proportions within FSF and BBF, suggesting a difference in FSF and BBF affinity to membrane receptors. The perception and recognition of elicitors from pathogens, plants and algae called damage- or pathogen-associated molecular pattern molecules (DAMPs, PAMPs) by Pattern Recognition Receptors (PRRs) in plants

induces a signalling cascade in the host cell through their cytosolic domains leading to the induction of defence mechanisms in the host plant [39]. This could be the cause of the early induction of PAL activity after treatment with FSF compared to BBF. In addition, following the induction of PAL activity, the phenolic metabolites were accumulated in the treated roots, as well as lignin deposition. Phenols and lignin are among the most involved defence elements during date palm–Foa interactions [15–20]. The results obtained are similar to the reaction observed in tobacco plants pretreated with fucoidans and sulfated oligofucoidans, in which PAL activity was also induced [14]. In addition, such tobaccos demonstrate an increase in the activity of lipoxygenase (LOX) and Pathogenesis-related protein (PR), as well as transient defence reactions such as acidification of the cytoplasm and accumulation of H2O2 [14]. Based on this, it is possible to assume similar effects in the case of date palm root response to FSF and BBF, including also the induction of glutathione-S-transferase (GST) activity, as shown for tobacco [15]. In addition, a fucoidans pre-treatment of carrot leaves protects them against *Alternaria radicina* and *Botrytis cinerea* attacks by stimulating the accumulation of phenolic compounds and by inducing peroxidase (POD) and polyphenol oxidase (PPO) activities [40]. Thus, fucoidans-treated date palm roots, besides phenolic compounds accumulation, may also activate the POD and PPO enzymes. Otherwise, we have shown in recent work, that alginates extracted from the same brown algae *F. spiralis* and *B. bifurcata* stimulate the natural defenses of date palm in the same way as fucoidans, but the latter seem to be more active at low concentrations (0.5 g/L) compared to 1 g/<sup>L</sup> of alginates [41]. This could be explained by the structural difference between alginates and fucoidans, in particular the presence of sulfated groups and the degree of sulfation. The structure–function relationship governing the induction of plant defence mechanisms in response to fucoidans stile less elucidated. However it was reported that sulfation of polysaccharides alters their affinity for receptors located in cell walls [42]. In addition, the desulfation of sulfated polysaccharides reduces or eliminates their eliciting effect on natural defenses in tomatoes [43], whereas oligo-carrageenans (λ) with a higher degree of sulfation reduce the impact of various viral, bacterial and fungal diseases [44]. Furthermore a sulfated ulvan and oligo-ulvans improve the induction of PAL activity and therefore the accumulation of the phenols in tomato leaves [43], apple fruit [3] and olive tree [45]. Numerous works on the other biological activities of fucoidans relate their effectiveness to their structural characteristics, notably sulfation and molecular weight. It has been widely documented that fucoidans owe their broad spectrum of biological activities to their sulfated nature and molecular weights [6,27,28,46,47]. Based on previous work, fucoidans with a molecular weight (Mw) between 10 and 300 × 10<sup>3</sup> g/mole showed significant anticoagulant activity than those with higher Mw > 850 × 10<sup>3</sup> g/mole [48–50]. Likewise, immune-regulation activities increased with low molecular weight fucoidans of *Laminaria japonica* [51]. It has been shown also that the immune-regulatory potential of fucoidan was influenced by the sulfate group as well as the acetyl one [52]. In short, the biological efficiency of fucoidans was modulated by several parameters, in particular the proportion of SO4<sup>2</sup>− groups and their position, the molecular weight, the acetylation degree and monosaccharides composition [6,53].

**Figure 5.** Effect of sulfated polysaccharides from *F. spiralis* (FSF) and *B. bifurcata* (BBF) on the accumulation of lignin in date palm roots. Means values ± SE. Based on Tukey's test at 12 h, 24 h, 48 h and 72 h \* Control vs. FSF: *p* < 0.05, at 12 h, 48 h and 96 h \* Control vs. BBF: *p* < 0.05.
