Seaweeds and algae

Seaweeds have been used in agriculture for centuries and were among the first source materials used to produce agricultural biostimulants (Craigie, 2011; Khan et al., 2009). Since the introduction to commercial agriculture, seaweed extracts (SWEs) have been subject to the extensive characterization of critical components and potential mode(s) of action (Abbott et al., 2018; Shukla et al., 2019; Halpern et al., 2015; Ertani et al., 2018). The determination of SWE composition and the identification of biological effects are, however, greatly complicated by the substantial diversity in sources of available SWE and the manner in which SWE extracts are prepared (Khan et al., 2009). Even within seaweed species, studies have found a widely varying composition of amino acids, secondary/exogenous metabolites, hormones and minerals and consequently have observed varied effects on plant growth, stress tolerance and mineral acquisition. Recognizing this diversity of composition and effect, several recent reviews have appropriately focused on the analysis of the composition and function of individual species of seaweeds rather than attempting to provide a unified explanation for the function of SWEs as a whole (Shukla et al„ 2019; Ertani et al., 2018). The complexity and varied nature of plant responses to SWEs was nicely illustrated in the recent work of Ertani et al. (2018) who examined six commercial SWEs from Ascophyllum nodosum and Laminaria sp. While all SWEs tested resulted in varied but significant increases in root growth and esterase activity, the effects on mineral concentrations and sugar contents were inconsistent (Ertani et al., 2018).

Despite the wide diversity in species, origin and preparation for various SWE products, it is widely observed that SWEs enhance root growth and less frequently shoot growth, with consequent effects on nutrient content and concentration and hence AE-NUE. Given the effects on root and shoot growth, it is frequently unclear if the effects of SWEs on NUE are a consequence of increased nutrient demand, increased root exploration, increased activity of root transport processes and/or more efficient internal utilization of nutrients.

Modes of action relevant to nutrient use efficiency

Root growth

SWEs have long been observed to enhance root growth with explanations, including the beneficial effects of SWEs on soil structure by virtue of improved aggregate formation, hydrophilic characteristics of alginates and other gellike components and positive effects on the soil microbial composition and function (Shukla et al., 2019). These beneficial effects are frequently greater when applied to young plants or during periods of drought stress and when applied as root drenches orthrough targeted deposition in the root rhizosphere through drip irrigation (Khan et al., 2009). Given the small volumes of SWEs typically applied under commercial field conditions and recognizing that SWEs are highly biologically available and hence would rapidly be degraded (Craigie, 2011), it is perhaps not surprising that applications need to be carefully timed and precisely placed to achieve positive effects. While the SWE would not be expected to persist long in field soils, the benefits of the enhanced root growth and improved establishment of tolerance of stress may have long-term benefits.

Investigations of the mechanism by which SWEs improve root growth have largely been conducted under highly controlled environment assays, a condition that effectively eliminates the possibility of benefits to soil health and soil structure of soil microbial dynamics. Nevertheless, under these conditions, benefits to root production are frequently observed, suggesting that SWEs positively influence endogenous phytohormone production (Craigie, 2011; Ertani et al., 2018; Shukla et al., 2019). Positive effects from SWE application are not always seen, which may be a consequence of the concentrations used; dose dependency is also a characteristic of many phytohormone responses (Wally et al„ 2013). Despite this apparent contradiction, a majority of literature supports that SWEs increase root growth across various phenological stages (Jannin et al„ 2013; Sharma et al., 2014).

Because of the similarity in plant response to SWEs, with responses seen as a result of phytohormone applications, it has long been suggested that SWEs function by virtue of the phytohormone content within SWE extracts (Craigie, 2011; Khan et al., 2009; Kurepin et al., 2014). More recent examinations have suggested, however, that at the rate of SWEs used and given the frequently modest levels of phytohormone concentrations it is unlikely that phytohormone content in SWEs is responsible for any observed phenotypic changes; rather, it appears likely that SWEs stimulate phytohormone pathways through a process that is not well understood (Shukla et al., 2019; Ertani et al., 2018), perhaps by virtue of other molecules within SWEs that interact with endogenous phytohormone production.

Nutrient transport

SWE-induced root growth responses are likely a primary driver of enhanced AE as a consequence of increased root absorptive surface area and hence better utilization of soil reserves; however, it has also been shown that some formulations of Ascophyllum nodosum can enhance nutrient uptake through mechanisms other than root stimulation (Ertani et al„ 2018). Indeed, there are many reports of altered nutrient content and nutrient concentration in experiments conducted in well-stirred solution culture with the abundant provision of soluble nutrients, conditions under which root growth would not be expected to have a substantial effect on nutrient acquisition.

Modulation of transporter gene expression has been observed in a number of experiments (Jannin et al., 2013; Billard et al., 2014). On occasions, however, changes in gene expression have not resulted in predictable changes in tissue nutrient concentrations.

Despite no significant differences in shoot N concentration of Brassica napus, microarray analysis revealed that both BnNRT1.1 and BnNRT 2.2 were significantly upregulated by 68 fold and 16 fold, respectively, relative to control (Jannin et al„ 2013).

While there are a large number of reports of SWE effects on nutrient concentration and content, many such reports have not adequately controlled the interactive effects of enhanced growth on nutrient dilution, both within plant and growth media on the final measured nutrient concentration. The frequent reports of simultaneous increase in the concentration (often incorrectly reported as content) of a diverse range of nutrients, both monovalent and divalent and cationic and a nionic for which there would have to be simultaneous upregulation of a wide diversity of transporter and assimilation processes, suggest that many observed effects of SWEs on NUE are likely secondary in nature occurring as a consequence of growth promotion.

Nutrient assimilation and storage

Nitrate reductase (NR), putatively a key rate-limiting step in nitrogen assimilation, is increased by SWE application (Billard et al., 2014; Chouliaras et al., 2009; Jannin et al., 2013; Rouphael et al., 2017c, 2018; Sabir et al., 2014; Zodape et al., 2011; Suzuki et al., 2010). Increases in NR activity, however, do not always result in predictable changes in plant nitrogen status, perhaps due to growth-induced nutrient dilution. SWEs have also been shown to increase the expression of glutamine synthetase (GS1) in spinach (Fan et al., 2013), which plays an important role in the regulation of plant nitrogen cycling (Masclaux-Daubresse et al., 2010).

A large body of research has established that SWEs can effectively mitigate plant stress caused by salinity, heatorwaterdeficit(Shukla etal., 2019). Enhanced plant senescence caused by stress will reduce the duration during which nutrient remobilization can occur and hence will reduce physiological NUE (Uauy et al., 2006; Erenoglu et al., 2011; Masclaux-Daubresse et al., 2010). Positive effects of SWEs on stress-induced senescence, which have been widely observed, will therefore have beneficial effects on internal NUE and realize a greater yield (Brown and Saa, 2015). This is a promising but underexplored use of SWEs.


SWEs have been clearly demonstrated to improve NUE under both field and controlled growth conditions. The most compelling mechanism by which this could occur is through stimulatory effects on root growth and thus enhanced exploration of the soil volume which would result in improved agronomic NUE. While SWEs also clearly influence the expression of nutrient transport and assimilation processes, there is an inconsistency between observed upregulation of these processes and tissue nutrient content, and it remains uncertain if these effects are direct or a consequence of gross stimulatory effects of SWEs on plant metabolism.

The greatest promise for SWE use to positively influence NUE in crop plants is likely to occur through the enhanced establishment of roots in seedlings and the mitigation of plant stress events that compromise root uptake and result in enhanced tissue senescence and hence compromised remobilization of nutrients. Since SWEs are likely to be rapidly broken down in the environment, there is an imperative to time the application of SWEs to mitigate these stressors and to place SWEs precisely in the root zone or as a targeted foliar application.

As is the case with many classes of biostimulants, there is dramatic diversity in source and preparation techniques utilized to prepare commercial SWE biostimulants; given this diversity, it is unlikely that a single unified explanation for the beneficial effects of SWEs will be identified, and hence there is an imperative that each commercial SWE undergo careful well-replicated field testing.

Protein hydrolysates

Protein hydrolysates (PHs) are recycled plant/animal waste products that may enhance plant growth and improve plant mineral nutrition. Given the diversity and inconsistency of source materials used for these products, validation of these products as a class faces considerable challenges. Wide variations in processing, application method, cropping systems use, management practices and analytical methodology further complicate this analysis. PHs can be divided into two general categories: animal (typically derived from connective tissues) and plant (typically derived from unharvested plant parts of by-products of agricultural processing). The use of agricultural and industrial by-products as a source of plant biostimulants has been proposed as a potentially important aspect of the circular-economy concept (Xu and Geelen, 2018). A distinguishing characteristic of PHs is their relatively high concentration of soluble proteins, peptides and both essential/non-essential and free or peptide-bound amino acids. Additionally, PHs frequently contain significant concentrations of carbohydrates but negligible quantities of mineral nutrients, phenols, phytohormones and other organic compounds (Colla et al., 2017a; Tejada et al., 2018; Vesela and Friedrich, 2009).

Despite the wide array of source materials and preparation methods, PH studies have generally shown that PHs can affect mineral nutrition in plants. Results are, however, frequently inconsistent, perhaps due to differences in experimental designs, crop, methodology, environmental conditions or PH composition. The variation in responses is observed in both types of PHs (i.e. animal and plant-derived) and across a wide range of application rate/method.

Regardless of the underlying cause(s), these 'outliers' should be seriously considered (Lucini et al„ 2015; Cerdan et al., 2009; Tsouvaltzis et al., 2014).

Examples of enhancements of physiological processes from PH application are sparse and primarily limited to measurements of photosynthetic parameters such as chlorophyll concentration and quantum efficiency. PH interaction with photosynthetic parameters is not fully understood, given the variability in findings across studies, with approximately 40% of the surveyed studies finding no significant difference in chlorophyll concentration (Colla et al., 2014; Ertani et al., 2013; Grabowska et al., 2012; Lucini et al., 2015; Paradikovic et al., 2011; Rouphael et al., 2017a, 2018; Sestili et al., 2018).

Modes of action relevant to nutrient use efficiency

Direct effects of nutrient form in soils

Depending upon environmental conditions, plants can extract nutrients from variable forms in the soil. Several studies have postulated that organic nutrient forms present in PHs or resulting from PH application may explain the observed increases in NUE. To test this hypothesis, Teixeira et al. (2018) investigated the effect of isolates and combinations of cysteine, phenylalanine, glycine, glutamate on nitrate and total N uptake. In both PH and amino acid combinations, they found results similar to those reported earlier, notably no consistent effect on leaf NR activity but an increase in leaf nitrate concentration, suggesting that amino acids may be involved in the modulation of assimilation/ storage networks observed with PH application. This relationship is still inconclusive, given that PH and amino acid isolates are yet to be tested on full cropping cycles (Teixeira et al., 2018).

At the rates of PHs utilized in most commercial applications, the total N applied and the content of putative nutrient complexing amino acids are far too low to influence gross nutrient dynamics, particularly for full-growth cycle experiments. PH applications directly to seeds or transplants or injected precisely into root rhizosphere may indirectly increase full-season nutrient use by stimulating early phenological states where minute amounts of nutrients can significant effects.

Root growth/morphology

Root growth increase is a commonly reported effect of PHs. Increases in root growth translate to increased surface area and root zone exploration for nutrient absorption - effectively increasing the total uptake of nutrients and improving AE. Field-based and season-long studies are, however, limited, with most studies being conducted for short time frames under solution culture and in greenhouse studies. PH applications increased root growth in maize and lettuce under both optimal and limiting conditions (Rouphael et al., 2017b; Lucini et al., 2015; Santi et al., 2017; Ertani et al., 2009, 2013). Studies in tomato suggest that the root growth response interacts with the mode of application (i.e. root vs foliar application) but was unaffected by nutrient conditions (Sestili et al., 2018).

Microarray analyses suggest that the effects of PHs on root growth are related to an array of factors, including the expression of transcription factors controlling lateral root formation, cell wall components and hormone metabolism/synthesis/signaling (Colla et al., 2017b; Paul et al., 2019). Regardless of the specific pathway(s), microarray analyses have demonstrated that PHs are mechanistically distinct from isolated amino acid components in affecting root growth and ultimately distinct in how they affect nutrient acquisition (Santi et al., 2017).

In addition to root growth stimulation, it has been shown that soybean- derived PHs can modulate root morphology through root hair-promoting peptides (RHPPs) that activate root hair formation by increasing the density of trichoblasts (root hair cells) and atrichoblasts (root hairless cells). RHPPs increased the root surface area 16.6 times relative to control and altered root morphology through mechanisms physiologically distinct to other plant hormones (Matsumiya and Kubo, 2011; Matsubayashi and Sakagami, 2006). A subtilisin-like alkaline protease in B. circulans HA12 produces RHPP from soybean meal, which is also a component in the commercial PH 'Trainer' with several sources corroborating its beneficial root growth effects (Matsumiya and Kubo, 2011; Rouphael et al., 2018; Lucini et al., 2015; Colla et al., 2017a, 2014; Sestili et al., 2018). In these studies, PH 'Trainer' significantly increased both shoot and root growth - ultimately increasing nutrient uptake, which would result in increased AE. Inconsistency in the effects of PHs on nutrient concentration in plants suggests that either limiting factors exist upstream from uptake or nutrient uptake is diluted by growth -the latter likely the predominant factor (Cerdan et al., 2009; Colla et al., 2014, 2017a; Ertani et al., 2009, 2013; Lucini et al., 2015; Rouphael et al., 2017a,b, 2018; Santi et al„ 2017; Sestili et al., 2018; Tejada etal., 2018).

Several research groups have demonstrated that PHs can modulate salt stress response through enhanced production of compatible solutes of low molecular weight that have been documented as tools for osmotic adjustment and/or involved in stress signaling during salt stress (Colla et al., 2017b; Van Oosten et al., 2017; Ertani et al., 2013; Lucini et al., 2015; Petrozza et al„ 2014; Rouphael et al., 2017a). Microarray analyses have found that stress-related transcripts are shared and differentially expressed between PH and equivalent amino acid treatments, suggesting that PH stress-related response is partially linked to free amino acid components yet also due to different components (Santi et al., 2017).

Improved growth under salt or drought stress conditions have been observed and will generally increase use/uptake efficiency as continued plant growth results in greater nutrient uptake and hence an improvement in agronomic nitrogen use efficiency (AE).

Nutrient transporter and assimilation

Microarray analyses have shown that PHs affect the gene expression of transporters and related transcription factors (TFs). In maize roots, it was found that the TCP family, putatively related to the regulation of dual-affinity NRT1.1 expression, was differentially expressed between PHs, equivalent amino acid treatment and equivalent inorganic N treatment (Sestili et al., 2018; Santi et al., 2017). These findings suggest that different forms of N differentially affect TF expression, thereby modulating transporter expression and nutrient uptake. However, the analysis of transporter/TF expression must consider that intrafamily regulation of transporter proteins and posttranslational modifications prevent causal extrapolation of expression variation to observed differences in nutrient uptake. Putative effects on transporter networks are yet to be proven across multiple species, suggesting that a general mechanism for enhancing nutrient transport is yet to be proven for higher plants. Furthermore, modulation of transporter expression is network specific. This distinction is evident in the observed down-regulation of high- affinity nitrate transporters, NRT 2.1 and NRT 2.3, in tomato roots across the levels of N deficiency/sufficiency. Additionally, under N sufficiency, the root application of PHs was associated with increased expression of ammonium transporters while under N deficiency, the same treatment was associated with increased expression of amino acid transporters. These observations suggest that the PH not only modulates transporter expression but is so selectively dependent on conditions of nutrient availability (Santi et al., 2017; Sestili et al., 2018)

Concomitant with observed increases in nitrate and ammonium concentration of tomato leaves, NR in leaves was decreased after legume-derived PH application (Sestili et al„ 2018; Colla et al., 2018). Contrary to these results, increases in nitrate assimilation were observed in roots under N sufficiency regardless of the application method (Sestili et al., 2018), and PHs were observed to decrease leaf nitrate levels in a variety of vegetables (Colla et al., 2018). Interestingly, Ertani et al. (2009) found that under N-sufficiency nitrate levels significantly decreased in a dose-dependent fashion in maize roots, 14 days after seeding and 2 days after treatment with both alfalfa and meat-flour PHs. Observed decreases in root nitrate corresponded to increased NR and glutamine synthetase (GS) activity, also in a dose-dependent fashion. Furthermore, leaf nitrate concentration increased despite counteracting increases in biomass. These trends confirmed those found in tomato by Sestili et al. (2018) and suggest there may be a common mechanism for modulating assimilation/storage networks between legume-derived and animal-derived PHs (Ertani et al., 2009).

Given that both ammonium and nitrate concentration of leaves increased across multiple studies, N deficiency/sufficiency and the mode of application, it is likely that observed increases in total N are generally attributable to root growth effects and to a lesser extent depend upon assimilation/storage networks as explained earlier.

While the specific effects on N uptake and assimilation described earlier and non-specific increases on general nutrient uptake as a result of root and shoot growth have been widely reported, specific effects on the uptake or transport of other nutrients have not been widely observed. In iron-limited conditions induced by liming, Cerdan et al. (2009) found that PHs from plant origin applied as a root drench can increase the concentration of iron without increasing shoot or root growth of tomato. This increase in uptake efficiency was associated with increased Fe(lll) chelate reducatase (FCR) activity. In contrast to observed positive effects from soil drench, Cerdan et al. (2013) found that foliarly applied PHs from plant origin and all modes of application of PHs from animal origin significantly decreased iron concentration.


Given the wealth of studied components and positive effects associated with PH application, it is likely that the PH enhances NUE via multiple mechanisms. With the exception of consistent effects of PH on root growth response, there is a wide variability of findings with regards to the effects on specific uptake processes (transporters and transport regulation), the activity of assimilatory pathways and effects on tissue nutrient content and concentration. This may be a result of the great diversity in PH products present in the marketplace and may suggest that some PH components have specific effects on critical metabolic pathways. As with each of the biostimulants reviewed in this chapter, the effects of PH on root growth are clearly a primary driver of enhanced NUE.

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