Abstract
The growing population requires sustainable, environmentally-friendly crops. The plant growth-enhancing properties of algal extracts have suggested their use as biofertilisers. The mechanism(s) by which algal extracts affect plant growth are unknown.
We examined the effects of extracts from the common green seaweed Ulva intestinalis on germination and root development in the model land plant Arabidopsis thaliana. Ulva extract concentrations above 0.1% inhibited Arabidopsis germination and root growth. Ulva extract <0.1% stimulated root growth. All concentrations of Ulva extract inhibited lateral root formation. An abscisic-acid-insensitive mutant, abi1, showed altered sensitivity to germination- and root growth-inhibition inhibition. Ethylene- and cytokinin-insensitive mutants were partly insensitive to germination-inhibition. This suggests that different mechanisms mediate each effect of Ulva extract on early Arabidopsis development and that multiple hormones contribute to germination-inhibition.
Elemental analysis showed that Ulva contains high levels of Aluminium ions (Al3+). Ethylene and cytokinin have been suggested to function in Al3+-mediated root growth inhibition: our data suggest that if Ulva Al3+ levels inhibit root growth, this is via a novel mechanism. We suggest algal extracts should be used cautiously as fertilisers, as the inhibitory effects on early development may outweigh any benefits if the concentration of extract is too high.
Introduction
Plant growth, development and productivity is affected by various abiotic (physical) and biotic (biological) factors. Responses to these factors determine cropping pattern and plant distribution 1. Global demand for crops is predicted to increase ∼100% from 2005 to 2050, while ∼795 million people worldwide were undernourished in 2014–16 2,3.
Current global food challenges and pressure on the food production industry are due to the exponentially growing human population and increasing soil- and water issues compounding the pressure induced by anthropogenic climate change. The frequency of abiotic environmental stresses (flooding, drought, water limitation, salinity and extreme temperatures) is increasing 4 and causing crop losses worldwide 5-7. More intense, frequent droughts in Africa, southern South America and southern Europe and increased flooding in temperate regions will drive future crop yield decline 8-10. Soil salinity threatens agriculture and natural ecosystems 11-13. Intensive farming leads to unfavourable conditions for crop growth, development and survival 14.
Humans have used seaweeds (macroalgae) and seaweed-based products for centuries, for food, fuel, aquaculture, cosmetics, colouring dyes and therapeutic/botanical applications 14-16. The earliest written reference to using seaweed as a fertiliser is from Roman times 17. Applying seaweeds/seaweed extracts in modern agriculture leads to increased seed germination rates, improved plant development (flowering, leaf quality and root system architecture), elevated defence against pathogens and pests 18 and protection against nutrient deficiency and environmental stresses including salinity 19, cold or drought 20-23. Seaweed fertilisers have been used in agricultural programs to improve soil management, disease management, nutritional strategies, water efficiency and drought tolerance 23.
Several manufacturing practices are used to liquefy seaweed biomass 21,23,24. Seaweed extracts are marketed as liquid biofertilisers or biostimulants containing a variety of plant growth-promoting components – those identified include plant growth regulators (phytohormones), minerals and trace elements, quaternary ammonium molecules (e.g. betaines and proline), polyuronides (e.g. alginates/fucoidans) and lipid-based molecules e.g. sterols 23. Seaweed products are also available in soluble powder form. Depending on whether algal extract is applied as liquid fertiliser or seaweed manure to plant roots, or as a leaf spray, different plant responses to seaweeds occur 14,21. The mechanism by which seaweed fertilisers affect plant growth, development and yield is currently unknown. Crop plants treated with seaweed extracts showed similar physiological responses to those treated with plant growth-regulatory substances 20. Phytohormones detected in seaweed extracts are auxins, cytokinins, gibberellins, abscisic acid and brassinosteroids 25-27 but chemical components other than phytohormones, which elicit physiological responses reminiscent of plant hormones, have also been detected 28. One hypothesis is that the effects of seaweed fertilisers are due either directly or indirectly to phytohormones: seaweed extracts may themselves contain beneficial phytohormones, or may contain substances that trigger land plant signaling pathways that usually respond to these signals. Which, if either, of these scenarios occurs is not clear.
Although seaweeds could potentially benefit plant growth by providing macronutrients, including nitrogen (N), phosphorus (P), ammonium (NH4+) and potassium (K), studies have consistently shown that seaweed extracts’ beneficial effects are not due to macronutrients, particularly at the concentrations used in the field 20,29. Very dilute seaweed extracts (1:1000 or below) still have biological activity but the compound(s) involved are unknown: the beneficial effects may involve several plant growth-promoters working synergistically 25,30-32.
Understanding at a mechanistic level how seaweed fertilisers affect land plant growth and development is important. Previous studies have applied a diverse range of extracts from brown, green and red seaweeds to a heterogeneous range of crop plants 33,34. Generally, lower concentrations of an algal extract have beneficial effects on root- and shoot growth while higher concentrations have inhibitory effects 34-38. Thus, algal extract concentration is critical to its effectiveness. However, because of the range of plants, seaweeds and extraction methods used, “positive” concentrations of algal extract ranged from 0.002%-0.2% while inhibitory concentrations ranged from 0.1%-1%.
In this paper, we establish a “standardised” laboratory-based system to determine the molecular mechanisms by which seaweeds can affect land plant productivity, using model organisms. The extensively-studied model plant Arabidopsis 39, from Brassicaceae (cabbage) family, was the first plant with a sequenced genome 40 and extensive mutant collections are available, including mutants in hormone signaling and perception 41-45. Phytohormone biosynthetic/signalling pathways have been determined, yielding a broad understanding of plant responses to stimuli 46-48. Employing Arabidopsis as a model organism has enabled translation of the understanding of plant growth and development to crops and agriculture 49-51.
The green seaweed Ulva (sea lettuce; green nori) is an emerging experimentally-tractable model organism to study macroalgal development, growth, morphogenesis (reviewed in Wichard 2015). Ulva is a cosmopolitan macroalgal genus, the main multicellular branch of the Chlorophyte algae, and the most abundant Ulvophyceae representative 52,53. Ulvophyceae are multicellular algae with simple morphology compared to land plants. Distinctive features that make Ulva attractive as model systems are the small genome [100-300Mb; 54,55] (the established model system Ulva mutabilis is currently being sequenced), symbiotic growth with bacterial epiphytes, naturally-occurring developmental mutants (in Ulva mutabilis), simple organization of the thallus (body) consisting of three differentiated cell types (blade, stem and rhizoid), laboratory cultivation 56,57 and the ability to generate stable transgenic lines 58-60.
The species of Ulva chosen for this study was Ulva intestinalis, an intertidal alga found worldwide, which can be lab-grown similarly to Ulva mutabilis 56,61. We compared directly the growth- or inhibition parameters of different concentrations of Ulva intestinalis extract versus a control, applied to both wild-type and mutant Arabidopsis genotypes. By using two experimentally tractable organisms we have begun to understand the plant signalling pathways that can be triggered by algal extract. As Ulva genetic manipulation becomes better-established 59 this raises the possibility of future engineering of improved macroalgal fertiliser properties.
Results
Concentrations of Ulva extract of 0.5% and above inhibit wild-type Arabidopsis seed germination
Previous experiments demonstrated conflicting effects of different concentrations of algal extract on seed germination, e.g. in tomato 33,34. To investigate the effect of Ulva intestinalis extract on Arabidopsis germination, concentrations of Ulva extract ranging from 0 – 1.0% was tested (Fig. 1). All Ulva extract concentrations from 0.5% upwards delayed wild-type germination. The final germination percentage was reduced in 0.8% and 1.0% Ulva extract: only about half the seeds germinated in 1.0% Ulva extract after a week (Fig.1a). Concentrations of 0.3% Ulva extract and below had no effect on seed germination and no stimulatory effect of Ulva extract on germination was observed at any concentration tested (Fig. 1a).
Ulva extract stimulates Arabidopsis primary root growth at low concentrations and inhibits root growth at higher concentrations
Having demonstrated that seed germination is inhibited by Ulva extract, we sought to discover whether the next stage of development, primary root elongation, was also affected by Ulva extract. Seeds were germinated, and seedlings grown, on standard growth medium containing a range of Ulva extract concentrations ranging from 0 to 2%. Ulva extract significantly stimulated root growth at concentrations from 0.03-0.08% (∼80% stimulation at 0.06%), while concentrations of 0.3% and above had an inhibitory effect on root growth (∼68% inhibition at 2%) (Fig. 1b). The stimulatory effect of Ulva extract was similarly present when seedlings were grown on non-nutrient-containing agar (Fig. 1c).
To ascertain whether the inhibitory effect of Ulva extract concentrations ≥0.3% on root growth was simply a consequence of delayed germination (Fig. 1a), we conducted an experiment where seedlings were germinated on normal growth medium for three days before transferring to medium containing Ulva extract. Root growth was once again inhibited by Ulva extract, showing that higher concentrations of Ulva extract have an inhibitory effect on root growth, independent from any effect on germination (Fig. 1d).
Ulva extract inhibits Arabidopsis lateral root formation
Once the Arabidopsis primary root is established, it acquires branches, or lateral roots (LRs), as the seedling matures to secure anchorage and extract micro- and macronutrients from the soil 62,63. Having ascertained that Ulva extract affects primary root growth, we went on to investigate the effect of Ulva extract on LR formation. Increasing concentrations of Ulva extract show a progressive inhibition in the density of LR branching from the primary root, even at concentrations that stimulate primary root growth (Fig. 1e, f).
In summary, Ulva extract inhibits germination, has a biphasic effect on primary root growth (stimulatory at low concentrations; inhibitory at higher concentrations) and inhibits LR formation. Taken together, our data are reminiscent of the effect of the plant hormone abscisic acid (ABA) on germination and early root development, as ABA is a negative regulator of germination 64, shows a biphasic effect on primary root growth 65-67, and inhibits LR growth at concentrations that stimulate primary root growth but do not affect germination 68.
The germination-inhibitory effect of Ulva extract is not apparent in an ABA-insensitive mutant
We next sought to determine whether ABA signalling could mediate the effects of Ulva extract on Arabidopsis development to uncover the mechanism by which Ulva extract inhibits germination. Arabidopsis seeds from the ABA-insensitive mutant abi1 69,70 were assayed for their response to Ulva extract. abi1 seeds are unresponsive to the inhibitory effect of Ulva extract and behave similarly to untreated controls under all treatments (Fig. 2a-c). This suggests that the inhibition of Arabidopsis seed germination by Ulva extract depends on a functional ABA signalling pathway in the seeds.
The abi1 mutant’s root growth responds normally to low concentrations of Ulva extract and is slightly insensitive to higher concentrations of Ulva extract
Since the abi1 mutant is impaired in its germination response to Ulva extract and since ABA is known to have a biphasic effect on root growth 65, we tested the effect of Ulva extract on the root growth of the abi1 mutant. The abi1 mutant behaved similarly wild type plants at low concentrations (<0.1%) of Ulva extract (Figure 3 a, b). This suggests that the stimulatory effect of Ulva extract on root growth cannot be attributed to changes in ABA signalling in the plant. At higher concentrations (0.3% - 1%) of Ulva extract, the abi1 mutant showed some insensitivity to inhibition of root growth (Figure 3c), but this was of a much smaller magnitude than the abi1 mutant’s insensitivity during germination. This suggests that changes in ABA signalling in Arabidopsis may partially contribute to the inhibitory effect of Ulva extract on root growth.
The abi1 mutant is sensitive to lateral root-inhibition by Ulva extract
Since ABA plays an inhibitory role in LR development 71, we next tested the effect of 0.1-1% Ulva extract on lateral root development in the abi1 mutant. The abi1 mutant’s LR development was inhibited by Ulva extract more strongly than wild type controls (Figure 3d), including at 0.1% Ulva extract, which has no effect on primary root growth. This implies that the inhibition of LR development by Ulva extract is not mediated by the ABA signalling pathway and that LRs respond differently to Ulva extract compared to the primary root.
Elemental analysis of Ulva intestinalis
Although Ulva 72 and other seaweeds 73 are known to produce ABA, the Ulva extract used in our experiments is a water-soluble extract, so its effects on the Arabidopsis ABA signalling pathway are most likely indirect: ABA is soluble in organic solvents rather than water 74,75. We therefore measured the concentration of a panel of 31 water-soluble ions in our Ulva intestinalis samples using ICP-MS to determine whether (i) there were substances present in the tissue that were at markedly different levels to those in a land plant standard, (ii) whether any substances were present at substantially different levels to those in our standard Arabidopsis growth medium and (iii) whether the presence of any of the substances could explain the effects of Ulva extract on Arabidopsis seedling development. Our analysis identified 16 elements present at higher levels in Ulva intestinalis extract than in the land plant control, namely Boron, Sodium, Sulphur, Lithium, Aluminium, Vanadium, Manganese, Iron, Copper, Arsenic, Strontium, Silver, Caesium, Thallium, Lead and Uranium (Table 1). Of these, 12 elements (Sodium, Lithium, Aluminium, Vanadium, Copper, Arsenic, Strontium, Silver, Caesium, Thallium, Lead and Uranium) are present in 1% Ulva intestinalis extract at higher values than in Arabidopsis growth medium (Table 1). Out of these 12 elements, only three, namely Sodium, Aluminium and Copper, are present at levels known to have an effect on Arabidopsis development. The remaining 9 elements are present at micromolar (Lithium) or nanomolar quantities, while the published literature demonstrates their effects on Arabidopsis germination and root growth only in the micromolar to millimolar 76-83 range.
The level of Sodium in the 1% Ulva extract is 10.5mM, but Arabidopsis germination is inhibited only by concentrations of salt above 150mM 84. Thus, the level of germination-inhibition with Ulva extract at ≥0.3% is not attributable to the levels of Sodium in the extract. Arabidopsis root growth is inhibited by concentrations of 25mM Na+ and above 85. Thus, the inhibition of root growth seen in our experiments is unlikely to be attributable wholly to salt stress. This conclusion is in accordance with the fact that the abi1 mutant is not wholly insensitive to the root growth inhibition (Fig. 3c) since salt stress responses are mediated by ABA signalling 86,87.
Aluminium (Al3+) ions are present in 1% Ulva extract at >500µM, and 0.05% Ulva extract at 26µM. Previous literature shows that even 5µM Al3+ can slow root growth 88 while 500µM Al3+ can reduce root growth by around 80% 89. Thus, the elevated Al3+ levels in the Ulva extract could be contributing to the inhibition in root growth that we see at concentrations of Ulva extract ≥0.3%. Copper ions (Cu2+) are present in 1% Ulva extract at 2.1µM (Table 1). Copper levels of 1.6µM have previously been described as rhizotoxic 76 and 20-25µM Cu2+ inhibits root elongation in several studies 90. Higher levels of copper (500µM-2mM) inhibit seed germination 91,92. Thus, it is unlikely that the elevated copper levels in the Ulva extract are causing germination inhibition, but they could be partly contributing to the inhibition in root growth that we see in ≥0.3% Ulva extract.
Auxin, ethylene, cytokinin mutants respond similarly to wild-type Arabidopsis on Ulva extract with respect to root growth
Aluminium stress on roots is mediated by a combination of ethylene (via changes in auxin transport, at higher Al3+ concentrations) and cytokinin signalling (at lower Al3+ concentrations) 89. We tested mutants in auxin-, cytokinin and ethylene signalling for their root growth responses to Ulva extract (Supplemental Figure 1). The two auxin signalling mutants used were the receptor mutant tir1-1 93 and the auxin-resistant signalling mutant axr1-1 94. The two ethylene signalling mutants used were the receptor mutant etr1-3 and the signalling mutant ein2-1 95. The cytokinin mutant used was the receptor mutant cre1-1 45. All mutants responded similarly to wild type seedlings to “inhibitory” concentrations of Ulva extract ranging from 0.1-1%, equating to approximately 50-500µM Al3+ (Supplemental Fig. 1a). This suggests that the ethylene, auxin and cytokinin hormone signalling pathways do not participate substantially in root growth inhibition by Ulva extract. Moreover, none of the mutants were insensitive to the root growth-stimulatory effect of Ulva extract (Supplemental Fig. 1b), suggesting that these hormones do not participate in the root growth stimulation brought about by Ulva extract. Similar data was obtained when the quintuple della mutant 96 was assayed for root growth stimulation and inhibition: the mutant behaved as wild-type, showing that gibberellin-DELLA signalling, which is involved in multiple plant stress- and growth-responses 97 is not involved in the effects of Ulva extract on root growth (Supplemental Figure 2).
Cytokinin- and ethylene ethylene-signalling mutants show some insensitivity to inhibition of germination by Ulva extract
As Aluminium (Al3+) ions are present in 1% Ulva extract at >500µM, a concentration that causes very substantial decreases in root growth 89, and since two hormones involved in root responses to Aluminium, cytokinin and ethylene, are also regulators of seed germination 98,99, we also tested the germination behaviour of cytokinin receptor mutant cre1 and the ethylene receptor mutant etr1 on 0.1-1% Ulva extract. Both mutants’ seeds showed some insensitivity to germination-inhibition compared to wild type (Fig. 4a, 4b), but were not as insensitive as the abi1 mutant (Fig. 2b). Both cre1 and etr1 also showed a higher final germination percentage in comparison to WT germination on 0.8% and 1% Ulva extract over the same period of time (Fig 1c, d, e). This suggests that the inhibition of Arabidopsis seed germination by Ulva extract is influenced by the cytokinin- and ethylene signalling pathways in addition to the ABA signalling pathway.
Discussion
Ulva extract at concentrations of 0.5%-1% inhibits wild-type Arabidopsis seed germination. Ulva extract ≥0.3% reduces wild-type Arabidopsis primary root growth and the extract inhibits wild type LR formation even at concentrations below 0.1%, suggesting that LRs are more sensitive than the primary root to the inhibitory agent(s) in the Ulva extract.
Our results concur with other studies where seaweed extract at high concentrations inhibited seed germination and seedling growth. Reduced germination occurred in pepper seeds primed with brown seaweed (Ascophyllum) extract at 1:250 (0.4%) and at higher concentrations (10%) of Maxicrop (commercial seaweed extract) solution compared to control seeds 100. A higher concentration (1.0%) of water-extracts from the brown seaweeds Caulerpa sertularioides, Padina gymnospora and Sargassum liebmannii reduced tomato germination and seedling development 34. 2%-10% aqueous extracts from Sargassum johnstonii led to similar effects on tomato 101.
Concentrations of kelp waste extracts (KWE) from 10–100% inhibited germination of pakchoi (Brassica chinensis L.). This was attributed to high levels of NaCl 102, which are absent from our Ulva extract. Arnon and Johnson (1940) reported detrimental effects on early tomato development as a result of higher pH in the growth medium. In our experiments, the pH was adjusted to be the same for all concentrations of Ulva extract so the effects are not due to altered pH.
Ulva extract has a growth-stimulating effect on wild type Arabidopsis primary root elongation specifically at concentrations between 0.025-0.08%. This is in accordance with data from other species, suggesting that Arabidopsis is a good model for studying the effects of seaweed extracts. Seaweed extract may improve water and nutrient uptake efficiency by root systems 103 leading to enhanced plant growth and vigour. Commercial extracts from the brown seaweed Ecklonia maxima stimulated tomato root growth at low concentrations (1:600; 0.17%) while higher concentrations (1:100; 1%) strongly inhibited root growth 33. Root growth enhancement was seen in Arabidopsis plants treated with aquaeous Ascophyllum nodosum extracts (0.1gL-1; 0.01%), whereas plant height and number of leaves were affected positively at 1gL-1 (0.1%) 104. Lower concentrations (0.2%) of extracts of both Ulva lactuca (green seaweed) and P. gymnospora (brown) were more effective at enhancing tomato seed germination (Hernández-Herrera et al., 2014). We observed no boost in Arabidopsis germination with Ulva intestinalis extract under our growth conditions where we vernalise seeds to break dormancy before an assay, so this may explain the discrepancy between the experiments.
2% kelp waste extract (KWE) stimulated pakchoi seed germination 102. Pakchoi seedling growth (plumule length, radicle length, fresh weight and dry weight) was improved by treatment with 2-5% KWE 102. This data is in-line with our observed root growth stimulation at low concentrations. The KWE was prepared differently (cell wall digestion and centrifugation) to our Ulva intestinalis extract, which may explain why higher concentrations of KWE than Ulva extract give stimulatory effects; in addition, pakchoi is larger than Arabidopsis.
The stimulatory effect of KWE on pakchoi may be attributed to the combined effects of soluble sugars, amino acids and mineral elements 102. Sugars are immediate substrates for intermediary metabolism and effective signaling molecules: thus accessibility of sugars influences plant growth and development 105. The growth-enhancing potential of algal extract correlates with the presence of diverse polysaccharides, including unusual/complex polysaccharides not present in land plants 21,106. However, a role for macro- and microelements, vitamins and phytohormones is also suggested 20,27,32,107-109.
Since our Ulva extracts are water-based, it is unlikely that they contain high quantities of plant hormones, which are largely soluble in organic solvents. Our Arabidopsis mutant analysis demonstrates that germination-inhibition by Ulva extract is dependent on activation of the Arabidopsis ABA signaling pathway, with cytokinin- and ethylene-signaling also playing a role. This suggests that a substance(s) in Ulva extract activates endogenous plant hormone signaling to inhibit germination. Ulva extract-mediated inhibition of primary root growth is partly blocked in an ABA-insensitive mutant, while cytokinin-, auxin-ethylene- and gibberellin signaling mutants all respond similarly to wild type with respect to root growth. This implies that although ABA signaling plays a role in primary root growth inhibition by Ulva extract, additional pathways also contribute. Lateral root development is inhibited via a different mechanism to primary root growth, as the ABA-insensitive abi1 mutant’s LR development is inhibited by Ulva extract to a greater extent than wild-type (Fig. 3d).
Our elemental analysis of Ulva tissue suggests that the most likely cation contributing to the inhibitory effects of Ulva extract is Al3+, which is present in quantities known to inhibit Arabidopsis primary root growth 88,89. Al3+ may not be the only inhibitory substance present: previous research has demonstrated a role for auxin, ethylene and cytokinin in root responses to Al3+ stress 89 and this is not apparent from our mutant root assays. Conversely, there may be other hormones involved in seed- and root responses to Al3+ stress: the effects of Al3+ on germination and lateral root development in Arabidopsis has not previously been studied. The toxic effect of Al3+ in the Ulva extract may be partially countered by the relatively high levels of Mg2+ also present in the extract (In 1% Ulva extract, 4x that present in Arabidopsis growth medium - Table 1; 79).
Al3+ stress has a range of physiological effects that could affect root growth and development. Al3+ stress alters membrane potentials, which affects transport of ions, including Ca2+, across membranes. This can result in changes in cytoplasmic Ca2+ homeostasis, which controls cell signaling, metabolism and cell-growth processes including root development 110. Al3+ stress induces changes in the expression and activity of the plasma membrane H+-ATPase that controls cytosolic pH and membrane potentials 111.
Seaweeds contain high levels of particular cations: macroelements (Na, P, K, Ca) and microelements (Fe, B, Mn, Ca, Mo, Zn, Co) that have critical roles in plant development and growth 112,113. In many vegetable crops, the accumulation of sodium ions restrains embryo or seedling development, leading to reduced germination, uneven morphogenesis and loss of crop production e.g. 114. Our data suggests that the only macroelement present at higher concentrations in Ulva extract than in plant tissues (or indeed plant growth medium) is Na+, but Na+ is not present at high enough concentrations to explain the inhibition of germination, root growth and lateral root development that we see. Ulva species tolerate low salinity despite being marine algae. Our Ulva sampling site is where a river meets the sea: the salinity of the seawater is low (F. Ghaderiardakani, unpublished). A reduction in germination rate and growth of tomato attributable to salt (and perhaps reduced imbibition of water by seeds) was suggested upon applying brown seaweed (Caulerpa sertularioides and Sargassum liebmannii) liquid extracts, but not with U. lactuca and P. gymnospora with a lower salt concentration 34.
Some seaweed extracts alleviate salt stress: the survival of Kentucky bluegrass (Poa pratensis L. cv. Plush) treated with a proprietary seaweed extract (38Lha-1) increased significantly, under various levels of salinity, with improved growth and promotion of rooting of the grass at a soil salinity of 0.15Sm-1 19. Application of seaweed extract activated a mechanism reducing the accumulation of Na+ in plants; grass treated with seaweed extract had less sodium in the shoot tissue 115,116.
The microelements B and Fe are present at higher concentrations in Ulva tissue than in our land plant control, but at levels that are very similar to that found in our Arabidopsis growth medium, so cannot be contributing to the observed stimulatory or inhibitory effects. The content of minerals in Ulva intestinalis is in-line with values for Ulva spp. reported previously, e.g. Ulva lactuca 34 and Ulva reticulata 38,112.
Using seaweed extracts as biofertilisers due to their direct or indirect stimulatory impacts on plant metabolism has been suggested as one of their key beneficial applications 23. Taken together, our results and others’ suggest that for plants to benefit optimally from algal extracts, only a small quantity should be used or could be mixed with commercially available fertilisers for a synergistic effect on crop yield and a reduction in quantities and costs of chemical fertilisers applied 117.
Our data demonstrates that Ulva extract can inhibit Arabidopsis seed germination, early root growth and lateral root development, even at concentrations below 1%, by activating endogenous plant hormone signaling pathways. Could this in itself be useful? One of the top priorities in organic agriculture is the eradication of weeds from the production area 118. Concerns about improvements in agriculture focus on diminishing weeds’ adverse effects on the environment and improving the sustainable development of agricultural systems. New approaches are required to integrate biological and ecological processes into food production and minimize the use of practices that lead to the environmental harm 119. Considering the observed biological inhibitory effects resulting from the action of seaweed extracts on crops’ germination and early development particularly at high concentration, it might be worthwhile to employ seaweed extracts as organic herbicides. The evidence at hand establishes that there are benefits to be obtained from utilizing macroalgal products in agricultural systems. Further translational studies are required to define the appropriate algal sources for commercial biostimulants (considering inherently different algal extracts and also the availability of seaweed biomass in a particular area), their application form and frequency, the timing of applications in relation to plant development and the optimal dosages needed to maximise both agricultural productivity and economic advantages.
In conclusion, water-soluble algal extracts from Ulva intestinalis were effective at stimulating the primary root growth of Arabidopsis thaliana only when applied at low concentrations. High concentrations of Ulva extract inhibit germination and root development, perhaps in part due to Al3+ toxicity, with endogenous plant ABA signalling playing a role in this inhibition. The effects of algal extracts on Arabidopsis development are likely mediated by a complex interplay of hormones. Future work targeting candidate genes in Ulva 60 may uncover how Ulva extracts exerts their effects on plant hormone signalling. Although if used sparingly, seaweed extracts are potential candidates to produce effective biostimulants, they may be just as beneficial as organic herbicides by targeting plants’ ABA signalling mechanisms. Cross-disciplinary research could help farmers to benefit optimally from the use of algal extracts in the future, particularly for cost-effective organic farming and an environmentally-friendly approach for sustainable agriculture.
Methods
Collection and Identification of Algal Samples
Vegetative and fertile U. intestinalis blades were collected from the intertidal zone at low tide, three times between March 2015 and April 2016, from the coastal area of Llantwit Major beach, South Wales, UK (51°40’ N; 3°48’ W). Excess water and epiphytic species were removed at the site by blotting the sample’s surface before storage on ice for transport back to the laboratory. Epiphyte-free samples were subjected to a molecular identification using plastid-encoded rbcL (large unit ribulose bisphosphate carboxylase) and tufA (plastid elongation factor) markers as identification solely by morphological characteristics is not reliable 61.
Preparation of water-soluble Ulva Extract
Ulva samples were washed with tap water to remove surface salt, shade dried for 10 days, oven-dried for 48h at 60 °C, then ground to a fine powder using a coffee grinder (Crofton, China) to less than 0.50mm. 10g of this milled material was added to 100mL of distilled water with constant stirring for 15 min followed heating for 45 minutes at 60°C in water bath 38. The contents were filtered through two layers of muslin cloth. This Ulva extract was designated as 10% stock solution and added to MS solution to make up specific concentrations and autoclaved. 1% Ulva extract stock was subjected to pH measurement and elemental analysis. All measurements were performed in triplicate.
Digestion of plant material for elemental analysis
Ulva samples were digested using a microwave system, comprising a Multiwave 3000 platform with a 48-vessel MF50 rotor (Anton Paar GmbH, Graz, Austria); digestion vessels comprised perfluoroalkoxy liner material and polyethylethylketone pressure jackets (Anton Paar GmbH). Dried samples (∼0.2g) were digested in 2mL 70% Trace Analysis Grade HNO3, 1 mL Milli-Q water (18.2 MΩ cm; Fisher Scientific UK Ltd, Loughborough, UK), and 1mL H2O2 with microwave settings as follows: power =1400 W, temp = 140 C, pressure = 20 Bar, time = 45 minutes. Two operational blanks and two certified reference material of leaf (Tomato SRM 1573a, NIST, Gaithersburg, MD, USA) were included in each digestion run. Following digestion, each tube was made up to a final volume of 15mL by adding 11mL of Milli-Q water and transferred to a universal tube and stored at room temperature.
Elemental analysis
Sample digestates were diluted 1-in-10 using Milli-Q water prior to elemental analysis. The concentrations of 28 elements were obtained using inductively coupled plasma-mass spectrometry (ICP-MS; Thermo Fisher Scientific iCAPQ, Thermo Fisher Scientific, Bremen, Germany); Ag, Al, As, B, Ba, Ca, Cd, Cr, Co, Cs, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, S, Se, Sr, Ti, U, V, Zn. Operational modes included: (i) a helium collision-cell (He-cell) with kinetic energy discrimination to remove polyatomic interferences, (ii) standard mode (STD) in which the collision cell was evacuated, and (iii) a hydrogen collision-cell (H2-cell). Samples were introduced from an autosampler incorporating an ASXpress™ rapid uptake module (Cetac ASX-520, Teledyne Technologies Inc., Omaha, NE, USA) through a PEEK nebulizer (Burgener Mira Mist, Mississauga, Burgener Research Inc., Canada). Internal standards were introduced to the sample stream on a separate line via the ASXpress unit and included Sc (20µgL-1), Rh (10µgL-1), Ge (10µgL-1) and Ir (5µgL-1) in 2% trace analysis grade HNO3 (Fisher Scientific UK Ltd). External multi-element calibration standards (Claritas-PPT grade CLMS-2; SPEX Certiprep Inc., Metuchen, NJ, USA) included Ag, Al, As, B, Ba, Cd, Ca, Co, Cr, Cs, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, S, Se, Sr, Ti (semi-quant), U, V and Zn, in the range 0 – 100µgL-1 (0, 20, 40, 100µgL-1). A bespoke external multi-element calibration solution (PlasmaCAL, SCP Science, Courtaboeuf, France) was used to create Ca, K, Mg and Na standards in the range 0-30mgL-1. Boron, P and S calibration utilized in-house standard solutions (KH2PO4, K2SO4 and H3BO3). In-sample switching was used to measure B and P in STD mode, Se in H2-cell mode and all other elements in He-cell mode. Sample processing was undertaken using Qtegra™ software (Thermo Fisher Scientific) with external cross-calibration between pulse-counting and analogue detector modes when required 120. Differences between seaweed and tomato control were analysed using a Welch’s t- test.
Germination Bioassay
Arabidopsis thaliana wild-type Col-0 and mutant lines abi1-1, tir1-1, axr1-3, cre1-12, etr1-3, and ein3-1 were obtained from the Nottingham Arabidopsis Stock Centre (Loughborough, UK). Arabidopsis seeds were sterilised in 20% Parozone™ bleach on a turning wheel for 10 minutes and subsequently washed 2-3 times in sterile water. Seeds were vernalized at 4°C for 48h and placed on 1% agar, containing 0.5MS and Ulva extract. Plates were transferred to the growth room for 7-10 days and incubated at 22 ± 2°C with a 16-h-light photoperiod and light intensity of 120µmolm-2s-1. Germination was observed daily as in 121. A seed was scored as germinated when its radicle had emerged from within the seed coat. Germination percentage (GP) was calculated as follows: GP = (the number of germinated seeds/total number of seeds) ×100). Data from three independent biological repeats (n=30-90 seeds per genotype and treatment) were combined. To identify significant differences between treatments and genotypes, Kruskal-Wallis one-way ANOVA on ranks followed by Dunn’s post-hoc tests were performed using SigmaPlot 13 software (Systat Software, San Jose, CA).
Root Bioassay
Experiments were conducted using 10cm square agar plates. 20 seeds were placed individually on the agar following a line across the top of the plate. The plates were sealed with Micropore tape (3M), taped together and incubated vertically in standard growth conditions as in 121.
From day 7 to 14 the seedlings were photographed and primary root (PR) lengths were measured with ImageJ open-source software (http://rsb.info.nih.gov/ij). For some assays, the number of visible emerged lateral roots (LR) on each primary root was also counted and the lateral root density was calculated by dividing the number of LRs present by the length of that root. To identify significant differences between treatments and controls in wild-type plants, data were first checked to confirm normality, then appropriate two-tailed t-tests (normal data) or Mann-Whitney U-tests (non-normal data) were performed in Excel using an Excel template from Gianmarco Alberti’s lab (xoomer.alice.it/Exceltemplates.pdf), comparing the results of each Ulva extract concentration to the control (without Ulva extract). To identify significant differences between treatments and genotypes, Kruskal-Wallis one-way ANOVA on ranks followed by a Dunn’s post-hoc test were performed using SigmaPlot 13 software (Systat Software, San Jose, CA). All experiments were repeated a minimum of two and a maximum of four times with similar trends observed in each biological repeat.
Author contributions
FG and JCC conceived the study and designed experiments. All authors performed experiments and analysed data: FG - Fig 1a,d,e, Fig 2, Fig 3a,b,c,d; Fig 4; EC - Fig 1a,c, Supplemental Fig 1; Fig 4; DKD – Fig 1e, Fig 3c,d, Supplemental Fig 1; KT – Fig 1a; Fig 2; NSG - Table 1, JCC – Supplemental Fig 2. FG supervised EC, DKD, KT in the lab; JCC supervised FG, ED, DKD, KT. FG, NSG and JCC wrote the paper.
Competing interests
The authors declare no competing interests.
Data availability statement
The datasets generated during the current study are available from the corresponding author on reasonable request.
Acknowledgements
This work was funded by an Islamic Development Bank PhD scholarship to FG and University of Birmingham funds for JC to host EC, DKD and KT in the lab.
Footnotes
↵* J.C.Coates{at}bham.ac.uk
References
- 1.↵
- 2.↵
- 3.↵
- 4.↵
- 5.↵
- 6.
- 7.↵
- 8.↵
- 9.
- 10.↵
- 11.↵
- 12.
- 13.↵
- 14.↵
- 15.
- 16.↵
- 17.↵
- 18.↵
- 19.↵
- 20.↵
- 21.↵
- 22.
- 23.↵
- 24.↵
- 25.↵
- 26.
- 27.↵
- 28.↵
- 29.↵
- 30.↵
- 31.
- 32.↵
- 33.↵
- 34.↵
- 35.
- 36.
- 37.
- 38.↵
- 39.↵
- 40.↵
- 41.↵
- 42.
- 43.
- 44.
- 45.↵
- 46.↵
- 47.
- 48.↵
- 49.↵
- 50.
- 51.↵
- 52.↵
- 53.↵
- 54.↵
- 55.↵
- 56.↵
- 57.↵
- 58.↵
- 59.↵
- 60.↵
- 61.↵
- 62.↵
- 63.↵
- 64.↵
- 65.↵
- 66.
- 67.↵
- 68.↵
- 69.↵
- 70.↵
- 71.↵
- 72.↵
- 73.↵
- 74.↵
- 75.↵
- 76.↵
- 77.
- 78.
- 79.↵
- 80.
- 81.
- 82.
- 83.↵
- 84.↵
- 85.↵
- 86.↵
- 87.↵
- 88.↵
- 89.↵
- 90.↵
- 91.↵
- 92.↵
- 93.↵
- 94.↵
- 95.↵
- 96.↵
- 97.↵
- 98.↵
- 99.↵
- 100.↵
- 101.↵
- 102.↵
- 103.↵
- 104.↵
- 105.↵
- 106.↵
- 107.↵
- 108.
- 109.↵
- 110.↵
- 111.↵
- 112.↵
- 113.↵
- 114.↵
- 115.↵
- 116.↵
- 117.↵
- 118.↵
- 119.↵
- 120.↵
- 121.↵