Application of seaweed and pruning residue as organic fertilizer to increase soil fertility and vine productivity

Algae have an indisputable role in coastal ecosystems, but their accumulation and uncontrolled proliferation cause severe damage for the local municipalities. Fertilization with seaweed has been shown to increase soil fertility and crop production reducing ultimately the need for inorganic fertilizers. However, contradictory results of the compost effect have been reported. In the present work, we aimed at testing the suitability of three composted algae materials obtained in a previous study as soil amendments for vines. The composted materials consisted of pruning waste (P) and seaweed (S) mixed (henceforth, P2S1, P1S1 and P1S2, referring the number to the ratio of P to S). Overall, we observed an increase in soil organic matter, phosphorus (P) and potassium (K) in the soil treated in comparison with soils with inorganic fertilization. A moderate N soil enrichment (ca. 20%) was also detected. The leaf analysis reflected generally the greatest concentrations of NPK for the organic treatments, but this was remarkable only during the first year after seaweed application. A noticeable improvement in grape production was detected especially with the P1S1 compost without compromising grape quality although a decrease in sucrose content was noted with the compost with higher productivity (e.g. P2S1 and P1S1). This slight sucrose drop could be attributable to a greater water availability mediated by the compost or to a dilution factor of the sucrose content caused by a greater number of berries in those vines. These findings suggest that although monitoring of the long‐term effects is needed, the use of seaweed amendments for agriculture could offer a cost‐effective method for coastal municipalities to reduce excessive algae debris while also minimizing the impacts of inorganic fertilization.


| INTRODUCTION
Algae constitute a characteristic element of coastal ecosystems and provide important ecological benefits including erosion control, provision of breeding grounds, food and shelter for a variety of fauna (Deniz & Ersanli, 2018;Kaur, 2020). However, their accumulation and uncontrolled proliferation (seaweed blooms) often generate serious economic and environmental problems regarding water quality and the recreational use of beaches, especially in touristic places (Sembera et al., 2019).
Previous studies have confirmed the suitability of seaweed composted materials as natural fertilizers that contribute to increase and maintain soil quality and fertility because of the stimulation of soil microbial activity (Renuka et al., 2018). Specifically, they are gaining significance in sustainable agriculture because of the role they play in increasing crop biomass, improving yield and fruit quality and the resistance to biotic and abiotic stressors (Cole et al., 2016;Sabir et al., 2014;Yuvaraj & Gayathri, 2017). In addition, seaweed compost might improve water and nutrient retention by delivering bioactive substances such as vitamins, minerals, growth regulators and organic compounds to the soil medium (Górka et al., 2018;Michalak et al., 2016). However, contradictory results of the agronomic effect are reported depending upon the algae species used, the amount of compost, supplemental mixing substrate, C/N ratios and soil and crop type (Eyras et al., 2008;Madejón et al., 2021;Michalak & Chojnacka, 2013).
Some studies conducted in the European region identify vineyards as the most vulnerable areas subjected to the highest runoff and soil losses which might lead to reduce the quality of the grapes and wine, especially because of the soil organic matter loss (Ramos & Martínez-Casasnovas, 2004;Rodrigo-Comino et al., 2018). This is particularly important for countries such as Spain owing to the economic importance of grape production and its wine-growing and wine-making heritage (Alston & Sambucci, 2019;Guesmi et al., 2012). Therefore, finding new sustainable alternatives that allow preserving this sector without compromising the production is a priority (Mac Monagail et al., 2017). In this sense, seaweeds could offer a sustainable and cost-effective alternative to mineral fertilizers owing to the frequent proximity of vineyards to coastal areas that save the high costs of drying and transportation. Thus, making use of all seaweed biomass that more and more frequently drift ashore would allow local municipalities to tackle what could become a serious economic and environmental problem and in turn to prompt a circular economy strategy of zero waste. However, the issues of its exploitation and management still need to be worked out (Kaur, 2020).
In a previous study, Madejón et al. (2021) demonstrated the feasibility of co-composting excess of macroalgae biomass with garden prune waste at pilot scale using different ratios of garden prune waste to algae. In the present work, we first aimed at testing the suitability of the composted materials obtained by Madejón et al. (2021) as soil amendments for vineyards. Then, it investigated the most appropriate ratio of seaweed to pruning waste to obtain the best agronomic benefits. We hypothesized that the composted algae materials in combination with garden prune waste are a valuable organic amendment that can improve soil quality and by extent vines nutritional status and yield. To test this hypothesis soil properties (pH, electrical conductivity (EC), nutrient and organic matter content), plant nutritional status as well as grape quality and yield in plots were measured for two consecutive years.

| Study area and vine cultivation
The field experiment was conducted on a horticultural farm named 'La Viña del Gato' (36°39′19″N; 6°22′26″W) located near the town of Rota (Cádiz, Southern Spain). The municipality of Rota is characterized by a Mediterranean climate; this is hot and dry summers with a yearly annual temperature of 17.8°C and an average cumulative precipitation of 588 mm ( Figure S1). The main characteristics of the soil in the study area which is classified as Arenosol (IUSS Working Group WRB, 2015) are summarized in Table 1. In this locality, a native grapevine (Vitis vinifera L.) variety called 'Tintilla' characterized by its high sugar content can be found. The main structure of the trellis is formed by metal posts of 40 mm in diameter for the header posts and between 30 and 40 mm for the intermediate posts. The total length of the entire row is 35 m, and the distance between posts is 7 m. The rootstock is taken from the Paulsen variety resulted from the crossbreeding of Vitis berlandieri cv. Rességuier number 2 and Vitis rupestris cv. Lot. Its cultivation has remained residual in a few and small vineyards of the Cadiz province, although it is recently gaining prominence in the production of great red wines within the VT Cádiz appellation.

| Experimental design
The experiment was conducted using a linear randomized design of four treatments including a control with inorganic fertilization and three replicates ( Figure 1). The treatments consisted of adding to the soil three different composted materials from pruning waste and seaweed mix (henceforth, P2S1, P1S1 and P1S2, referring the treatment names to the ratio (W/W) of pruning waste (P) to seaweed (S) used to prepare the composted amendments in this study). A dose of 80 tons ha −1 of each type of compost, which delivered 0.51, 0.44 and 0.38 kg of C m −2 for P2S1, P1S1 and P1S2, respectively, was randomly applied in lines of approximately 35 m along the ridge of the vines in January 2020. A detailed description of the key parameters of the compost used in this study can be found in Table 2. A control treatment with mineral fertilization consisting in 200 kg ha −1 of 8N-15P-15K was established. Lines without compost application or mineral fertilizer were interspersed in between the compost treatments. Each line consisted of ca. 30 vines of ca. 3-4 years old.
Crop management tasks included mainly pruning, shoot thinning, sucker and watersprout removal, tipping and shoot positioning. Phytosanitary treatments consisted of the application of wettable sulphur and penconazole 10% p/v as a fungicide to treat powdery mildew and one application of an insecticide (based on cipermetrine) to treat the grape moth (Lobesia botrana).
The composted materials were produced using the windrow method as explained in detail by Madejón et al. (2021). In brief, pruning and garden waste and seaweed removed from beaches near the field experiment were composted in windrow piles, and the result was a material nonphytotoxic, which could be used as soil amendment for agriculture. The macroalgae collected were F I G U R E 1 Location of the experimental site and treatments applied typically found on the intertidal region of the Western coast of Cádiz included a variety of red, brown and green algae among which Halopithys incurva, Laurencia obtusa (Hudson) and Halopteris scoparia (Linnaeus) were particularly noteworthy.

| Soil and leaf analyses
Three soil samples (0.5 L) within the lines treated were collected in June 2021. Each sample was air-dried and <2 mm sieved before analysis. Soil pH was measured in a 1 M KCl extract (1:2.5, m/v) after shaking for 1 h (Hesse, 1971) using a pH meter (CRISON micro pH 2002). EC was determined in the water extract (1:5, m/v) after shaking for 1 h using conductivity meter (CRISON micro CM 2201). Soil organic matter (SOM) was calculated by dichromate oxidation and titration with ferrous ammonium sulphate (Walkley & Black, 1934). Total Kjeldahl-N (TN) was determined by the method described by Hesse (1971). Available-P was determined after extraction with sodium bicarbonate at pH 8.5 (Olsen et al., 1954), and available-K was determined after extraction with ammonium acetate at pH 7.5 (Freitas, 1970).
At each plot, a representative sample of leaves of each plant was collected in June 2020 and June 2021 (coinciding with the greatest phenological crop development). Vegetal material (leaves) was washed with a 0.1 N HCl solution for 15 s and subsequently with distilled water for 10 s. Washed samples were oven dried at 70°C. Dried plant material was ground and passed through a 500μm stainless-steel sieve prior to preparation for analysis. Macronutrients (P, K, Ca, Mg and Na) and trace elements were determined by ICP-OES after digestion of plant tissues by wet oxidation with concentrated HNO 3 in a Digiprep MS block digester (SPS Science). We assessed the quality of the analyses using the reference sample INCT-OBTL-5 (tobacco leaves), and confident recovery rates (ranged between 90% and 105%) were obtained for all the elements included here.

| Grape harvest
The harvest of each campaign was determined in September 2020 and August 2021. Each vine line with the different treatments was divided into three subplots, and grapes were weighed for the vines of each individual subplot.
In 2021, a representative sample of grapes was transferred to the laboratory, and after a light washing with distilled water, the grapes juice was extracted by handpressing and its pH and sucrose content (brix degrees) were measured as grape quality parameters. Brix degrees were determined using a hand-held refractometer (ZaZi 300 series) with a Brix range of 0%-32%. T A B L E 2 Main parameters of compost used in this study (mean values ± SEM). Treatment names (i.e. P2S1, P1S1 and P1S2) refer to the ratio of pruning waste (P) to seaweed (S) used to prepare the composted amendment in this study.

| Statistical analyses
Analysis of variance (one-way ANOVA) with treatment as the main factor was performed on soil chemical and grape harvest and quality parameters. Two-way ANOVA with sampling time and treatment as main factors followed by Tukey's post hoc test was performed to explore the effect of compost application on macronutrients and trace elements in the leaves. For all statistical tests, p < .05 was selected as the significance cut-off value. Statistical analysis was performed with SPSS v25 for Windows (IBM Corp.).

| Changes in soil chemical parameters
Overall, amendment addition increased soil pH in comparison with the control (6.86 pH value) by 0.1-0,5 units but statistical differences were only observed for the composted material with a seaweed ratio equal or higher than the pruning waste ratio (P1S1 and P1S2) ( Table 3). By contrast, the EC decreased after amendment application between 0.020 and 0.040 dS m −1 that is 10%-20% less than the control (0.22 dS m −1 , p < .05). Organic matter increased in all amended treatments between 13% and 47%, but significant differences with respect to the inorganic control were solely observed for the lowest seaweed ratio (i.e. P2S1). For macronutrients (N, P and K), greater concentrations were generally observed in amended treatments compared with the control. A larger increase in macronutrients was observed for those treatments amended with unequal proportions of algae and prune waste (P1S2 and P2S1) than with equal proportions (P1S1). The macronutrient concentrations followed the order: P1S2 > P2S1 > P1S1 ≥ control except for P concentration. The organic amendment effect was most visible for N and K with a net maximum increase of 35%-40% with P1S2. For P, the maximum increase was 10% also with P1S2.

| Leaf response to compost addition
In general, greater concentrations of N, P and K in leaves were observed for the organic treatments compared with the inorganic fertilization during the first year (Table 4) with a maximum net increase of ca. 10%. The largest effect was observed for the P1S2 treatment (largest algae to prune waste ratio). For the second year, significant differences were only observed for K contents. For Ca, Mg and Na, no significant differences among treatments were observed during the first nor the second year (Table 4). In general, macronutrient concentrations were lower in the second year compared with the first year with few exceptions (i.e. Ca in control and P1S1). For micronutrients, no clear treatment effect was observed, that is, there were neither substantial differences between organic and inorganic fertilization nor between organic amendments with different ratios of prune waste and algae (Table 5). For certain micronutrients, interannual differences were observed between the years 2020 and 2021. For B and Fe, concentrations in leaves decreased from 2020 to 2021, whereas a significant increase was found for Cd, Mn and Ni. These trends were observed for all treatments.

| Grape productivity and quality
In 2020, grape harvest was less than 500 kg ha −1 , with no differences between treatments (Figure 2). For the 2020 campaign, results for grape productivity and quality have to be considered with caution since the vineyard maintenance works (i.e. pruning, phytosanitary control and tipping) were limited because of the sanitary COVID-19 situation, thus affecting grape production substantially. This is evident when results for 2020 and 2021 are compared (Figure 2). In 2021, grape harvest was 2-4 times greater than in 2020. Grape harvest in P1S1 and P2S1 almost doubled in 2021 that in the inorganic control, although statistical differences were not observed.
T A B L E 3 Soil parameters for inorganic (control) and organic (composted amendments) treatments during the 2021 campaign. Productivity in the P1S2 treatment was about 33% greater than in the inorganic control. Grape quality parameters (pH and sucrose content) did not differ statistically between organic treatments and the inorganic control (Table 6), yet control plants displayed a slightly higher content of sucrose (ca. 9%) than the plants treated with the composted amendments.

| DISCUSSION
Seaweed components are shown to have positive impacts on the growth and development of crops, the yield and also soil conditions (Battacharyya et al., 2015;Padam & Chye, 2020). In fact, they have been particularly useful under stress conditions, for example, drought and salinity, which are of vital importance in Mediterranean ecosystems (Górka et al., 2018;Yuvaraj & Gayathri, 2017). In our study, the addition of composted materials with the highest seaweed ratio slightly increased pH values compared with the control, which can be ascribed to the marine origin of the raw material rich in Na, which was incorporated in greater proportion. Other authors have suggested that this slight increase in the pH values, particularly in soils with a low pH (i.e. 6.8), could cause greater nutrient availability (mainly P and K; Eyras et al., 2008;Michalak & Chojnacka, 2013). Regarding N content, soils treated showed a noticeable improvement compared with the control soils. Our results provide evidence that composted seaweed can in fact maintain but also moderately enhance soil N storage contributing to long-term soil quality and fertility without compromising productivity. Roussis et al. (2022) found that manure and seaweed compost as opposed to inorganic fertilization had the greatest influence on soil total nitrogen, which was attributed to the clear relationship with the increase in SOM as our results corroborated as well.
In the case of the available-K, only the compost with the highest seaweed ratio (i.e. P1S2) differed significantly from the control which it is likely because of the natural high K content in this type of algae (Shehata et al., 2011;Turan & Köse, 2004). Although it has been observed that excess K levels in soils can have a detrimental effect on wine quality owing to the resulting high juice pH (Assimakopoulou & Tsougrianis, 2012), previous studies have shown that an additional source of K can directly affect cluster number and weight and consequently grape yield, being this an added value of the compost worth considering (Amiri & Fallahi, 2007;Basak & Sarkar, 2017). Additionally, this significant K increase did not cause any salinization so its use as fertilizer would not present a risk in well-drained soils as it has been observed before with other types of crops (Cole et al., 2016;López-Mosquera & Pazos, 1997).
Leaves response to the organic composted materials showed a significant variation of N and P contents, in the interaction of time and treatments. The percentage of N and P in leaves suggests that there is an initial positive enrichment just after compost application and also that there is a greater use of these nutrients in treated vines than in the controls if the initial-final percentages are compared. Indeed, it is interesting to note that the accumulation pattern in leaves of some of these nutrients (e.g. N, P and K) kept a direct relationship with the ratio of seaweed applied (i.e. higher ratio greater concentrations) whereas others T A B L E 4 Macronutrients in the leaves for inorganic (control) and organic (composted amendments) treatments.
such as Ca, Mg or Na were seaweed ratio independent. Organic fertilization had a major impact on K concentration in the leaves that unlike in the soil had an effect even with the lowest seaweed ratios. López-Mosquera and Pazos (1997) attributed this effect to the fact that the K present in seaweed was in a readily available form and that had a direct impact also on the yield as our results seemed to indicate as well. Eyras et al. (2008), however, ascribed this yield enhancement to the increase in P availability because of slight changes in the pH after compost addition.
Our results seem to be consistent with these observations detecting an improvement in both K content and P. In the literature, nutrients in seaweed compost are shown to be highly dependent on the seaweed species used, the environment they live in, the composting method, the concentration used, the intrinsic soil characteristics and the specific crop requirements (Cole et al., 2016;Gibilisco et al., 2020;Michalak & Chojnacka, 2013). In our experiment, although the specific crop needs seemed to be fully met, it would be useful to assess the effect of seaweed compost using different species in order to intensify the enrichment of some of the most beneficial macronutrients. T A B L E 5 Trace elements in leaves for inorganic (control) and organic (composted amendments) treatments.
One of the main concerns with respect to the use of seaweed compost is the potential of algae as accumulating sources of heavy metals (Deniz & Ersanli, 2018;Michalak & Chojnacka, 2013). In our case, the analysis of trace elements reflected no evidence of heavy metal contamination. In fact, their concentration in the leaves remained far below the limits established by the EU legislation (Commission Decision (EU) 2015/2099). This fact is of particular importance as the concentration of trace elements in seaweed compost has been indicated to be highly related not only to the seaweed species used but also to their environment (Foday et al., 2021). In agreement with our study, other authors have reported low heavy metal contents in compost made from seaweed but the accumulation rate of these elements is rarely explored (Illera-Vives et al., 2013;López-Mosquera et al., 2011). Here, most of the trace elements in plants, such as B and Fe, experienced a noticeable decrease over time, indicating that their residence time within vegetal biomass is limited. However, elements such as Cd, Mn or Ni tended to increase over time, suggesting that their turnover rate is significantly slower, and therefore, their release/uptake should be monitored if the continuous use of the compost is desired. Some studies focussed on metal removal by seaweed biomass related this phenomenon to the particular biosorption strength of the seaweed cell walls that interact with metal ions differently (Ortiz et al., 2017).
Apart from the key role of seaweed as biosorbent for heavy metals, their favourable effects on agriculture as an alternative to conventional fertilizers have been empirically proven from ages ago (Cole et al., 2016;Illera-Vives et al., 2020;López-Padrón et al., 2020). Specifically, in grapes, beneficial effects range from early fruit ripening, higher number of fruits per plant, larger berry size and weight and stimulation of several phenolic compounds (Arioli et al., 2021;Gutiérrez-Gamboa et al., 2020;Norrie & Keathley, 2006). Here, we observed a noticeable improvement in grape production especially with the P1S1 compost without compromising grape quality as it has been identified before (Genesio et al., 2015). This yield increase suggests that the P1S1 offered a suitable and preferential C/N and other nutrients ratio for the development of this specific crop. Winberg et al. (2013) determined that the compost made from green waste and seaweed with lower algae percentage attained the highest crop yields, since higher doses of certain algae compounds can inhibit plant growth. López Varela et al. (2015) did not find any improvement in grape yield production after compost amendment, because of a more conditioning effect of the soil water availability and the photosynthetic rate than the enhancing effect of compost. By contrast to the results presented in Cole et al. (2016), in which a C/N ratio of the compost less than 20 was recommended to avoid nitrogen limitations and obtain better yields, our findings showed that the P1S1, which had the highest C/N ratio (i.e. 27.3, Madejón et al., 2021), outperformed the other treatments in terms of grape productivity without any clear N deficiency F I G U R E 2 Grape quality parameters for inorganic (control) and organic (composted amendments) treatments. Treatment names (i.e. P2S1, P1S1 and P1S2) refer to the ratio of pruning waste (P) to seaweed (S) used to prepare the composted amendment in this study. Same lower-case letters indicate no significant difference (p > .05) between treatments. Data are mean values ± standard error of the mean (SEM) T A B L E 6 Grape quality parameters under different compost treatments. Treatment names (i.e. P2S1, P1S1 and P1S2) refer to the ratio of pruning waste (P) to seaweed (S) used to prepare the composted amendment in this study. Data are mean values ± standard error of the mean (SEM) neither in the soil nor in the leaves. Our results are in line with the studies of Han et al. (2014) and Michalak et al. (2016) in which a C/N of the composted materials between 25 and 30 were highly recommended to enhance microbial activity. However, in the light of the above mentioned, it would be highly recommendable to test our results with repeated compost applications over time, in order to determine the specific processes and descriptors of this beneficial effect for this crop. The evaluation of grape quality parameters did not reflect any detrimental effect of the use of seaweed amendment to the grape quality. Values of pH and sucrose were within the normal range for this variety (Heras-Roger et al., 2016). However, the trend towards lower sucrose contents found for P2S1 and P1S1, the two treatments with the highest grape yields, may indicate that plants growing on soil amended with those composts produce berries with higher water contents and/or less sucrose concentration. This could be related to higher water availability as it has been described before (Medrano et al., 2003;Yuvaraj & Gayathri, 2017) or related to a diluting factor because the sugar content in those plots is distributed among a higher number of berries. Since optimal sucrose content has a key role in wine production, while higher water content of the berries increases fruit yields, a trade-off between these two grape berries parameters has to be taken into account (Dai et al., 2011). However, facing a climate change scenario, improved water efficiency should be listed as a primary target for the vineyard sustainability in Mediterranean areas (Borsato et al., 2020).

| CONCLUSIONS
The present study highlights the potential of seaweed organic amendments to maintain soil fertility and meet vine nutrient requirements. Among the studied ratios of pruning waste and seaweed, the mixture with an equal ratio (1:1) of both materials was more effective in enhancing grape production compared with the control. However, despite this yield increase, the mixture with the highest pruning waste ratio (2:1) caused the greatest increases in soil properties compared with the control, especially in terms of carbon storage. We conclude that the resulting materials from composting seaweed and pruning wastes are suitable organic amendments for agricultural use in vineyards, adding an agronomic value to a bioresource that otherwise will turn into a residue. However, future work should investigate these effects in a wider range of crops, soils and time extent in coastal areas. In this context, the promotion of eco-friendly management of marine by-products appears to be essential in the context of the climate challenge, helping to reduce greenhouse gas emission, the reliance on mineral fertilizers, and achieving the EU landfill reduction target of 10% by 2030.