Response of Hops to Algae-Based and Nutrient-Rich Foliar Sprays

: Over recent years, some hopyards of northeast Portugal have presented poorly developed plants and reduced productivity. In this study, an attempt was made to improve the homogeneity of hop ﬁelds and restore their productivity by using plant biostimulants as foliar sprays. The experimental apparatus included four ﬁeld trials carried out in four plots of different plant vigour, as evaluated by farmers over previous years (weak, fair, good and very good). The experiments were arranged as a factorial of foliar treatment (two plant biostimulants containing extracts of seaweed algae and an untreated control) and year (2017 and 2018). The plot and the year inﬂuenced greatly almost all the measured variables related to tissue nutrient concentration and crop performance. In the control plots, cone dry matter (DM) yield varied from 83.3 to 394.4 g plant − 1 from the weak to the very good plots. In 2018, cone DM yield was signiﬁcantly higher than in 2017. The use of foliar sprays inﬂuenced less the elemental composition of plant tissue than the plot or the year. The use of foliar sprays only increased signiﬁcantly crop yield in the plot of weak plant vigour. The foliar treatments did not increase α - and β -acid concentration in the cones; in the control treatment of the most productive plot, the values were, respectively, 11.2 and 3.9%. Although seaweed extracts tend to help plants cope with several abiotic and biotic stresses, they showed to be effective in mitigating the stress that is affecting these plants, which probably is poor soil drainage caused by the ﬂooding irrigation system, only under conditions of severe stress.


Introduction
A common way to meet crop nutritional requirements and improve crop productivity is through the application of conventional fertilizers to the soil. However, nutrient uptake can also take place via leaf surface, stomata and other specialized cells, which allows the use of foliar sprays as fertilizing materials [1]. In general, both macro and micronutrients can be applied as foliar sprays. However, the restricted amounts of nutrients that can be supplied by foliar sprays make this strategy more attractive for the application of micronutrients, especially when the application to the soil is of little effect, such as in acidic or alkaline soils [2].
The range of fertilizer formulations for foliar application is currently huge, and their use is expected to increase at a compound annual growth rate of 4% until 2028 [3]. In addition to macro and micronutrients essential to plants, many products for foliar application contain substances of differing natures with the potential to have a biostimulating effect on plants. A plant biostimulant has been defined as a substance or microorganism applied to the soil, seeds or plants with the aim of enhancing nutritional efficiency, abiotic stress tolerance and/or crop quality, regardless of its nutrient content [4,5]. Some commercial products can, however, be formulated as mixtures of more than one plant biostimulant The hop plots are arranged in a 7 m conventional high trellis system, with concrete poles, connected with cables, in a "V" design system. The plantations were installed about 20 years ago. In the original plantation, the rhizomes were spaced 2.8 m × 1.6 m between rows and within rows, respectively. From each position of an original rhizome, a double tutor thread was placed in "V" connecting the plants (groups of 3 to 4 stems) to the upper wire structure, which set up a density of ~2,232 plants ha −1 .
The hop plots were selected according to their yield potential as recorded in the previous seasons by the farmers. Four extreme situations were considered adequate for this study, from the poorer to the better plots found in the region. With the help of the farmers, the plots of different yield potential were named as weak (Plot 1), fair (Plot 2), good (Plot 3) and very good (Plot 4). The soils of the different plots used in this experiment were sampled before the trial started for characterization of the growing conditions. Three composite soil samples (15 sampling points) were taken between the rows on 2 June 2016, at 0-20 cm depth in each one of the plots. The soil textures varied from clay loam (Plots 1 and 2), to sandy clay loam (Plot 3) and sandy loam (Plot 4). More details on the chemical soil properties determined from these samples are provided in Table 1.  The hop plots were selected according to their yield potential as recorded in the previous seasons by the farmers. Four extreme situations were considered adequate for this study, from the poorer to the better plots found in the region. With the help of the farmers, the plots of different yield potential were named as weak (Plot 1), fair (Plot 2), good (Plot 3) and very good (Plot 4). The soils of the different plots used in this experiment were sampled before the trial started for characterization of the growing conditions. Three composite soil samples (15 sampling points) were taken between the rows on 2 June 2016, at 0-20 cm depth in each one of the plots. The soil textures varied from clay loam (Plots 1 and 2), to sandy clay loam (Plot 3) and sandy loam (Plot 4). More details on the chemical soil properties determined from these samples are provided in Table 1.

Experimental Design and Treatment Application
Four similar and independent field trials corresponding to the plots of weak, fair, good and very good vigour plants were arranged as a factorial design, to accommodate two experimental factors, foliar fertilization (three levels) and year (two levels, 2017 and 2018), in six replicates. As foliar fertilization, two commercial plant biostimulants were used as foliar sprays to which a non-fertilized control was added.
One of the plant biostimulants is particularly rich in nutrients (Folivex Crescimento ® ) and was named in this study as Fnut. It combines several macro and micronutrients and a small portion of an extract of the algae A. nodosum (1.4% w/w). Fnut contains (w/w) 12% N, 6% P 2 O 5 , 4% K 2 O, 0.025% B, 0.1% Fe-Ethylenediaminetetraacetic acid (EDTA), 0.05% Cu-EDTA, 0.05% Zn-EDTA, and 0.05% Mn-EDTA. The second plant biostimulant (Fitoalgas Green ® ) was selected due to its high content (15% w/w) of A. nodosum, and the treatment was named 'Algae'. The foliar sprays were applied at the rates recommended by the manufacturers. Fnut was applied at a rate of 3.5 L ha −1 , diluted in 1500 L water, three times during the growing season (20 June, 10 July and 27 July 2017, and 20 June, 8 July and 24 July 2018). The first foliar treatment was done when the plants of Plot 4 had reached 80% of the top wire height, the second at the end of bine growth, and the third during the enlargement of inflorescence buds. Algae was applied at a rate of 2 L ha −1 , also diluted in 1500 L of water, on the same dates mentioned for Fnut.
Each replication consisted of six twin canopies of three plants (in the "V" design system). These plants were marked when those in Plot 4 (very good) were~3 m tall. In the other plots, plants with height within the plot pattern were selected.
All the plots received the basal fertilization plan usually used in the region, consisting of a compound NPK (7:14:14) fertilizer applied late in winter at a rate of~500 kg ha −1 , and two side dress N applications performed during the growing season, the first with 200 kg ha −1 of nitromagnesium (27% NH 4 NO 3 + 3.5% MgO + 3.5% CaO) and the second with~450 kg ha −1 of calcium nitrate (15.5% NO 3 − + 27% CaO). The farmers manage their fields with a surface irrigation system, consisting of regular flooding of the space between rows. Several tillage passes (3 to 4) were performed every year to remove the crusts and allow water infiltration.

Laboratory Analyses
The soil samples were firstly oven-dried (40 • C) and sieved (2 mm). Thereafter, they were analysed for pH (H 2 O and KCl) (soil: solution, 1:2.5), cation-exchange capacity (am-Agriculture 2021, 11, 798 5 of 15 monium acetate, pH 7.0), organic C (wet digestion, Walkley-Black method) and extractable P and K (Egner-Rhiem method). Extractable P was also determined by the Olsen method. Soil B was extracted by hot water and the extracts analysed by the azomethine-H method. For more details on these analytical procedures, the reader is referred to Van Reeuwijk [29]. The availability of other micronutrients (Cu, Fe, Zn, and Mn) in the soil was determined by atomic absorption spectrometry after extraction with ammonium acetate and EDTA, according to the method described by Lakanen and Erviö [30].
Elemental tissue analyses were performed by Kjeldahl (N), colorimetry (B and P), flame emission spectrometry (K) and atomic absorption spectrophotometry (Ca, Mg, Cu, Fe, Zn and Mn) methods after nitric digestion of the samples [31]. Bitter acids (α and β) in hop cones were extracted with methanol and diethyl ether by high performance liquid chromatography (HPLC), according to the Analytica European Brewery Convention (EBC) 7.7. method [32].

Data Analysis
Data was analysed for normality and homogeneity of variances using the Shapiro-Wilk and Bartlett's test, respectively. The analysis of variance was performed according to the experimental design as a two-way ANOVA. When significant differences were found between experimental treatments, the means were separated by the Tukey HSD test [for the factor of three levels (Foliar treatment)] or Student's t-test [for the factor of two levels (Year)] (α = 0.05).

Plant Dry Matter Yield
The total aboveground DM yield, and DM yield of the different plant parts (stems, leaves or cones), varied between plots of different plant vigour, the former from~300 (weak) to~1200 (very good) g plant −1 (Figure 2). The result was expected since the plot selection took into account the vigour of the plants in previous years. The nutrient-rich foliar spray did not significantly influence the total DM yield or any of its components, including the DM yield of the cones. Algae gave significantly higher values of cone and total aboveground biomass in comparison to the other treatments only in the plots of weak vigour plants. In 2018, the DM yield was significantly higher for all plant parts in comparison to 2017. Significant interaction between the two factors was not usually observed, thus not deserving particular attention.
Plant vigour had little influence on the size of the cones ( Figure 3). The foliar treatment also did not significantly influence the size of the cones in most of the plots. However, in the plot of good vigour plants, cone dry weight was found to be significantly lower in the Fnut treatment. The year had a great influence on the size of the cones. Significant differences were found in the plots of weak, fair and good vigour plants, with 2018 showing the higher values. selection took into account the vigour of the plants in previous years. The nutrient-rich foliar spray did not significantly influence the total DM yield or any of its components, including the DM yield of the cones. Algae gave significantly higher values of cone and total aboveground biomass in comparison to the other treatments only in the plots of weak vigour plants. In 2018, the DM yield was significantly higher for all plant parts in comparison to 2017. Significant interaction between the two factors was not usually observed, thus not deserving particular attention. Plant vigour had little influence on the size of the cones (Figure 3). The foliar treatment also did not significantly influence the size of the cones in most of the plots. However, in the plot of good vigour plants, cone dry weight was found to be significantly lower in the Fnut treatment. The year had a great influence on the size of the cones. Significant differences were found in the plots of weak, fair and good vigour plants, with 2018 showing the higher values. Plant vigour had little influence on the size of the cones (Figure 3). The foliar treatment also did not significantly influence the size of the cones in most of the plots. However, in the plot of good vigour plants, cone dry weight was found to be significantly lower in the Fnut treatment. The year had a great influence on the size of the cones. Significant differences were found in the plots of weak, fair and good vigour plants, with 2018 showing the higher values.

Tissue Nutrient Concentrations
The concentration of nutrients in the leaves varied greatly between plant vigour plots in the leaf samples taken at~2 m height in July ( Table 2). The leaf concentrations of N, Ca, and particularly K, were remarkably higher in the plot of very good plant vigour. In contrast, average leaf P and Mg concentrations were significantly lower in the plot of high vigour plants. The results of the plots of fair and good plant vigour were very close to those recorded in the plot of weak plant vigour. The effect of the foliar treatments was smaller for macronutrients, in spite of significant differences being found for P and Mg in the plot of weak vigour plants and for Ca in the plot of very good vigour plants. Significant differences between foliar treatments in fair and good plots were not common, nor did they show a consistent trend with the results of weak and very good plots (data not shown). The years showed a large variation in the concentration of the macronutrients in the leaves, particularly for K, Ca and Mg. Leaf K levels were particularly high in 2018 and Ca levels in 2017. Significant interactions between foliar treatment and year was neither frequent nor consistent between plots for a given nutrient.
The micronutrients Mn, Zn and B were particularly high in the leaves of the plots of higher vigour plants in comparison to the others ( Table 2). The foliar treatments had little effect on the concentration of micronutrients in the leaves. Only leaf B levels were found to be significantly higher in the Algae in comparison to the other treatments. The year significantly influenced the concentration of most of the micronutrients in the leaves, although some were higher in 2017 and others in 2018.
The plant tissues analysed at harvest (top and bottom leaves and stems) showed many differences in comparison to the leaves sampled in July, related to the date of sampling, the position in the canopy and type of tissue, but maintained the trend of the effect of the treatments of the samples taken in July (data not shown). In comparison to the July samples, the leaves at harvest from the bottom half of the plants showed low levels of N and P and higher levels of Ca, Mg and B. As observed for the July samples, leaf concentrations of all nutrients varied greatly between plant vigour plots. Leaf concentrations of some nutrients also varied significantly with the foliar treatments. However, in general terms, the control did not show lower values than the fertilized treatments. The year effect was statistically significant for most of the nutrients, some being higher in 2018 and others in 2017. In the top half leaves, the concentrations of N, K and B were markedly higher and P and Mg markedly lower in the very good vigour plants, in comparison to the weak vigour plants. Although significant differences between foliar treatments were found for some nutrients, a clear pattern distinguishing between the results of the fertilized and the non-fertilized plots was not observed. The year again showed a marked influence on leaf nutrient concentrations. Stem nutrient concentrations were markedly lower for the majority of macro and micronutrients in comparison to the values found in the leaves. Significant differences between foliar treatments were also often found for some nutrients, but the control did not display significantly lower values than the fertilized treatments. The effect of the year was statistically significant for most of the nutrients, as observed for leaf analysis.
The concentration of the majority of the macro and micronutrients in the cones was markedly different from leaves and stems (Table 3). The levels of P and K were markedly higher in the cones in comparison to those of leaves or stems. The levels of Ca, Mg and B, for instance, were lower in the cones in comparison to the leaves. The cone levels of N were similar to that of the leaves. The range of variation for each nutrient seemed to be lower than that observed for leaves.
However, as observed for leaves, great differences between plant vigour plots were found for all nutrients. Very high vigour plants showed lower levels of N, P, Mg and Cu in the cones in comparison to the weaker plants. K and B, for instance, were significantly higher in the very good vigour plants than in the weaker plants. Table 2. Leaf concentrations of macro and micronutrients (average ± standard error) in July from samples taken at 2 m height in the plots of weak and very good vigour plants as function of foliar treatment (Fnut, nutrient-rich foliar spray; Algae, algae-rich foliar spray; and Control), and year. Means followed by the same letter are not statistically different (α = 0.05) by Tukey HSD (Foliar treatment) or t-Student t (Year) tests.  The effect of the foliar treatments on cone nutrient concentration was small, and not consistent between the different plots.

Nitrogen
Once again, the year showed a marked influence on tissue plant composition. Significant differences were found for almost all the nutrients, with the concentration of some to be found higher in 2017 and others in 2018.

SPAD Readings, NDVI and Chlorophyll Fluorescence
SPAD, NDVI and chlorophyll a fluorescence varied greatly among plant vigour plots (Table 4). A tendency for a significant increase was found in these tests from the weaker to the higher vigour plants.
In turn, the foliar sprays did not have a great effect on these indices of nutritional status and photosynthetic performance of plants, the most important exception being the higher SPAD values in the Algae treatment in the plot of weak vigour plants. The year seemed to influence some of those variables, but with little consistency between plots and DM yield. NDVI, however, showed higher values in 2018, the most productive year.

Concentration of Bitter Acids in the Cones
The concentrations of αand β-acids in the cones were higher in the plants of very good vigour than in the plants of lower vigour (Figure 4). Foliar sprays did not significantly increase the concentrations of αand β-acids in the cones in comparison to the control. The year had a marked effect on the cone composition; the values of αand β-acids were significantly higher in 2018 in comparison to 2017. Significant interaction of foliar treatment × year was found to α-acid concentrations in the fair and good vigour plots and β-acid concentration in good vigour plot.

Concentration of Bitter Acids in the Cones
The concentrations of α-and β-acids in the cones were higher in the plants of very good vigour than in the plants of lower vigour (Figure 4). Foliar sprays did not significantly increase the concentrations of α-and β-acids in the cones in comparison to the control. The year had a marked effect on the cone composition; the values of α-and β-acids were significantly higher in 2018 in comparison to 2017. Significant interaction of foliar treatment × year was found to α-acid concentrations in the fair and good vigour plots and β-acid concentration in good vigour plot.

Discussion
The plots identified by farmers as the most productive in previous years (very good vigour plants) produced more biomass in all the plant parts (stems, leaves and cones) than the other plots. The cone size, however, did not differ between the different plots. The concentration of nutrients in plant tissues varied greatly with the vigour of the plants.

Discussion
The plots identified by farmers as the most productive in previous years (very good vigour plants) produced more biomass in all the plant parts (stems, leaves and cones) than the other plots. The cone size, however, did not differ between the different plots. The concentration of nutrients in plant tissues varied greatly with the vigour of the plants. Some nutrients were found at higher levels in the most productive plots, but the concentrations in plant tissues of many other nutrients were higher in the less productive plots. However, no evidence was found that the nutritional status of plants had been an important factor influencing DM yield, since most of the nutrients were found within or close to their sufficiency ranges as reported by Bryson et al. [33]. These variations in tissue nutrient concentrations were probably the result of dilution and concentration effects, which have been well-known for a long time [34], and/or antagonism and synergism in plant nutrient uptake [35]. SPAD and NDVI usually provide a good indication of the greenness of the leaves and general plant health [36,37]. In this study, however, only NDVI values were associated with the most productive plots. Chlorophyll fluorescence ratios (F V /F M and F V /F 0 ) and OJIP transient are usually seen as important tests to assess stresses that affect the function of photosystem II, and are also usually related to crop productivity [38,39]. In this study, these tests showed little sensitivity in discriminating between the different plant vigour plots.
In 2018, the DM yield of all plant parts was significantly higher than in 2017. The spring and summer of 2017 were warmer and drier than in 2018, accentuating the Mediterranean characteristics of the regional climate, which usually reduce the performance of hop crops [23,24]. The concentration of almost all the nutrients analysed varied significantly with the year, irrespective of plant tissue and sampling date. This great dynamic in tissue nutrient concentrations depends on diverse factors; one of the most relevant of these is the dilution/concentration effect related to nutrient uptake and carbon assimilation, and plant growth [34,40], which does not justify further development here. SPAD readings and chlorophyll fluorescence and OJIP transient variables presented inconclusive results, they too are probably related to the concentration of nutrients in the tissues and dilution/concentration effects [37]. NDVI, in turn, showed consistently higher values in 2018, the most productive year. The most productive year displayed significantly higher αand β-acid concentrations in the cones, showing that the conditions promoting plant growth also enhanced the accumulation of bitter acids in the hop cones. Hop-acids are soft resins produced by hop plants as secondary metabolites. The impact of the weather conditions during the two-week period before harvest have a strong influence on the accumulation of α-acids [41]. This is probably the reason explaining the greatest effect of the year in comparison to the plot or the foliar treatment.
The foliar sprays did not produce a significant effect on total DM yield or on DM yield of any of the different plant parts, including the cones in fair, good and very good vigour plots. However, in the plot of weak vigour plants, cone and total DM yields were significantly higher in the Algae in comparison to the other treatments. Due to the high amount of tissues analysed for their elemental composition, sometimes significant differences between treatments were observed but, in these cases, the control treatment never showed lower values. The fertilizer treatments also did not significantly influence the variables related to the photosynthetic performance of the plants, nor did they influence the levels of αand β-acids in the cones in comparison to the control. Other studies can be found in the literature in which the application of algae extracts did not increase productivity or improve the quality of products [42][43][44]. The efficacy of plant biostimulants can vary greatly depending on the conditions of application (concentration of active ingredients, phenological state of plant, etc.), including the competition for uptake by microorganisms in the phyllosphere (or rhizosphere, if the products are applied to the soil), an aspect that is recognized as needing further investigation [4,45]. However, it is usually under stressful conditions that the use of algae extracts tend to give better results [13][14][15], which is in agreement with the observations in this study. The subject has been under intense investigation during the last decades with numerous reviews updating the current knowledge [5][6][7]. The use of seaweed extracts has been highlighted since these products are the most commonly used and those that usually present favourable results on crop growth [8][9][10].
Plant biostimulants have been shown to act as elicitors, enhancing plant growth and triggering stress responses by activating molecular and biochemical pathways [5,11], so it should be emphasized that, in this study, significant effects on the elemental composition of plants by the application of foliar sprays were not observed. Extensive research on the topic has shown that the beneficial role of plant biostimulants, and in particular those made from algae extracts, has been observed mainly under harsh environmental conditions, such as drought [14,46,47], heat [15] or saline [13] stress. In a first analysis, none of these stresses can be identified in these hop fields, which may help to explain the absence of a significant effect by the application of the plant biostimulants on crop productivity in the plots of fair, good and very good vigour plants. A previous study in this region reported poor soil drainage and aeration as the main reason for the heterogeneity observed in hop fields, which is exacerbated by the irrigation method, consisting of flooding the spaces between the rows [28]. Thus, under these conditions, the algae extract was only beneficial on highly stressed plants.

Conclusions
Under the conditions of this experiment, two plant biostimulants containing seaweed algae extracts did not positively influence the mineral composition of hop plants, indices of crop nutritional status and photosynthetic efficiency or the αand β-acid concentrations in the cones. Crop yield was only significantly increased with the use of algae extract in the plot of weak plant vigour. However, almost all those variables changed greatly with the effect of plot and year. This poor effect on crop performance might have been due to the type of stress that limits plant growth, which is probably poor drainage and deficient soil aeration. Although the effectiveness of seaweed extracts in improving plant performance under conditions of drought, heat and salt stress is well known, they had little effect under the conditions of this experiment. Only on highly stressed plants did the positive effect of the algae extract prove to be significant. Thus, to help farmers to overcome the problem, other avenues should be explored, such as the use of different plant biostimulants and/or testing different conditions of application.