*2.2. Experimental Designs*

The experimental design was divided into four field trials arranged as factorial designs with six replications (six twin canopies of three plants). The plants were randomly selected in the corresponding experimental plots when they reached 3 m in height in the plots of higher vigour plants.

Experiment 1 consisted of a factorial design (two factors) including plots of plants of different vigour (weak, fair, good and very good) and years (2016, 2017 and 2018). The classification of the vigour of the plants was made with the farmers help as above mentioned. The plots were planted with the Nugget cultivar and were installed ~20 years ago.

Experiment 2 consisted of a factorial design (three factors) of plots of different plant vigour (weak, fair, good and very good), foliar sprays (algae- and nutrient-rich foliar sprays and control) and years (2017 and 2018).

The algae-rich foliar spray (Algae) is a solution containing 15% (*w*/*w*) the algae *Ascophyllum nodosum* (L.) Le Jolis, applied at a rate of 2 L ha−<sup>1</sup> (diluted in 1500 L of water) three times during the growing season, at the phenological stages of inflorescence

emergence, flowering, and beginning of the development of cones (on 20 June, 10 July and 27 July 2017, and 20 June, 8 July and 24 July 2018, respectively). In spite of the differences on plant vigour of the different plots, the phenological stage of the plants was similar.

The nutrient-rich foliar spray (Fnut) is a mixture of *A. nodosum* (1.4% *w*/*w*) enriched with macro- and micronutrients containing ( *w*/*w*) 12% N, 6% P2O5, 4% K2O, 0.025% B, 0.1% Fe-EDTA, 0.05% Cu-EDTA, 0.05% Zn-EDTA, and 0.05% Mn-EDTA. This fertilizer was applied at a rate of 3.5 L ha−<sup>1</sup> (diluted in 1500 L of water) on the dates reported for Algae. In each plot the foliar sprays were applied in four rows and the six twin canopies of each treatment were sampled in the two interior rows. The plots where this experiment was carried out were the same reported for experiment 1, although in a different part of the plots.

Experiment 3 was arranged as a factorial design (three factors) and included hop plots (two) of good vigour plants (Plots 5 and 6), liming (limed and not limed) and years (2017 and 2018). The limestone (55% CaCO3, 28% CaO and 20% MgO) was applied at a rate of 1000 kg ha−<sup>1</sup> in February 2017 and incorporated into the soil with a cultivator. Both fields, ~2 ha each, are of the Nugget cultivar and they are ~20 years old. As the study was carried out as on-farm research, liming was carried out in a larger part of the area, with only four rows of ~150 m remaining for the control treatment, and the plants were sampled from the internal rows of the treated or untreated plots.

Experiment 4 was a factorial of two factors: cultivars (Nugget, Cascade and Columbus) and year (2017 and 2018). This experiment was carried out in Plot 4, in which part of the plot was installed with several different cultivars, each one occupying a row of ~150 m. This hop field was planted in 2014. An overview of the experimental design is shown in

#### *2.3. Plant Sampling and Tissue Analysis for Elemental Composition*

Plant material was collected at harvest and subsamples of fresh cones were carried to the laboratory, oven-dried at 70 ◦C and thereafter ground for laboratory analysis. Tissue analysis for elemental composition was performed by Kjeldahl (N), colorimetry (P and B), flame emission spectroscopy (K), and atomic absorption spectroscopy (Ca, Mg, Cu, Fe, Zn, and Mn) methods [27], after the samples were digested with nitric acid in a microwave (MARSXpress CEM).

### *2.4. Analysis of Total Phenolics*

Hop cone samples were ground in a Cyclotec mill, with a 1 mm mesh screen, to obtain a fine powdered sample. Infusion preparation was performed by using 1 g of fine powdered hop sample, which was added to 100 mL of boiling distilled water and left to stand at room temperature for 5 min, and then filtered. Total phenols were determined in a total of 204 samples (36 samples from Experiment 1, 72 samples from Experiment 2, 72 samples from Experiment 3 and 24 samples from Experiment 4). The extracts obtained were diluted 1:1. Folin–Ciocalteu's assay, briefly, 0.5 mL of each diluted extract was mixed with the Folin–Ciocalteu reagen<sup>t</sup> (2.5 mL). After 3 min, they were saturated with sodium carbonate solution (2 mL) and the reaction was kept in a water bath at 40 ◦C for 30 min. The absorbance was read at 765 nm (PG Instruments T80 UV/VIS Spectrophotometer, QLabo, Portugal). Gallic acid was used to prepare the standard curve and the results were expressed as mg of gallic acid equivalents (GAEs) per g of dry matter of hop cones. The analysis of total phenols in each sample was carried out in triplicate.
