**3. Results**

*3.1. Plant Growth Traits, Production Features, NUE and Fruit Firmness*

ANOVA for plant height at 10, 20, 30 DAT and for number of leaves at 10 and 20 DAT showed no significant interaction between SwE doses and genotype (Table 1).


**Table 1.** Effect of the seaweed extract treatments (SwE) and genotypes (G) on plant height at 10, 20 and 30 DAT and on the number of leaves at 10 and 20 DAT of *L. siceraria* plants.

Values present in a column and followed by different letters are significantly dissimilar at *p* ≤ 0.05. NS, \*\*\* non-significant or significant at 0.001, respectively.

> Regardless of the genotype, SwE meaningfully enhanced the aforesaid plant growth parameters. On the other hand, irrespective of the SwE application, G4 and G5 genotypes revealed the highest plant height at 10, 20 and 30 DAT and the highest number of leaves at 10 and 20 DAT, whereas, G3 landrace had the lowest values (Table 1).

> Statistic on the number of leaves at 30 DAT displayed a significant interaction between SwE and genotype (Figure 2).

> Overall, plants treated with SwE revealed a higher number of leaves at 30 DAT compared with the untreated ones. G4 and G5 landraces treated with SwE showed the highest values, followed by G1 and G2 landraces treated at 3 mL L−<sup>1</sup> SwE. G3 untreated plants had the lowest value (Figure 2).

> ANOVA for first female flower emission did not have a significant interaction between SwE and genotype (Figure 3).

Regardless of the genotype, SwE application delayed the first female flower emission (Figure 3). Disregarding the SwE treatment, G3 landrace had the earliest female flower emission, followed by G2 landrace. G4 and G5 landraces revealed the latest female flower emission (Figure 3).

ANOVA for yield and yield-related traits did not reveal a significant influence of the interaction SwE × G (Table 2).

**Figure 2.** Effect of the seaweed extract treatments (SwE) and genotypes (G) on the number of leaves at 30 DAT of *L. siceraria*. Values with different letters indicate significant differences at *p* ≤ 0.05. \*\*\* significant at 0.001. Bars represent the standard error.

**Figure 3.** Effect of the seaweed extract treatments (SwE) and genotypes (G) on first flower emission of *L. siceraria*. Values with different letters indicate significant differences at *p* ≤ 0.05. NS, \*\*\* non−significant or significant at 0.001, respectively. Bars represent the standard error.


**Table 2.** Effect of the seaweed extract treatments (SwE) and genotypes (G) on fruits total yield, fruits marketable yield, No. of marketable fruits, fruit mean mass, young shoot yield and number of *L. siceraria*.

Values present in a column and followed by different letters are significantly dissimilar at *p* ≤ 0.05. NS, \*\*, \*\*\* non−significant or significant at 0.01 or 0.001, respectively.

> SwE application did not affect total fruit yield (Table 2). Conversely, notwithstanding the biostimulant treatment, G1 had the highest total yield, followed by G3, which in turn had a higher total fruit yield than the G2 landrace. The lowest total fruit yield value was recorded in the G4 landrace.

> When averaged over genotype, fruit marketable yield was increased by SwE treatment (Table 2). Regardless of the SwE application, data collected on fruit marketable yield followed the trend recognised for total fruit yield.

> SwE non-treated plants showed a greater number of marketable fruits compared with the treated ones (Table 2). Averaged over the SwE application, G3 landrace showed the highest number of marketable fruits, followed by G1 landrace. Whereas G2 plants did not meaningfully diverge neither from G3 plants nor from G1 plants. G4 and G5 landraces had the lowest number of marketable fruits.

> SwE treatment significantly increased fruit mean mass compared with the control (Table 2). Regardless of the SwE application, the G1 genotype gave the highest fruit mean mass compared with the other genotypes.

> Young shoot yield in SwE treated plants was higher by 22.2% compared to untreated control (Table 2). Averaged over SwE application, the G4 genotype showed the highest young shoot yield, whereas the G3 landrace revealed the lowest one.

> SwE application boosted the number of young shoots (Table 2). G4 landrace gave the highest number of young shoots, followed by G1, G2 and G5. The genotype G3 gave the lowest value.

Statistic for NUEys underlined no significant interaction SwE × G (Figure 4).

Regardless of the genotype, SwE treated plants displayed the highest NUEys value (Figure 4). Averaged over SwE treatment, G4 and G5 landraces gave the highest NUEys value. However, the G5 landrace did not significantly differ neither from the G4 landrace nor from the G2 landrace. The lowest values were observed in the G3 landrace (Figure 4).

Statistic on fruit firmness revealed no significant interaction SwE × G (Figure 5).

Fruits from plants treated with SwE revealed a higher firmness than fruits from untreated plants. When averaged over SwE treatment, G2 and G3 landraces displayed the highest firmness, followed by G1 landrace. G4 and G5 landraces had the lowest values (Figure 5).

**Figure 4.** Effect of the seaweed extract treatments (SwE) and genotypes (G) on shoot nitrogen use efficiency of *L. siceraria*. Values with different letters indicate significant differences at *p* ≤ 0.05. NS, \*\*\* non−significant or significant at 0.001, respectively. Bars represent the standard error.

#### *3.2. Young Shoot Nutritional Properties, Mineral Profile and Functional Components*

Statistical analysis for SSC did not show a significant interaction between SwE and G (Figure 6).

Averaged over genotype, SwE treatment did not affect young shoot SSC (Figure 6). Contrariwise, when averaged over SwE application, G2, G4 and G5 landraces had the highest SSC, followed by G1 genotype which in turn had a higher SSC value than G3 landrace (Figure 6).

The mineral profile was mainly influenced by SwE application and genotype. However, ANOVA for N, P, K, Ca, and Mg concentrations highlighted no significant interaction SwE × G (Table 3).

**Figure 6.** Effect of the seaweed extract treatments (SwE) and genotypes (G) on young shoot soluble solid content of *L. siceraria*. Values with different letters indicate significant differences at *p* ≤ 0.05. NS, \*\*\* non−significant or significant at 0.001, respectively. Bars represent the standard error.

**Table 3.** Effect of the seaweed extract treatments (SwE) and genotypes (G) on nitrogen (N), phosphorous (P), potassium (K), calcium (Ca) and magnesium (Mg) concentrations of *Lagenaria siceraria* young shoots.


Values present in a column and followed by different letters are significantly dissimilar at *p* ≤ 0.05. NS, \*, \*\*\* non−significant or significant at 0.05 or 0.001, respectively.

> Averaged over genotype, the highest N concentration was observed in young shoots from untreated plants (Table 3). Disregarding the SwE treatment, genotype G5 revealed the highest N concentration, followed by G4 landrace which in turn displayed a higher N concentration than the G1 landrace. The lowest value was observed in young shoots from the G3 genotype.

> P, K, Ca, and Mg contents in plants exposed to 3 mL L−<sup>1</sup> of SwE was higher by 3.1%, 2.3%, 0.9% and 4.8%, respectively compared with untreated plants (Table 3). Averaged over SwE application, G4 and G5 landraces revealed the highest P concentration, followed by G1 landrace. However, young shoots from the G5 landrace did not show a significant difference in terms of P concentration. The lowest P concentration was recorded in young shoots from the G3 genotype. The highest K concentration was found in young shoots from G1, G4 and G5 landraces (19.80, 20.13 and 19.82 g kg−<sup>1</sup> dw, respectively), whereas, G2 and G3 genotypes had the lowest values. Data on Ca concentration followed a similar trend to that described for K concentration. Regardless of SwE application, G4 and G5 landraces had a higher Mg concentration compared with the other genotypes.

ANOVA for shoot ascorbic acid concentration showed a significant interaction between SwE and G (Figure 7).

**Figure 7.** Effect of the seaweed extract treatments (SwE) and genotypes (G) on the ascorbic acid concentration of *L. siceraria* young shoots. Values with different letters indicate significant differences at *p* ≤ 0.05. \*\*\* significant at 0.001. Bars represent the standard error.

Overall, SwE treatment enhanced ascorbic acid concentration in G1, G2, G3, G4 and G5 young shoots by 29.5%, 35.9%, 79.6%, 53.4% and 71.6%, respectively (Figure 7). The combinations 3 mL L−<sup>1</sup> SwE × G3 and 3 mL L−<sup>1</sup> SwE × G4 gave the highest ascorbic acid content. However, when G3 landraces were exposed to SwE did not significantly differ neither from G4 nor from G5 in terms of ascorbic acid concentration. The lowest ascorbic acid concentration was detected in young shoots from the 0 mL L−<sup>1</sup> SwE × G2 combination.

ANOVA for polyphenols concentration showed a significant interaction SwE × G (Figure 8).

**Figure 8.** Effect of the seaweed extract treatments (SwE) and genotypes (G) on polyphenols concentration of *L. siceraria* young shoots. Values with different letters indicate significant differences at *p* ≤ 0.05. \*\*\*, \*\* significant at 0.01 or 0.001, respectively. Bars represent the standard error.

As for ascorbic acid concentration in all tested genotypes, polyphenols were significantly enhanced by SwE treatment. The highest values were recorded in G3 and G4 landraces supplied with 3 mL L−<sup>1</sup> of SwE, followed by G2 and G5 landraces (Figure 7). The lowest values were observed in young shoots harvested from G1 and G4 untreated plants.

#### *3.3. Heat Map Analysis of the Whole Data Set*

A data heat-map analysis of all assessed features (agronomic, nutritional and functional) was performed to realise a graphical appraisal of the influences determined by the experimental factors on *L. siceraria* (Figure 9).

**Figure 9.** Heat map assessment including all *L. siceraria* plant traits in response to seaweed extract treatments (0 or 3 mL <sup>L</sup>−1) and genotypes (G1, G2, G3, G4 or G5). The heat map picture was produced via https://biit.cs.ut.ee/clustvis/ (accessed on 14 September 2021).

The heat-map output consisted of two dendrograms, Dendrogram 1 sited on the top containing all the combinations of SwE doses and genotypes and Dendrogram 2, located on the left side, comprising all traits that influenced this distribution. Dendrogram 1 presented two main clusters, the first on the left included the G4 and G5 landraces both treated and untreated with SwE. The other site on the right side contained G1, G2 and G3 landraces both treated and untreated with SwE. Expressly, in the left cluster of Dendrogram 1, the G4 and G5 landraces treated with SwE were parted from the other G4 and G5 untreated controls due to the higher P, K, Mg, plant height (at 10, 20 and 30 DAT), number of leaves (at 10, 20 and 30 DAT), first female flower emission, NUEys, young shoot yield, number of young shoots, fruit mean mass, ascorbic acid and polyphenols. The group on the left included G4 and G5 landraces treated with SwE. Within this group, the G4 × 3 combination was clearly separated by lower total yield, marketable yield, number of marketable fruits, plant height at 10 and 20 DAT, number of leaves at 20 and 30 DAT and N. While, the group on the right side included the G4 and G5 landraces untreated plants. Inside this cluster, the G4 × 0 combination was separated by higher P, K, Mg and ascorbic acid and lower total yield, marketable yield, fruit firmness, N, fruit mean mass and polyphenols.

On the right of the Dendrogram 1, two main groups were documented, the one on the left included the combinations G1 × 0 and G1 × 3, separated from the G2 and G3 landraces treated with 0 or 3 mL L−<sup>1</sup> of SwE, that had, in particular, lower fruit total yield, fruit marketable yield, Ca, P, K, N, Mg, plant height at 20 DAT and fruit mean mass, but the higher number of marketable fruits, firmness and polyphenols.

The group on the left side comprised G1 untreated control and G1 SwE treated plants; the G1 × 0 combination was separated from G1 × 3 by lower fruit marketable yield, fruit firmness, P, K, Mg, plant height (at 10, 20 and 30 DAT), number of leaves (at 10, 20 and 30 DAT), first female flower emission, NUEys, young shoot yield, number of young shoots, fruit mean mass, ascorbic acid and polyphenols.

The cluster on the right comprised G2 and G3 untreated and SwE treated combinations. In this group, the G2 × 3 combination was evidently parted from G2 × 0, G3 × 0 and G3 × 3 combinations by higher Ca, P, K, plant height (at 10, 20 and 30 DAT), number of leaves (at 10, 20 and 30 DAT), first female flower emission, NUEys, young shoot yield and number of young shoots. The right side of this cluster included G2 × 0, G3 × 0 and G3 × 3 treatments; the G2 landrace untreated control was parted by higher Ca, P, K, Mg, plant height (at 10,20 and 30 DAT), number of leaves (at 10 and 30 DAT), first female flower emission, NUEys, young shoot yield, number of young shoots, N and SSC. The right part of this group comprised G3 landraces SwE treated and untreated control. G3 × 0 combination was separated from G3 × 3 combination by lower fruit marketable yield, fruit firmness, Ca, P, K, Mg, plant height (at 10, 20 and 30 DAT), number of leaves (at 10, 20 and 30 DAT), first female flower emission, NUEys, young shoot yield, number of young shoots, fruit mean mass, ascorbic acid and polyphenols.
