Stand-Alone or Combinatorial Effects of Grafting and Microbial and Non-Microbial Derived Compounds on Vigour, Yield and Nutritive and Functional Quality of Greenhouse Eggplant

The current research investigated the effects of endophytic fungi such as Trichoderma atroviride (Ta) or Ascophyllum nodosum seaweed extract (An) and their combination on growth, yield, nutritive and functional features, and mineral profile of ‘Birgah’ F1 eggplant either ungrafted, self-grafted or grafted onto the Solanum torvum rootstock. Eggplant exposed to An or An+Ta had a significant increase in root collar diameter 50 days after transplanting (RCD50), total yield (TY), marketable yield (MY), ascorbic acid (AA) content, Mg, Cu, and Zn concentration, and a reduction in glycoalkaloids (GLY) compared with the control. Furthermore, grafted plants had a higher TY, MY, number of marketable fruits (NMF), RCD50, AA, Cu, and Zn and a lower SSC, GLY, and Mg than the ungrafted plants. The combination of grafting and An+Ta significantly improved mean weight of marketable fruits (MF), plant height 50 days after transplanting (PH50), number of leaves 50 days after transplanting (NL50), fruit dry matter (FDM), chlorogenic acid (ClA), proteins, and K and Fe concentration. This combination also produced fruits of high premium quality as evidenced by the higher AA and ClA concentration, the lower GLY concentration, and an overall improved mineral profile.


Introduction
The fast growth of the world population will cause a boost in the world-wide food requirement, estimated to double in 2050 [1]. Enhancing the agricultural production seems to be the only approach to face the rapid increase in food need [1]. This strategy demands the use of high-input resources such as protected cultivation systems, which, however, can negatively affect the agroecosystem [2]. Indeed, greenhouse techniques use a high quantity of synthetic fertilizers, which can cause damage to both the environment and human health [3,4]. The leafy and fruiting vegetable production sector can adopt specific agronomic practices to minimize its impact on the ecosystem [5][6][7][8]. In this scenario, a promising strategy, which could encourage a change from a fertilizers-intensive agriculture to an eco-friendlier system, is the integrated use of biostimulants [9][10][11][12][13][14][15][16]. Biostimulants are products that can stimulate plant growth and development due to several bioactive

Plant Performance and Production
Data on plant growth parameters are presented in Figure 1. ANOVA for PH 50 revealed a significant influence of the interaction plant type (T) × biostimulant (B) ( Figure 1A). Plants grafted onto S. torvum rootstock and supplied with both biostimulants (An+Ta) had the highest height, followed by those self-grafted and treated with both An and Ta. The lowest PH 50 values were recorded in ungrafted non-biostimulated plants.
ANOVA for NL 50 exhibited a significant effect of the T × B interaction ( Figure 1B). 'Birgah' F 1 grafted eggplants treated with An or An+Ta had the highest NL 50 , followed by those grafted and supplied with Ta. The lowest NL 50 was found in untreated plants (control) and in ungrafted plants treated with Ta. ANOVA for RCD 50 did not display a significant effect of the T × B interaction ( Figure 1C). Regardless of the biostimulant application, grafted plants had the highest RCD 50 value, whereas ungrafted plants had the lowest. Irrespective of T, plants treated with An+Ta showed the highest RCD 50 values, followed by those exposed to An. The lowest values were detected in control plants and in those exposed to Ta.
Yield features are shown in Figure 2.  No significant T × B interaction was found for TY ( Figure 2A). Grafted plants revealed the highest TY, followed by self-grafted plants, whereas ungrafted plants had the lowest TY. Irrespective of the type of plant, plants treated with An+Ta revealed the highest TY, followed by those supplied with An. Untreated plants and those treated with Ta had the lowest TY.
ANOVA for MY did not show a significant effect of the T × B interaction ( Figure 2B). Regardless of the biostimulant supply, data on MY followed the trend established for TY. Disregarding the type of plant, those treated with An or An+Ta showed the highest MY, followed by the non-treated (control) or supplied with Ta.
For NF, ANOVA did not reveal a significant effect of the T × B interaction ( Figure 2C). Notwithstanding the biostimulant application, data on NF followed the same tendency recorded for TY and MY. Plants subjected to An application had the highest NF, whereas those exposed to Ta revealed the lowest values.
ANOVA for MF revealed a significant effect of the interaction between T and B ( Figure 2D). Plants grafted onto S. torvum and treated with An or An+Ta showed the highest MF, followed by those ungrafted or self-grafted and supplied with An+Ta. The lowest MF was recorded in the combinations ungrafted × control, ungrafted × Ta, selfgrafted × control, and self-grafted × Ta eggplant.
All plant types (ungrafted, self-grafted, or grafted), non-biostimulated or inoculated with Ta, showed the highest firmness. Supplying grafted eggplants with An+Ta resulted in reduced fruit firmness compared to the other treatments.
Grafted plants supplied with An or An+Ta had the highest ClA, protein, and Fe concentrations, followed by the combinations of grafted × control and grafted × Ta. The lowest ClA, protein, and Fe concentrations were detected in fruits from ungrafted × control, ungrafted × Ta, self-grafted × control, and self-grafted × Ta eggplant combinations.
Self-grafted plants treated with An or An+Ta showed the highest TA concentration, followed by the combinations of ungrafted × An, ungrafted × An+Ta, self-grafted × control, and self-grafted × Ta eggplants. The lowest TA concentration was detected in fruits from untreated plants or Ta inoculated grafted plants.
Grafted plants treated with both biostimulants showed the highest fruit K concentration, followed by the grafted and inoculated with Ta eggplants. The lowest fruit K concentration was recorded in fruits from the combination of ungrafted × control eggplants.
ANOVA for SSC, AA, GLY, P, Ca, Mg, Cu, and Zn did not display a significant effect of the T× B interaction ( Table 2).
Regardless of the biostimulant supply, ungrafted plants showed the highest fruit SSC, whereas grafted plants exhibited the lowest values. Conversely, independent of the type of plant, biostimulants did not affect fruit SSC.
Grafted eggplants showed the highest fruit AA, Cu, and Zn concentrations, whereas fruits from self-grafted and ungrafted plants had the lowest ones. Irrespective of the type of plant, fruits from plots treated with An and An+Ta had the highest AA, Cu, and Zn concentrations. The lowest fruit AA, Cu, and Zn concentrations were recorded in the control plants and those supplied with Ta.
Ungrafted and self-grafted plants showed the highest fruit GLY concentration, whereas grafted plants had the lowest one. Without regard to the type of plant, both control and Ta inoculated plants displayed the highest fruit GLY concentration. The lowest GLY values were recorded in fruits from plots treated with An or An+Ta.
ANOVA showed that both main factors did not significantly affect fruit P and Ca content.
Regardless of the biostimulant, the fruit from ungrafted and self-grafted plants had the highest Mg concentration. Irrespective of the type of plant, fruits from plots treated with An+Ta exhibited the highest Mg content, followed by those from plots sprayed with An, which in turn had a higher Mg concentration than those from plots inoculated with Ta. The lowest fruit Mg concentration was detected in the control eggplants.

Heat Map Analysis of All Eggplant Traits
A heat-map analysis of all tested parameters, comprising growth, yield, and fruit quality traits, was performed to produce a graphical evaluation of the respective experimental factors effect on 'Birgah' F 1 eggplant ( Figure 3). Heat-map analysis revealed two dendrograms. Dendrogram 1 was placed on the top, encompassing the groupings of type of plant and biostimulant, and Dendrogram 2, sited on the left, involving the studied parameters that affected this distribution. Dendrogram 1 showed two main groups; the group on the left side involves all combinations with grafted plants (grafted × control, grafted ×Ta, grafted × An, and grafted × An+Ta). The other, located on the right, includes all combinations with ungrafted and self-grafted plants. Explicitly, in the left cluster of Dendrogram 1, the grafted × control and grafted × Ta combinations were separated from the combinations grafted × An and grafted × An+Ta due to the lower P, Ca, PH 50 , RCD 50 , MF, AA, Cu, proteins, Zn, ClA, Fe, K, NL 50 , TA, and Mg. The cluster on the left comprised the grafted × control and grafted × Ta combinations. Inside this cluster, the grafted × control combination was evidently parted by lower PH 50 , FDM, K, NL 50 , SSC, and Mg. The cluster on the right enclosed the grafted × An and grafted × An+Ta combinations. Within this group, the grafted × An+Ta combination was separated by a higher PH 50 , RCD 50 , MF, AA, FDM, K, NL 50 , and Mg.
On the right side of Dendrogram 1, two chief clusters were documented. The cluster on the left incorporated the combinations self-grafted × An and self-grafted × An+Ta, divided from the self-grafted control plants or self-grafted plants treated with Ta and all combinations involving the ungrafted plants, that had, particularly, a higher P, NF, PH 50 , RCD 50 , MF, K, NL 50 , TY, and MY, but a lower firmness, SSC, GLY, TA, and Mg. The cluster on the left included the self-grafted × An and self-grafted × An+Ta combinations; the self-grafted × An combination was separated from the self-grafted × An+Ta combination by lower P, Ca, PH 50 , RCD 50 , MF, TA, and Mg. The cluster on the right comprised selfgrafted untreated and Ta inoculated plants and all the combinations including ungrafted plants. In this group, the self-grafted × control and self-grafted × Ta combinations were clearly separated from all the ungrafted combinations by the lower P, PH 50 , SSC, and Mg. The left side of this group included the self-grafted × control and self-grafted × Ta combinations; the self-grafted × control combination was separated due to the lower Ca, PH 50 , MF, AA, Zn, and K. The right side of the cluster included all the ungrafted eggplant combinations. The ungrafted × An and ungrafted × An+Ta combinations were divided by higher P, PH 50 , RCD 50 , MF, AA, Cu, proteins, Zn, ClA, K, NL 50 , TY, MY, and Mg. Inside this cluster, ungrafted An treated plants were parted by lower PH 50 , RCD 50 , MF, FDM, K, TY, and Mg. The right side of this group enclosed the ungrafted × control and ungrafted ×Ta combinations; particularly, the ungrafted × control combination is divided by lower PH 50 , MF, K, and Mg.

Discussion
Currently, Trichoderma is recognized as a valuable plant biostimulant due to its direct and/or secondary benefits related to the Trichoderma-plant molecular crosstalk and interchange of various elements and small peptides. These mechanisms elicit numerous plant responses, mineral absorption improvement, and tolerance to mineral distress in vegetables [36,37]. At the same time, seaweed extract is an additional conspicuous category of biostimulants that has displayed positive effects on overall plant performance, mineral uptake, and tolerance to different abiotic stresses affecting vegetable crops [9,10,26]. In addition to biostimulants, herbaceous grafting is a profitable tool for safeguarding and augmenting production, nutritional, and functional features of fruiting vegetables under optimal, sub-optimal, or unfavourable cultivation conditions [7,28,[38][39][40][41]. Thus, in the current research, grafting and biostimulant effects-alone or combined-were appraised on plant growth and productivity, as well as fruit nutritional and nutraceutical components of 'Birgah' F 1 eggplant. Results showed that grafting combined with An or An+Ta treatments significantly increased plant growth and yield traits. Similarly, Ascophyllum nodosum extract application increased spinach productivity [42] and sweet pepper growth [43]. Moreover, a significant increase of growth parameters of eggplant supplied with boric acid and seaweed extract was recorded by El-Gawad and Osman [44]. The increase of growth and yield of vegetable crops seems to be related to the cytokinins and auxin precursors included in A. nodosum extract, which stimulate cell division of plant tissues [42,44]. Moreover, there are reports that the higher plant yield of An-treated plants could be associated with the polysaccharides (alginates, fucoidan, and laminarins) in the A. nodosum seaweed extracts, which in turn trigger endogenous hormones homeostasis [45,46]. Blunden and Gordon [47] found that A. nodosum extracts contain betaines and betaine-like compounds. These components could lead to improved leaf chlorophyll and photosynthetic rates, resulting in plant productivity increase.
Thricoderma inoculation was previously demonstrated to have positive influences on tomato yield and growth [48]. Our findings are supported by Rouphael et al. [49], who found a yield increase of inoculated lettuce plants treated with Thricoderma virens and a biopolymer-based biostimulant. These results could be linked to the release of molecules with hormone-like activity, which upsurge plant nutrient bioavailability, uptake, translocation, and accumulation [22,50]. Moreover, it has been confirmed that Trichoderma inoculation stimulates root growth and modifies its structure, which in turn enhances nutrient uptake [23,51]. In this study, grafting onto S. torvum significantly increased eggplant productivity, thereby confirming the results of Consentino et al. [41]. The positive influence of grafting on plant growth and production was also described in tomato [40]. These results were associated to a boosted mineral and water uptake prompted by grafting [52,53]. In our study, we may assume that the growth and yield increase was related to the synergetic effect of grafting and biostimulants.
Findings revealed that FDM was highest in grafted plants treated with Ta or An+Ta, whereas, in ungrafted plants An did not significantly improve FDM. Thus, our data partly agree with those appraised by Rouphael at al. [42], who observed that A. nodosumbased extract did not significantly affect dry matter in spinach plants, although an increasing trend was recorded. Conversely, our findings highlighted a positive impact of Trichoderma inoculation, alone or combined with An. These results are corroborated by those of Rouphael et al. [49], who showed an upsurge of plant dry biomass in Trichoderma-treated plants. Thus, we may hypothesize that the FDM increase of grafted Ta and An+Ta treated plants was due to their enhanced plant nutritional status.
Seaweed extract alone or combined with Trichoderma reduced fruit firmness. These results overlap with those of Ali et al. [54], who found that A. nodosum extract decreases skin and pulp firmness in tomato. Concomitantly, in our study grafting improved fruit firmness. This outcome agrees with that of Sabatino et al. [30] who, by studying the combined effect of grafting and arbuscular mycorrhiza on eggplant performance and fruit quality, found that fruits from untreated controls have a lower firmness than those from S. torvum grafted plants. Therefore, combining grafting with the application of both biostimulants resulted in a relevant synergistic effect.
Grafted eggplants treated with An or An+Ta had the highest fruit ClA and AA concentrations. A considerable increase in total phenols and ascorbic acid in lettuce plants treated with A. nodosum extract, as observed in this study in eggplant, was reported by Rouphael et al. [42]. Ertani et al. [55] and Rouphael et al. [42] report that the enhanced biosynthesis of these metabolites could be connected both to the direct and/or indirect influences of the key enzyme activity included in phytochemical homeostasis and to the modification of plant nutritional status. Comparable results were accomplished by Fan et al. [56] who, by assessing the influence of A. nodosum extract application on spinach phenolic antioxidant content, detected an increase in flavonoids compared to the nonbiostimulated plants. Furthermore, as reported by Fan et al. [57], the enhancement in biosynthesis of bioactive molecules could be intermediated via the stimulation of chalcone isomerase, which is a key enzyme in the flavanone precursor biosynthesis. Our data highlighted that combining Ta and An trigger a high production of AA and ClA and-as speculated by Rouphael et al. [42]-glutathione, which is a metabolite that acts synergistically with AA to make an antioxidant plant response, protecting the plants from the reactive oxygen species (ROS) [58,59].
Results indicated that grafting significantly increased fruit ClA concentration. This is in contrast with the finding of Darré et al. [60] who, by studying the effects of a coldtolerant rootstock on eggplant fruit susceptibility, found a reduction of ClA in grafted plants. However, our data concur with those reported by Sabatino et al. [61], who noticed that grafting can increase or reduce total phenolics in relation to the scion-rootstock combination. This is also confirmed by other reports [62,63]. Data showed that fruits from grafted plants had a higher AA content than those from ungrafted or self-grafted plants. These findings are in line with those of Sabatino et al. [30], who observed an increase of AA in fruit from plants grafted onto S. torvum compared with those from ungrafted or self-grafted plants. In this respect, Oztekin et al. [64] pointed out that grafting does not significantly influence AA concentration in tomato fruits. These divergent outcomes could be ascribed to the diverse rootstock adopted [65].
The outcomes of this research revealed that self-grafted plants treated with An or An+Ta had the highest fruit TA concentration. These results are coherent with those of Roussos et al. [66] who, by investigating the influence of A. nodosum application on strawberry fruit quality, observed that plants treated with the biostimulants had a higher TA content than the untreated ones. Similarly, by studying the effects of genetic factors and A. nodosum application on eggplant production and quality, Pohl et al. [67] found that biostimulant supply significantly enhances fruit total anthocyanin concentration.
The use of S. torvum as rootstock resulted in reduced TA concentration, similar to the results of Consentino et al. [41] and Sabatino et al. [30]. As it is well known that anthocyanins biosynthesis and accumulation is strongly related to light exposure [68,69] and appraising that grafting technique boosted plant vigour parameters, we can assume that the TA reduction in grafted plants was the result of more shaded fruits.
Overall, our results showed that grafting combined with An or An+Ta significantly increased proteins and mineral concentration in eggplant fruits with the exception of P and Ca. According to Ali et al. [54], Rouphael et al. [42] and Ertani et al. [46], A. nodosum extract enhanced mineral concentration in tomato fruits, spinach, and maize, respectively.
The recorded upsurge of mineral concentration in fruits from plants treated with seaweed extract was also reported by several other authors [70][71][72]. This effect seems to be due to an up-regulation of genes involved in mineral transporters and/or in the stimulation of root development. Furthermore, grafting significantly augmented fruit mineral concentration, except for Mg. These findings are corroborated by those of Sabatino et al. [30], who reported a higher mineral concentration in fruits from grafted plants compared to those from selfgrafted or ungrafted ones. According to Oztekin et al. [64], the higher mineral concentration observed in fruits from grafted plants could be ascribed to an improved mineral and water absorption of grafted plants. Interestingly, our data also pointed out that the combined application of grafting and An+Ta positively influenced mineral profile and protein content in eggplant fruits, suggesting that both experimental factors had a synergetic action.
In our study, SSC was not affected by biostimulant treatments, whereas it was reduced by grafting. Consentino et al. [41] and Sabatino et al. [30] also found an SSC reduction in fruits from plants grafted onto S. torvum compared to those from ungrafted or selfgrafted plants. A similar pattern of results was obtained by Riga [73] on tomato. Results showed that both An and An+Ta significantly reduced GLY fruit concentration. Similarly, Mystkowska [74] found a lower glycoalkaloids concentration in biostimulated potato plants when compared to the untreated control. The use of S. torvum as rootstock, as well, resulted in reduced glycoalkaloids concentration. As Papathanasiou et al. [75] reported that unfavourable growing conditions and cultivation techniques enhance glycoalkaloids concentration in potato, we might assume that grafted plants supplied with An or An+Ta were less stressed than others due to a lower content of GLY. These considerations are entirely coherent with the results of Sabatino et al. [30], who stated that grafting combined with AMF significantly reduced GLY concentration in eggplant fruits.

Experimental Site, Plant Material, and Cultivation Conditions
The trial was accomplished in 2019 at Marsala, Sicily (37 • 44 54.7 N 12 • 33 05.2 E) during the winter-spring period. Three types of 'Birgah' F 1 eggplant were tested: ungrafted, self-grafted, or grafted onto S. torvum rootstock. The grafting technique was performed as reported by Sabatino et al. [28] and Sabatino et al. [39] using a tube grafting method. On 12 February, at the end of the healing phase, plants were transplanted (2 plants m −2 ) in a polyethylene-covered tunnel. The soil was characterized by sand (<80%) at 8.5 pH and limestone (8.8%), 65 mg kg −1 of exchangeable K 2 O, 70 mg kg −1 of phosphorous, 2% of total nitrogen, and 10 t ha −1 of organic matter. The soil was mulched with black polyethylene film (20 µm) under which a drip irrigation system was installed. During the whole cultivation cycle, all plant needs were fulfilled based on the recommendations of Tesi [35].
For the non-microbial biostimulant, every 10 days, starting from 14 th DAT to the end of the harvest, the seaweed extract was supplied via foliar spray at a dosage of 0 and 3 (optimal dose) mL L −1 , supplying 100 mL of solution per plant.

Plant Growth, Yield, and Yield Components
Growth and yield parameters were measured on all plants. Plant height (PH 50 ), number of leaves per plant (NL 50 ), and root collar diameter (RCD 50 ) were recorded 50 DAT. After each harvest, total yield (TY) was measured, and the production was divided into marketable (MY) and non-marketable; total yield and marketable yield were expressed as kg plant −1 . The number of marketable fruits (NF) was also recorded, and mean mass of marketable fruits was calculated (MF) and presented as g fruit −1 .

Fruit Quality Traits
All quality parameters were determined on five fruits per replicate. All fruits were harvested at the same maturity stage (40 days after flower labelling). Fruit dry matter (FDM) was assessed by dehydrating fruits at 105 • C until they reached a constant weight. The values were expressed as percentages. Fruit firmness was evaluated through a penetrometer (model 53205, TR Snc., Forlì, Italy) by stinging the fruit in the middle part; its value was presented as newtons (N). Soluble solids content (SSC) was recorded on fruit juice via a refractometer and the values were reported as • Brix. The ascorbic acid (AA) concentration was measured as reported by Sabatino et al. [76]; values were expressed as mg 100 g −1 of fresh weight (fw). Chlorogenic acid (ClA) concentration was determined following the procedure of Stommel and Whitaker [77], with slight modification. The final quantification was accomplished by HPLC (Sigma-Aldrich, St. Louis, MO, USA) with an absorbance of 325 nm. Data were reported as mg 100 g −1 of dry weight (dw). Total anthocyanins content (TA) was measured using the method of Mennella et al. [78]. Briefly, the measurement was accomplished on 200 mg of lyophilized and powdered samples; then, for HPLC analysis, the flow rate was 0.8 mL min −1 and the absorbance was 0.1. The values were expressed as mg 100 g −1 dw. For fruit glycoalkaloids (GLY) determination, the extraction procedure of Birner et al. [79] was used. HPLC analysis was used, adopting solasonine and solamargine as standard. The measurement values were reported as mg 100 g −1 of dw.

Proteins and Mineral Profile in Fruit Tissue
All assessments on mineral profiles were performed on five fruits, casually chosen from each replicate. Nitrogen concentration in fruits was appraised via the Kjeldahl method, then the value was multiplied by 6.25 to obtain the protein content. Phosphorous (P) concentration was measured using the method of Fogg and Wilkinson [80]. Potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), copper (Cu), and iron (Fe) concentrations were determined through atomic absorption spectroscopy as described by Sabatino et al. [8] and Subramanian et al. [81]. Macro element concentrations (P, K, Ca, and Mg) were expressed as mg 100 g −1 dw, whereas microelements (Zn, Cu, and Fe) were reported as µg g −1 dw.

Experimental Design and Statistics
The 12 treatments were tested in a factorial experiment [three types of plant (ungrafted, self-grafted, or grafted) and four biostimulant treatments (control, An, Ta, or An+Ta)] using a completely randomized block design. Treatments were replicated three times and included 10 plants per replication, rendering 360 plants. The data were analysed using the SPSS v.20 software package via a two-way analysis of variance (ANOVA), setting type of plant and biostimulant as main factors. Means separation was accomplished using the Tuckey HSD test at p ≤ 0.05. Furthermore, a heat map displaying all the agronomic and qualitative traits of eggplant was realized using the online program package clustvis (https://biit.cs.ut.ee/clustvis/, accessed on 1 March 2022).

Conclusions
The continuous necessity of the horticultural sector to maximize yield and the concomitantly increasing consumer demand of premium quality foods pose a crucial challenge for researchers in their search for eco-friendly tools to enhance quantitative and qualitative crop traits. Biostimulants represent a key method in this exertion. In the current study, A. nodosum application elicited vigour traits, yield, and fruit quality of 'Birgah' F 1 eggplant; however, the combined applications of Trichoderma and A. nodosum-seaweed extract further enhanced plant performance in terms of vigour features, productivity, nutritional and functional traits, and mineral profile. Moreover, the combination of both plant biostimulants and grafting onto S. torvum rootstock represents an encouraging, proficient, and environmentally friendly agronomic approach for increasing production and quality of eggplant grown in a protected environment.