1. Introduction
Tomato (
Solanum lycopersicum Mill.) (Solanaceae) is one of the most widely cultivated and consumed vegetables globally. Because of its popularity, nutritional and antioxidant properties, tomatoes are one of the most profitable horticultural crops [
1]. In 2023, the market size reached USD 174.7 billion and is expected to grow to USD 186.46 billion in 2024, with a compound annual growth rate (CAGR) of 6.7% (Tomatoes Global Market Report 2024). However, its production faces increasing challenges such as rising input costs, substantial crop losses from pests and diseases, and growing demands for sustainable agricultural practices [
2]. Furthermore, climate change exacerbates these issues, with projections indicating a potential 6% reduction in tomato yields by 2050 in key regions like Italy and California due to rising temperatures [
3].
In this context, open-field cultivation, particularly for processing tomatoes, is increasingly vulnerable to environmental stresses compared to greenhouse-grown salad tomatoes. This vulnerability underscores the need for effective strategies to enhance crop resilience and productivity. Additionally, transplantation is a critical stage in the growth period, as it causes root damage, promotes the formation of a fibrous root system, and requires the seedlings to adapt to new environmental conditions [
4]. The stressful shock of transplanting may slow down tomatoes’ growth and development, and sometimes lead to seedling death. So, the development of a functional and strong root system will allow for the efficient absorption of water and nutrients, together with faster growth, in transplants.
Biostimulants have emerged as a promising solution to mitigate these challenges. A wide variety of substances of natural origin have been used as biostimulants since they have a beneficial effect on plants [
5,
6,
7]. These substances, derived from natural sources, can improve plant growth, stress tolerance, and overall quality. A plant biostimulant, as defined by Du Jardin [
8], is “any substance or microorganism that applied to plants, regardless of its nutrients content, is able to enhance nutrition efficiency and also abiotic stress tolerance and quality traits” [
8]. The current knowledge suggests that biostimulants potentially function in regulating and modifying physiological processes in plants to promote growth, alleviate stress, and improve plant yield and fruit/flower quality [
8,
9,
10].
Given the growing need for sustainable solutions in tomato cultivation, our study focuses on evaluating Nurspray®, a biostimulant based on hydroxycinnamic acid oligomers (HAOs). It was selected for this study due to its unique properties and potential benefits in tomato cultivation.
Unlike many commercial biostimulants that consist of complex mixtures, Nurspray
® contains a single well-defined molecule. Nurspray
® is a biostimulant based on hydroxycinnamic acid oligomers (HAOs). HAOs are naturally present in plant cell walls and play a vital role in cell wall strength and extensibility [
11]. Despite these advantages, the use of HAOs in tomato cultivation is unknown. Therefore, our present work is a first report of the use of this compound as a biostimulant in open-field tomato cultivation. Open-field trials are particularly important because they offer realistic assessments of biostimulant effectiveness across diverse environmental conditions, which are essential for making practical recommendations for agricultural practices [
8]. However, relatively few articles have explored the use of a biostimulant in open-field tomato cultivation [
12,
13,
14,
15].
This study evaluates the effectiveness of Nurspray® in enhancing tomato plant development and yield under open-field conditions. We focused on optimizing application strategies to maximize its impact at different growth stages of tomato plants. Our approach aims to provide practical recommendations for integrating the biostimulant into both conventional and organic farming practices, contributing to a more sustainable approach to improving tomato cultivation.
2. Material and Methods
2.1. Plant Growth, Experimental Design, and Treatments
The field experiment was conducted at Sele Agroresearch S.r.l., located in Napoli, Italy, on tomato plants (
Solanum lycopersicum cv. Taylor F1) provided by Nunhems (BASF), during the growing season of 2020. The geographic coordinates for the trial location are latitude Nord 40°98′06.39″ N, longitude East 14°06′48.69″ E, and altitude 30 m.a.s.l. During the whole growing period, climatic data were registered (
Table 1).
The tomato seedlings were purchased as transplants from a nursery. The seedlings were first grown in germination trays and transferred to individual pots when they had 2–4 true leaves. Light irrigation was applied two days prior to transplanting to facilitate the process. The seedlings were mechanically transplanted into an open field on 24 April 2020, at a rate of 32,000 plants per hectare. Four types of treatment, including the untreated control, which did not receive any biostimulant, were evaluated in a randomized block with four repetitions. There were three treatments where the biostimulant was applied at different moments using the phenological growth scale [
16] (
Table 2). The commercial biostimulant Nurspray
®, from the Fyteko Company (Brussels—Belgium; [
17]), was used. This is a liquid formulation containing 0.015% (
w/
v) of HAO grafted on a natural modified polysaccharide (Belgian derogation EM622.B). Their effectiveness starts from 24 h after spray application, and they are compatible with microorganisms and other biostimulants, biodegradable, and free of harsh chemicals. The Nurspray
® solutions were applied mechanically via foliar spray at a dosage rate of 1.0 L/ha, or tap water was applied for the control treatment.
The application timings were strategically selected based on the BBCH growth stages to target critical phases of vegetative and reproductive development. These timings were designed to assess the effectiveness of the use of Nurspray® during the vegetative growth, lateral shoot development, and reproductive stages, with the aim of optimizing plant growth, flowering, and fruit setting.
Each experimental plot had an area of 39 m2. A double row system was used with a spacing of 2.4 m between each row and 0.25 m between plants in each row. Two meters from the top were excluded in the assessment zones and two meters at the end of the plots. Soil had a silty loam texture (156, 543, 301 g/kg−1 clay, silt, and sand, respectively), a pH of 7.6 (in H2O), 17.9 g kg−1 organic matter, and 0.5 dS m−1 at 25 °C cation exchange capacity. Prior to the trial, the soil nutrient analysis revealed a nitrogen (N) content of 14.0 g/kg, phosphorus (P) content of 12.7 g/kg, and potassium (K) content of 17.9 g/kg, reflecting moderate fertility levels.
Before transplanting, the nitrogen, phosphorus, and potassium (NPK) fertilizer (Agribios Italiana s.r.l., Padua, Italy), at a 14-11-22 ratio, was placed 15 cm from the plants via a slight milling process. The fertilizer was embedded at a depth of five centimetres. Weeds were controlled by using herbicide (Metribuzin), fungus was controlled by using fungicides (Metalaxil M, Dimetomorf, Oxathiapiprolin, Amisulbrom), and plagues were controlled by using insecticides (Clorantraniliprole, Acetamiprid, Bacillus thuringiensis) (Merck KGaA, Darmstadt, Germany). These applications were carried out with careful adherence to the manufacturer’s recommendations to ensure efficacy and safety for the plants.
Two days before the transplant, a small amount of irrigation was carried out to wet the soil for easy transplantation. At the point of application, soil and crops were in good condition.
Two weeks after the transplanting procedure, a root system assessment was conducted across all plots. To assess the condition and integrity of the root systems, plants were excavated at regular intervals following each treatment application. This process involved carefully removing the soil around the root zone to enable a visual inspection of both the roots and leaves, allowing for the detection of any signs of phytotoxicity. Observations were systematically recorded to monitor the effects of the treatments on root development. Additionally, plants were excavated on different days to further assess any phytotoxicity effects, ensuring comprehensive monitoring throughout the trial.
In the field where the test was performed, no pre-transplantation was carried out and no biostimulant was used. The irrigation timing was maintained over the entire field. During the complete trial period, different fertilizations were carried out using simple mineral fertilizers to ensure not only consistent nitrogen (N) levels across all treatments but also uniform applications of phosphorus (P) and potassium (K). This balanced fertilization strategy was aimed at minimizing nutrient variability and isolating the effects of Nurspray® on plant growth and performance. Also, throughout the test period, the tomato plants were treated with the classic fungicides.
2.2. The Studied Traits
An assessment of the phytotoxicity of Nurspray
®, including both efficacy and selectivity trials, was carried out for the crop during the entire trial period [
18]. For all the treatments, including the untreated control, the measurements of root systems and the percentage of canopy and flowering were made 23 and 30 days after application (DA-A). A rating scale of 1–9 was used for the measurement of root systems (
Supplementary Figure S1). The relative leaf chlorophyll content and the Normalized Difference Vegetation Index (NDVI) were measured after 29, 43, and 57 DA-A. The measurement of the relative leaf chlorophyll content was carried out using a Soil Plant Analysis Development (SPAD) 502 Plus (Minolta, Warrington, UK) portable chlorophyll meter and was given as SPAD units (ranging from 0 to 199.9) [
19]. NDVI index was measured using a FieldScout CM 1000 NDVI Meter (Spectrum Technologies, Inc., Aurora, IL, USA) [
20].
On 29 July 2020, the assessments of tomato production were carried out, and the following day, the harvest began. The tomatoes were harvested from individual plants, and the collected fruit was weighed and divided into ‘marketable’ and ‘unmarketable’ categories. Fruits were classified as unmarketable if they exhibited symptoms of Blossom-End Rot (BER), while fruits without BER were considered marketable. The assessments were composed of quantitative parameters, such as the number of fruits per plant, the weight of the fruit (g), the kg total of fruit per plant, divided into marketable fruit and unmarketable fruit with Blossom-end rot (BER), and the estimation yield (tons/ha), and qualitative parameters such as lycopene content (mg/kg) [
21], dry matter, pH, acidity, and firmness parameter [
22].
2.3. Statistical Analyses
All data management and statistical analyses used the Agriculture Research Management (ARM) software, version 2019 (GDM Solutions, Inc., Brookings, SD, USA). The effects of the biostimulant were significantly different if p-value < 0.05.
3. Results
The meteorological conditions recorded during the growing season showed a gradual increase in minimum temperatures, from 14.5 °C in April to 19.9 °C in July, while maximum temperatures peaked at 31.3 °C in July. Total rainfall varied, with the highest rainfall being recorded in July (105.0 mm) and the lowest in April (0.0 mm). Relative humidity ranged between 71.9% and 76.4% throughout the season. The recorded conditions, particularly the rising temperatures peaking at 31.3 °C in July, likely imposed heat stress on the tomato plants, potentially affecting fruit set and overall yield [
23]. While the relatively high humidity levels (71.9% to 76.4%) may have mitigated some of the transpiration stress, they also posed a risk for increased disease pressure, particularly fungal infections. The variability in rainfall, with no rain in April and a peak in July, could have further stressed the plants by creating fluctuations in water availability.
No phytotoxicity symptoms were observed on the crop during the entire trial period on any of the treated plots, either on the root system, flowers, or fruits, suggesting that all the tested treatments were completely selective for the crop.
The effects of Nurspray
® on the root system development, canopy, and flowering of tomato plant are shown in
Table 3. The development of the root system was increased by approximately 62.5% and 103% in plots T3 and T4 from the first assessment at 23 DA-A, respectively, compared with the control. After a few weeks, this difference disappeared. This advance in root growth was also observed for the flowering and canopy; both were anticipated in T3 and T4. After 30 DA-A, the percentage of canopy and flowering was 100% for both T3 and T4. Both treatments showed a significant increase in all the measurements compared to the untreated control (
p-value < 0.05). However, the best results were obtained with T4, when Nurspray
® was applied in BBCH 19–23–51.
Thanks to the much more balanced fertilization of the tomato plants, a slight difference in the colour of the tomato leaves were also found. This slight difference was not visible to the naked eye but was noticeable, with a significant difference in SPAD and NDVI value in leaves where Nurspray
® was applied compared to the untreated control (
Figure 1). The highest SPAD values compared to the control were observed in T4 during the first evaluation at 29 DA-A, showing increases of 6%. These elevated SPAD values were maintained at both 43 DA-A and 57 DA-A (
Figure 1a). For the NDVI value, no significant difference was detected in all treatments at 29 and 43 DA-A. At 57 DA-A, a reduction in NDVI values was observed in the untreated control (T1) and T2 compared to T3 and T4; however, this difference was not statistically significant in either treatment (
Figure 1b).
On the other hand, the results regarding the effect of Nurspray
® on quantitative parameters and on final yield and yield components are shown in
Table 4. A significant difference (
p < 0.05) was found for the number of fruits per plant, with higher values in the untreated control (T1) compared to plants treated with Nurspray
® (T2, T3, and T4). However, the weight per fruit (g) was significantly higher in T2, T3, and T4 compared to the untreated control, which led to a similar average in terms of the total kilograms of fruit per plant and total yield (tons/ha). Also, the number of marketable fruits was statistically similar in all the treatments, so the contribution of Nurspray
® to the increase in marketable yield was lower than expected. On the contrary, a significant reduction in the incidence of BER was detected in the plots where the Nurspray
® was placed and, consequently, in the number of unmarketable fruits (
p < 0.05) compared to the untreated control, where T3 and T4 showed the minimal value (0.00). Due to this difference, it was possible to estimate a higher yield per hectare, leading to greater profit for the owner of the farm.
From the analysis of tomato fruit, for some parameters, such as dry matter content (g), a significant increase in quality was found in the plots where Nurspray
® was applied (
p < 0.05) compared to the untreated control and the other treatments (
Table 5). No significant differences were found for the pH, acidity, and firmness parameters.
4. Discussion
This study demonstrates the significant effects of the foliar application of Nurspray
® on tomato plants at various developmental stages under open-field conditions, particularly under fluctuating climatic conditions. The application led to enhanced root development and improved fruit quality, as evidenced by the more voluminous fruit with better overall characteristics, as well as the more visible and healthy root systems (
Figure 2).
The plant cell-wall-derived oligomers in Nurspray
® effectively enhanced crop growth, flowering, fruiting, and yield quality, while also promoting plant defense mechanisms. These positive outcomes are likely due to Nurspray
® acting as a “plant priming” agent [
22], which accelerates the plant’s tolerance mechanisms when facing environmental stress. The plant cell-wall-derived oligomers in Nurspray
® effectively enhanced crop growth, flowering, fruiting, and yield quality, while also promoting plant defense mechanisms. These positive outcomes are likely due to Nurspray
® acting as a “plant priming” agent [
24], which accelerates the plant’s tolerance mechanisms when facing environmental stress. In open-field conditions, the varied climatic factors such as fluctuating rainfall, high temperatures, and relative humidity pose significant challenges to tomato cultivation, affecting plant growth and yield. The increasing temperatures, peaking at 31.3 °C in July, and variable rainfall patterns likely imposed stress on tomato plants, particularly during the critical fruit set stage. Despite these challenges, Nurspray
® demonstrated its potential to buffer against these stresses.
Successful tomato production begins with the sowing of seeds in protected environments under optimal germination and growth conditions to obtain seedlings that will later be transplanted to the final cropping site. Tomato plantlets treated with Nurspray
® showed enhanced root development, particularly during the critical transplanting phase, which is typically associated with root damage and stress. Previous studies have demonstrated that plant cell-wall-derived oligosaccharides promote root formation and elongation in species such as tomatoes, beans, and
Arabidopsis thaliana [
25,
26,
27]. Although the observed differences disappeared after a few weeks after transplant, the biostimulant had a positive effect on the vegetative growth of tomato plants. Probably, Nurspray
® is perceiving in the plant as signalling molecules stimulating the biosynthesis of endogenous phytohormones, thus promoting growth, flowering, and fruit setting. In agreement with this, flowering was anticipated in the plots where Nurspray
® was placed. Similar effects on tomato plant growth and development have been also reported in other studies using different biostimulants, such as a similar influence on the timing of plant flowering [
28,
29].
Nurspray
® also significantly increased chlorophyll content, as measured by SPAD, which is known to correlate with improved leaf physiology and photosynthesis [
30]. These results are consistent with another study using Nurspray
®, which demonstrated that the application of these oligomers stimulated photosynthesis in greenhouse-grown tomatoes under heat stress conditions [
31]. They found a significant increase in net photosynthesis that was accompanied by a significant increase in tomato yield compared with the control. The NDVI values were also slightly higher in the plants treated with a biostimulant in T4. Healthy vegetation leads to high NDVI values, so this indicator has a good correlation with several vegetation parameters, including the ability to predict yield [
32]. Altogether, the effects of Nurspray
® application are agronomically significant as prerequisites for increasing yield crop and fruit quality.
The positive effects of a biostimulant on yield parameters was demonstrated by a significant decrease in BER incidence, which is a devastating physiological disorder affecting vegetable production worldwide, including tomato [
33]. Many studies suggested that BER incidence initiates with Ca
2+ deficiency in young fruit and is influenced by different environmental factors, such as irregular watering conditions. The reduction in BER and the consequent increase in marketable fruit achieved with Nurspray
® suggest a positive effect on the transport of Ca
2+ to the fruit and/or the rate of the differential Ca
2+ concentrations between the proximal and distal end of the fruit (the higher the difference, the higher the incidence of BER). There was also a slight increase in the average fruit weight and the average number of fruits per plant that were marketable. Although the effect of Nurspray
® was not significant for either of these parameters, the combination of both of these and the high reduction in the number of unmarketable fruits per plant led to higher production and yield.
A positive effect of Nurspray
® was observed on fruit quality, with a significant increase in the dry matter content. However, the foliar application of Nurspray
® did not modify other parameters of interest in the fruit, such as firmness, pH, or lycopene. A possible reason for the higher dry matter content of the fruit achieved using T4 with Nurspray
® could be explained by the major photosynthetic capacity and vigorous vegetative growth, resulting in higher-quality fruit. Tomato fruit quality is assessed in terms of dry matter content (also known as total solids content) and is used as a reliable indicator of the harvest time for vegetables [
34]. An increase in dry matter is associated with fruit development and ripening, which is very important in tomatoes since they can ripen after harvest and need to be removed from the plant when fully ripe. These results may also increase tomatoes’ storage life post-harvest.
More remarkable than the effect of the biostimulant per se was the fact that its impact depended on the moment at which it was applied. The difference between treatments was related to the plant stage at which the foliar application of Nurspray
® was carried out. Different studies have focused on the use of biostimulants to improve fruit production and their predominant mode and time of application [
35]. Depending on the biostimulant, they can be applied only once, twice, or several times during the vegetative season. A very important criterion to consider is that biostimulants are mainly applied before the stressful event to prime the plant’s physiological defences, as previously mentioned. In our study, no stressful conditions were induced in the crop, although there were changing climatic conditions. Therefore, we focus on the moment of application, considering the vegetative state of the plants, to define the strategies that led to improvements in agronomic impact and yield. It is evident that the two foliar applications at BBCH 19 and BBCH 51 (T2) were not as effective as the three applications used in T3 and T4, BBCH 19–51–61 and BBCH 19–23–51, respectively. Notably, T4 showed the best results, demonstrating that the best biostimulant application strategy is three doses at different times: during the early, intermediate, and advanced stages of the crop (BBCH 19–51–61). Thus, the biostimulant was most effective in increasing yield and fruit quality when its application covered the phenological phases from leaf development and inflorescence emergence to flowering.
This study provides a strong basis for further research on the use of Nurspray® and other biostimulants to enhance crop resilience, growth, and quality. Nurspray® can be effectively integrated into agricultural practices, especially under stress conditions such as transplanting and fluctuating climates, making it a valuable tool in sustainable agriculture.
The observed increase in dry matter content and reduction in BER incidence highlight the biostimulant’s potential to improve both yield and marketable fruit quality. These results are consistent with metabolomic studies that demonstrate how biostimulants modulate a plant’s metabolic pathways to enhance stress tolerance and promote growth [
36]. In that sense, advanced metabolomics approaches could be developed to investigate the specific metabolic changes triggered by Nurspray
®, which would help elucidate the underlying molecular mechanisms. Additionally, further studies are also needed to evaluate Nurspray’s
® effectiveness against both biotic and abiotic stressors across various crops and environments.
Nurspray® demonstrates strong potential for use in both conventional and organic farming, promoting sustainable agricultural practices. Its ability to enhance root development and plant resistance during critical growth stages further highlights its value. From a consumer perspective, the use of biostimulants could lead to higher-quality, longer-lasting produce, aligning with the growing demand for environmentally friendly and health-conscious agricultural products.