Lentil Biorooting Agents: An Ecological Alternative to Improve the Growth and Development of Italian Zucchini in Sustainable Production Systems

Abstract

Zucchini (Cucurbita pepo L.) is a crop of global economic importance. Therefore, there has been a continuous search for alternative cultivation methods that contribute to improving its growth and development. In the context of sustainable agriculture, plant protection techniques involve the use of substances of natural origin (e.g., biorooters), which have a positive impact on the production cycle of plants. In this study, the effects of a lentil biorooter on the growth and development of Italian zucchini were investigated. Five treatments were analyzed in the study: 25 (T1), 50 (T2) and 75 (T3) mL of the biorooter; a chemical rooting agent (“Raixen PLUS ©”; T4); and a control (T0). The results showed that the application of the lentil biorooter (T3) increased the plant height by 166%, the number of leaves by 113%, the aerial fresh weight by 169%, the root length by 165%, the fresh and dry root weights by 102% and 116%, and the number of flowers and female flowers by 89% and 177%, respectively, compared to the control (T0). In the case of the conventional rooting agent “Raixen PLUS ©” T4, compared to (T3), the following agro-nomic variables were reduced: plant height by 13%; the number of leaves by 124%; fresh and dry aerial weight by 103% and 107%, respectively; fresh and dry root weight by 9% and 117%, respectively; and the number of total and female flowers by 112% and 160%, respectively. Pearson correlation coefficients were calculated to compare the heights of the plant between the five treatments, as well as to compare the number of leaves to the fresh aerial weight and the length of the root to its fresh weight. Furthermore, the principal component analysis (PCA) results indicated that the differences between components 1 and 2 appeared to be positively influenced by the root length. These results suggest that lentil biorooters could be an ecological alternative to conventional rooters, thus mitigating the adverse effects of their use in zucchini crop production. This is the first study to report on the induction of growth and development using a lentil biorooter in zucchini.

1. Introduction

Zucchini (Cucurbita pepo L.) is an annual herbaceous plant that is used for different purposes depending on the region and traditions, and is widely cultivated all over the world [1]. The global zucchini cultivation area is two million hectares, with an annual production of 28 million tons; China and India are the largest producers, with 5,746,048.69 and 4,101,981.09 million tons cultivated in these countries, respectively [2,3,4]. Given its economic relevance, there has been a constant search for strategies to optimize the growth and development of this crop.

To guarantee quality production, it is essential to consider all stages of the crop’s phenological cycle. The initial phase, in particular, is a critical stage as the transplant conditions and irrigation management during this stage can determine the success of plant establishment [5]. Proper care at this stage not only promotes robust root development, but also encourages vigorous growth, abundant flowering and higher fruit yields [6].

Sustainable agriculture practices aim to minimize environmental impacts and conserve natural resources, ensuring long-term productivity [7]. Among these practices, phytosanitary techniques based on substances of natural origin, such as biorooting agents, stand out; these agents can aid in the establishment of plants in the soil, strengthen their root system and increase their resistance to abiotic and biotic stresses [8]. Their application not only contributes to reducing the dependence on synthetic agrochemicals, but also promotes soil biodiversity by interacting with beneficial microorganisms, resulting in a positive impact on the productive cycle of plants [9]. Efficient water management and the use of organic fertilization can complement the use of bioproducts, ensuring a balance between productivity and the health of the agricultural ecosystem [10].

Rooters contain phytohormones such as auxins, cytokinins and gibberellins, which act in different ways [11]. The effect of auxin begins, for example, during the development of embryonic root and continues after embryonic development. Through influencing the growth of the primary root and the initiation and growth of the lateral and adventitious roots, auxins control all aspects of root architecture. Rooting agents containing these hormones are produced in a liquid or powder form. The most important hormonal agents are ANA (naphthaleneacetic acid), IBA (indolebutiric acid) and AIA (indole 3-acetic acid) [12]. Commercial formulations, which are mostly used in crop production, contain ANA, which is more effective than IBA and AIA. It is used in asexual propagation by introducing the lower part of the cutting into the rooting powder, such that the product adheres to the surface of the cutting. It is also used in solution as a foliar spray, which is used as a bioregulator for plant growth [13,14]. Another natural hormonal agent that promotes root formation is derived from lentil (Lens culinaris L.) as it contains a high concentration of phytohormones, mainly auxins. Experimental studies have demonstrated the power of this type of biostimulant; for example, in one research report, natural lentil roots were used for the propagation of fig trees (Ficus carica L.) [15], which resulted in a higher survival rate, longer root length and greater number of roots, as well as fewer days in root formation. In another study [16], lentil roots were used for the cultivation of coffee (Coffea canephora var. robusta), and significant increases in the lengths of the roots of seedlings and saplings were found. Finally, the application of a biostimulant in combination with an oak leaf powder extract was shown to improve the resistance of tomatoes to water stress, reducing lipid peroxidation, optimizing the fruit quality and increasing the content of bioactive compounds in the leaves and fruits [17]. However, there are no studies on the use of lentil roots to induce growth and development in the context of zucchini cultivation. Therefore, we investigated the effect of the application of three different concentrations of lentil roots (25, 50 and 75 mL) as a chemical rooting agent and a control on the growth and development of Italian zucchini seedlings (Cucurbita pepo var. Grey Zucchini), with the aim of offering an ecological alternative to the use of conventional rooting agents in zucchini production.

2. Materials and Methods

2.1. Study Area and Crop Establishment Protocol

This study was conducted at the Institute of Agricultural Sciences of the Autonomous University of the State of Hidalgo, Tulancingo de Bravo, Hidalgo (20°05′09″ N, 98°21′48″ W). The region is located 2157 m above sea level and has a temperate climate, an average temperature of 16 °C, 550 mm of precipitation annually and a relative humidity of 40% [18] (Figure 1). The experiment was carried out in a greenhouse with a polyethylene cover; the plant materials were obtained by planting Italian zucchini (Cucurbita pepo var. Grey Zucchini) in seedling trays containing a peat moss substrate. When the seedlings were 20 cm high and had 3 true leaves, they were transplanted into 12 L polyethylene bags containing 3 m² of a mixture of peat moss and perlite in a 1:1 (v/v) proportion. For crop nutrition, we used a Steiner solution adapted to the phenological stage (25% for the vegetative stage, 50% for the flowering stage, 75% during fruit setting, and 100% during fruit filling and harvesting [19]) and a drip irrigation system that adjusted the irrigation quantities for each growth phase, taking into account the management, environmental and development dynamics (100 mL for the vegetative stage and 500 mL for the flowering stage [20]). Pruning was performed to remove invasive plants, such as Verdolaga (Portulaca oleracea L.) and Zacate pluma (Leptochloa filiformis Lam Beauv), which were present in the study area and could compete with zucchini for space, nutrients and solar energy [21].

2.2. Preparation of Natural Rooting and Application of Treatments

The lentil seeds used in this study were donated by the Agricultural Chemistry Laboratory of the Autonomous University of the State of Hidalgo. The seeds were soaked in water in a ratio of 1:4 (500 g of lentil seeds in 2000 mL of water) for 24 h. Afterwards, they were covered with a dark cloth to promote germination. During this process, they were kept moist by applying water as needed. After 7 days of germination, the seedlings were processed in an industrial blender (Model VVCA-LI-12A, Cavimex, CDMX, Mexico City, Mexico) together with the soaking water, following the methodology in [22]. The chemical composition of lentil includes bioactive compounds such as flavones and flavonols (catechin, epicatechin and gallocatechin), as well as phenolic compounds such as ferulic and coumaric acids and hydroxybenzoic acids (4-hydroxybenzoic and protocatechuic acids) [23]. Lentil roots contain phenolic compounds such as 4-O-β-d-glucopyranosyl-2-methoxybenzoic acid, (αS)-4,4′-di-O-β-d-glucopyranosyl-α,2′-dihydroxydihydrochalcone, (αS)-4′-O-β-d-glucopyranosyl-α,2′,4-trihydroxydihydrochalcone and keto-2-hydroxyglycitein [24].

The experiment used a completely randomized design, considering five treatments: the application of different doses of a lentil biorooting agent (25 mL (T1), 50 mL (T2), and 75 mL (T3)), a chemical rooting agent (Raixen PLUS ©, CDMX, Mexico) (T4), and a control with only water (T0). Five repetitions per treatment were performed, with an individual Italian zucchini plant considered one repeat. The treatments were applied at the time of transplanting and then once a week for 7 weeks (75 mL per plant) in drech. The application of the biorooting agents was constant throughout the experiment to ensure their safety at the time of application.

2.3. Agronomic Analysis

To evaluate the effect of the treatments on the zucchini plants, the following agronomic variables were considered: plant height, which was measured from the base of the substrate surface to the growth apex (cm) using a flexometer (PRO-8-R Truper Model, Truper, CDMX, Mexico); the number of leaves, which was measured by visual counting; root length, which was measured using a tape measure (cm); the number of male and female flowers; and male flowering, which was measured as the number of days from planting until 50% of the plants were in the late phase. Female flowering, which was measured as the number of days from planting until 50% of the plants had receptive stigmas with a visible length of at least three centimeters [25]. At 49 days after transplanting (DDT), the aerial part (leaves, stem and flowers) of each plant was harvested and the dry weights of the aerial part and roots were measured. To determine the length and dry weight of the roots of the plants, the substrate was removed with water to separate the roots from the substrate. After drying in a drying oven (HFA-1000DP Model; Craft, CDMX, Mexico) at 80 °C for 4 days, the dry weights of the different parts of the plant were measured.

2.4. Statistical Analysis

For the analysis of the agronomic variables, the Shapiro–Wilk normality test and Levene variance homogeneity test were used (TS1). Subsequently, an analysis of variance (TS2, TS3 and TS4) and Fisher’s least significant difference (LSD) of means test (α ≤ 0.05) were carried out. All statistical procedures were performed using INFOSTAT 2020 software. Additionally, principal component analysis was performed, as well as Pearson correlation analysis, using the R 4.1.2 statistical software.

3. Results

3.1. Impact of Treatments on Height, Number of Leaves and Aboveground Biomass

An analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) of means test (α ≤ 0.05) revealed a significant treatment effect between T3 and T0 (Table S2; Figure 2) of the data obtained for the height and fresh aerial weight. The results for the plant height showed significant differences between treatments (α ≤ 0.05) (Figure 2A), and the plants treated with the lentil biorooter T3 reached a height that was 166% greater than that of the control T0 and 115 % greater than that of T4. On the other hand, plants treated with T2 showed a height reduction of 16% compared to those treated with T4. Likewise, plants in the T1 treatment group were 11% shorter than those in the T3 treatment group, which registered the greatest height among all the treatments evaluated. The number of leaves showed significant differences between treatments (α ≤ 0.05) (Figure 2B). The num-ber of leaves increased by 131% and 124% with T3 compared to T1 and T4, and T2 in-creased it by 5% compared to T1, while the lentil biorooter agents (T4 treatment) resulted in a 13% reduction compared to T0. The fresh aerial weight showed significant differences between treatments (α ≤ 0.05) (Figure 2C), as T3 increased by 169% compared to T0, 147% compared with T1, and 103% compared to T4; the T2 treatment increased the fresh aerial weight by 153% compared to T0, but reduced the fresh aerial weight by 7% compared to T4; and T4 increased the aerial fresh weight by 163% with respect to T0. The dry aerial weight (Figure 2D) was reduced by 14% and 17% with the T4 and T1 treatments compared to T0, while it increased by 107% with T3 compared to T4. Treatment T2 increased the dry aerial weight by 110% compared to T4, but T2 reduced the dry aerial weight by 4% com-pared to T0. In the case of treatment T4, the dry aerial weight was reduced by 14% com-pared to T0, while T1 reduced it by 17% compared to T0 (Figure 2D).

3.2. Effects of Different Treatments on Root Growth and Biomass

The analysis of variance (ANOVA) revealed significant effects of the different treatments on root growth and biomass (Table S3). The root length showed significant differences between treatments (α ≤ 0.05) (Figure 3A) The root length increased by 184% and 168% with T4 and T3 compared to T0, but T3 reduced this variable by 10% compared to T4. In the T1 group, the length was reduced by 33% compared to the T4 group, but T1 increased it by 123% compared to T0. In the (T2) group, the length increased by 124% compared to T0, but this same treatment T2 reduced the root length by 33% and 25% compared to T4 and T3. T3 increased the fresh root weight by 2% compared to T0 and by 116% compared to T1 (Figure 3B). While T4 reduced the fresh weight of the root by 9% compared to T3, the T2 treatment reduced this variable by 13% compared to T0 and by 7% compared to T4. With treatment T4, the fresh weight of the root increased by 106% compared to T2, but T4 reduced this variable by 7% compared to T2 and by 15% compared to T0. The dry weight of the root showed significant differences between treatments (α ≤ 0.05) (Figure 3C). T3 increased the dry weight of the root by 116% compared to T0 and 117% compared to T4 With treatment T2, there was an increase of 101% compared to T0 and 102% compared to T4. Treatment T1 showed the same dry root weight as T0, which was 101% higher than that of T4.

3.3. Effect of Treatments on Flower Production and Male and Female Flower Distributions

As shown in Table S4, the analysis of variance (ANOVA) revealed significant effects of the different treatment conditions on the Italian zucchini plants’ total flower production and distribution of male and female flowers. Figure 4 shows the results for the number of flowers with significant differences between treatments (α ≤ 0.05). Treatment T2 increased the number of flowers by 116% compared to T0 and T4 and by 118% compared to T1. Treatment T3 increased this variable by 112% compared to T4, and the T3 treatment also increased the number of flowers by 113% compared to T0 and by 114% compared to T1. Treatment T4 presented the same number of flowers as T0.

The number of flowers shows significant differences between treatments (α ≤ 0.05), which were reduced by 2% with T1 compared to T0 and T4. The number of male flowers shows significant differences between treatments (α ≤ 0.05) (Figure 4B); the number of male flowers increased by 115 % with treatment T2 compared to T0. With treatment T4, this parameter was reduced by 24% compared to T2. While T1 increased the number of male flowers by 109% compared to T3, it reduced this number by 12% compared to T0. The T3 treatment reduced the number of male flowers by 20% compared to T0 and by 9% com-pared to T4. Treatment T4 reduced the number of male flowers by 12% compared to T0. The number of female flowers shows significant differences between treatments (α ≤ 0.05) (Figure 4C). The number of female flowers increased by 176% with T3 compared to T0 and by 160% compared to T4; the T2 treatment increased this parameter by 106% compared to T0 and by 96% compared to T4. With T1, the number of female flowers was reduced by 9% compared to T2, 4% compared to T4, and 7% compared to T0. Treatment T4 increased the number of female flowers by 110% compared to T0 (Figure 4C).

3.4. Correlations Between Growth and Development of Italian Zucchini Under Different Treatments

The Pearson’s correlation coefficients calculated for all of the agronomic variables (Figure 5) showed that the plant height was strongly positively associated with the treatment (ρ = 0.67). The fresh aerial weight and dry aerial weight were positively associated with the number of leaves (ρ = 0.69 and ρ = 0.63) and weakly associated with the treatment (ρ = −0.05 and ρ = −0.17, respectively) (Figure 5). The fresh and dry weights of the roots were positively associated with the length of the roots (ρ = 0.53 and ρ = 0.57, respectively), and the dry weight of the roots was also moderately positively associated with the height of the plant (ρ = 0.43). The number of flowers was negatively associated with the number of leaves (ρ = −0.04), which was in turn strongly positively associated with the number of male (ρ = 0.53) and female flowers (ρ = 0.76) (Figure 5).

3.5. Treatment Differentiation Through Principal Component Analysis

The PCA of all the agronomic variables confirmed the variability between treatments (Figure 6,

Table 1. Loadings from the first 2 principal components of the PCA for Italian zucchini.

Variable	(60.64%)	Component 2 (29.19%)
Plant height	0.2882	0.0702
Number of leaves	0.3205	−0.4690
Fresh aerial weight	0.4222	−0.2522
Aerial dry weight	0.3658	−0.3103
Root length	0.3321	0.2996
Root fresh weight	0.3001	−0.1461

). The fresh aerial weight, dry aerial weight, and number of leaves had the strongest effects on the first component, accounting for 60.64% of the variance. The height of the plant and the length of the root had strong positive relationships with the second component, while the number of leaves, fresh aerial weight, and dry aerial weight had negative relationships with the second component. The differences between the treatments seemed to be strongly influenced by the length of the root.

4. Discussion

The leaves are the main organs during vegetative growth, which provide a flattened surface that allows for efficient light capture during photosynthesis [26]. In this study, the increase in the number of leaves (Figure 2B) in the plants treated with the lentil biorooter could be explained by the activation of genes present in simple leaves, such as JAGGED and NUBBIN, which regulate cell division and lateral organ growth [27]. The increase in the aerial biomass (Figure 2C,D) could be attributed to the activation of enzymes related to N and C metabolism to promote plant growth and biomass productivity [28].

The increase in root length (Figure 3) could be attributed to the presence of phytohormones such as auxins and cytokinins in the lentil-based biorooter. Auxins, such as indol-3-acetic acid (AIA) and the gibberellins (GA3), are associated with the promotion of plant growth [29,30]. Cytokinins are known for their multifaceted functions, which include promoting cell division, controlling meristem activity, facilitating organogenesis, controlling the direction of vascular differentiation and aiding in the acquisition of nutrients [31]. In addition, the presence of other bioactive compounds, such as amino acids, peptides, phenols and flavonoids, favor the expression of genes such as JAGGED, LYRATE and DTX41, which regulate cell growth in lateral organs and regulate the general development of plants through the transport of phytohormones and by controlling ionic homeostasis and tip growth processes, which are crucial processes in the development of plants [32]. This may explain the increases in length (Figure 3A), and fresh (Figure 3B) and dry root weight (Figure 3C) that were observed in the biorooter-treated plants in this study.

For example, the DTX41 gene, which belongs to the MATE family, is essential for plant development and growth. The genes in this family have been identified as voltage-gated chloride anion channels that facilitate the entry of chloride into the vacuole, leading to the regulation of turgidity pressure, cell expansion and the regulation of stomatic movements in the guardian cells [33]. Cell division and enlargement are the predominant processes during the initial growth phase, which are accompanied by the production and accumulation of organic acids, metoxipirazines and phenolic compounds [34], which are intimately linked to the transport of minerals and water absorption [35]. Additionally, it is hypothesized that DTX41 facilitates the sequestration of flavonoids in the vacuole of plant cells. This process is particularly important for the seed coating and also plays a crucial role in germination, resistance to environmental stress and protection against herbivores and pathogens. According to [32], the foliar application of a flavonoid biostimulant to tomato (Solanum lycopersicum L.) plants induced the expression of this gene. However, further studies are still required to obtain solid experimental evidence on the specific function of DTX41. Specifically, future research could focus on evaluating the expression of DTX41 at different stages of zucchini development, as well as its possible impact on seed quality and viability, plant growth and development, and tolerance to stress conditions.

The mechanism of biorooters in plants remains uncertain, but much of the evidence suggests that the most important molecules in the regulation of growth and development processes are amino acids and/or peptides, which also determine the shape of flowers and leaves, control the root length and stem thickness, and induce the biosynthesis of secondary metabolites [36]. Through the activation of numerous signaling pathways involving second messengers such as Ca²⁺, cyclic adenosine monophosphate (CAMP), cyclic guanosine monophosphate (cGMP), cyclic ribose ADP (cADPR), guanosine tryngosphate (GTPasa), 1,2-diacylglycerol (DAG) or inositol 1.4,5 triffosphate (IP3), amino acids translate different development signals to achieve a favorable end result in the plant [37]. These processes could explain the results obtained for the number of flowers (Figure 4).

Specifically, lentil biorooters optimize the uptake and utilization of nutrients through promoting the development of a root system, improving the efficiency of nutrient uptake, stimulating photosynthesis, and supporting favorable plant development. This results in greater aerial and root biomasses, which have a positive effect on plant growth and overall productivity. This observation was also confirmed by the positive correlation between plant height and the application of the lentil biorooter, as shown in the Pearson correlation analysis (Figure 5). Despite the correlation between the aboveground fresh and dry weights and the leaf number mentioned above, the lack of a significant correlation between the flower and leaf numbers (Figure 5) suggests that other factors may influence flowering, such as the environmental conditions. A plant can efficiently absorb nutrients but, if the environment is not conducive, it might fail to develop flowers. Furthermore, it is common for plants to follow developmental strategies that prioritize vegetative growth to ensure their survival before reproducing [38]. This aligns with the results of the Pearson correlation analysis (Figure 5), where it was observed that the length of the main root was most closely related to the fresh and dry weights of the roots, reinforcing the idea that root development also plays a key role in plants’ growth strategy. This fact coincides with the findings from other investigations such as [39], where the root length and fresh and dry weights of the root increased by an average of 14.77% when a biorooter based on aloe (Aloe vera L.) was applied in the cultivation of pomegranate (Punica granatum L.). Another investigation [40] reported a 39% increase in the dry weight of benjamina (Ficus benjamina L.) roots after applying a biorooter based on aloe gel and rice (Oryza sativa L.) husk. Finally, increases in the number of shoots, root length and plant height were reported when applying a lentil rooter (Lens culinaris L.) to a tomato crop [41]. These reports are consistent with the findings in this study (Figure 2, Figure 3 and Figure 4). However, it is important to note that, after an extensive literature search, this is the first study to report on the induction of growth and development using a lentil biorooter in zucchini cultivation.

5. Conclusions

The application of the lentil biorooter T3 significantly increased the plant height, the number of leaves, the aerial fresh weight, the root length, the fresh and dry root weights, as well as the total number of flowers and female flowers compared to the control T0. In con-trast, the conventional rooter “Raixen PLUS ©” T4 showed a reduction in some agronomic variables compared to the most efficient treatment T3, such as plant height, the number of leaves, fresh and dry aerial weight, fresh and dry root weight, as well as the total number of flowers and female flowers. The Pearson coefficients indicated positive associations between plant height and the five treatments, the number of leaves and aerial fresh weight, and root length and fresh root weight. Likewise, the principal component analysis (PCA) showed that the differences between components 1 and 2 seemed to be moderately influ-enced by the root length. Thus, this research is a starting point in the study of lentil-based biorooters, highlighting their potential as an ecological alternative in agricultural produc-tion. However, due to the experimental scale used, it is advisable to carry out additional studies to evaluate the behavior of this biorooter in different environmental conditions and production systems. With organic agriculture becoming increasingly important, the search for natural inputs to promote plant growth without compromising soil health or biodiversity is essential. Organic agriculture not only reduces the dependence on synthetic agrochemicals, but can also improve soil quality, promote ecological balance, and con-tribute to food security. The results obtained in this study reinforce the viability of using lentil biorooters as a sustainable strategy to optimize the development of crops such as zucchini. The improvements in the plant height, number of leaves, root biomass, and flower production suggest that such biorooters can play a key role in more resilient and sustainable agricultural systems. However, future research could consider larger-scale tri-als under different agroclimatic conditions to validate and expand these findings. In this sense, the present research opens a new line of study for the development of agricultural technologies that contribute to the transition towards sustainable and environmentally friendly production.