Report on the Influence of Homeopathic/Nosode Foliar Applications on Phaseolus vulgaris (L.): Agronomic and Phytochemical Changes and Control of Zabrotes subfasciatus (Boh.) and Diabrotica balteata (LeConte)

Abstract

Nosodes are solutions prepared from pests, capable of controlling those same pests in crops. The use of these solutions in agriculture is an emerging technology for producing eco-friendly and inexpensive plant-based foods free from toxic compounds. The effects of applying nosodes to bean (Phaseolus vulgaris L.) crops before and after harvest were evaluated. The experiment was conducted in the field, with nosode 200CH applied once, three times, biweekly, and weekly from the flowering stage of the “Delgado” bean plant. The application of nosodes resulted in a 108% higher yield, 26% greater biomass, a 32% increase in root length, 34% more pods, and up to a 227% decrease in damaged pods compared to untreated plants. Furthermore, compared to the untreated plants, treated plants exhibited a 37% higher membrane stability and a phenolic content that was up to 27% higher in seeds and 22% higher in leaves. Plants under conventional management showed 61% less Diabrotica balteata (LeConte) incidence than nosode-treated ones. Seeds treated with nosodes exhibited a mortality in Zabrotes subfasciatus (Boh.) of up to 80%. This study highlights the use of nosodes in bean cultivation as an agronomic strategy to enhance seed production and quality, aiding producers in informed decision making about their application.

1. Introduction

Homeopathy applied in agriculture, also known as agro-homeopathy, consists of using pests for the production of “nosodes”, which can control, in crops, the same pest with which they were made [1]. Agro-homeopathy is an ecologically and economically viable model with the potential to reduce agrochemical use [2]. Moreover, its implementation would increase environmental sustainability by 37% and reduce the potential for ecotoxicity by 91.4%, while enhancing socioeconomic indicators [3]. Homeopathy is primarily based on the scientific principle of therapeutic similarity (similia similibus curentur), which was described by several medical practitioners starting from Hippocrates in ancient Greece [4] and which Hahnemann elevated to the level of a “natural law of healing” [5]; it involves the use of secondary actions of the body to stimulate a healing response against diseases through the administration of infinitesimal doses [5]. Until recently, there was still doubt as to how an infinitesimal dilution surpassing the Avogadro limit (theoretically devoid of measurable remnants of the starting materials) could possess therapeutic properties. However, Chikramane and collaborators [6] demonstrated for the first time the presence of entities in these extreme dilutions in the form of nanoparticles of the starting metals and their aggregates using transmission electron microscopy (TEM), electron diffraction, and inductively coupled plasma atomic emission spectroscopy (ICP-AES).

In contrast, Montagnier and colleagues [7] reported that low-frequency electromagnetic signals are emitted in aqueous solutions with dilutions of DNA from certain bacteria and viruses; these solutions contain genetic information, which can be detected using polymerase chain reaction (PCR). Furthermore, Henry [8] explained through quantum field theory that the ability of water to store information is due to the two-dimensional coherence domains generated by water molecules during succussion (vigorous shaking). Thus, various studies have successfully demonstrated the effectiveness of nosode application in reducing pest populations. This success is achieved through the preparation of solutions using homeopathic nosodes, which are diluted to specific potencies denoted by CH (Centesimal Hahnemanniana). CH denotes a specific dilution process used in homeopathy, where one part of the original substance is mixed with 99 parts of a diluent (usually alcohol or water). Each CH represents one step in a series of dilutions. For example, in a 1CH dilution, the original substance is diluted as described, while in a 2CH dilution, one part of this already diluted solution is mixed with 99 parts of fresh diluent, and this process continues for each subsequent CH level. This sequential dilution process increases the potency of the solution, which enhances its effectiveness in treatment and contributes to effective pest management [5]. For instance, Dysaphis plantaginea Pass. (6CH) showed a 14% reduction in the number of juveniles on apple seedlings [9]. In the case of Acromyrmex spp., the application of nosodes at 30CH resulted in an 80% reduction in ant activity over 20 days [10]. Foliar damage on bean leaves caused by Epilachna varivestis Muls. was reduced by 50% with the application of 200CH [1]. Similarly, Meloidogyne enterolobii Yang & Eisenback (6, 18, and 30CH) showed a significant reduction in the reproduction factor. Yang and Eisenback (6, 18, and 30CH) showed a significant reduction in the reproduction factor [11]. Additionally, for Alternaria solani Sorauer (Ellis), treatments with 27CH and 28CH achieved an average disease control of 57% and 62%, respectively, showing a significant difference compared to water and hydroalcoholic solution [12]. Likewise, in Xanthomonas campestris pv. campestris Pammel (Dowson), a reduction in disease severity was observed when nosodes were applied via irrigation at 24CH and 6CH [13]. The use of other homeopathic preparations has also been proposed to reduce the incidence of Pseudomonas syringae van Hall compared to conventional treatments [14].

It is important to highlight that the use of nosodes in agricultural production offers an alternative for pest and disease control, as shown by various studies on natural products. However, their mechanism of action is different: While bioactive compounds in natural products act directly on pests [15], nosodes do not contain active ingredients. Instead, their effectiveness is based on the presence of electromagnetic signals, which result from structural changes in water at the quantum level. These signals are detected by the plant, activating its natural defense mechanisms [5,7,8].

The systemic agro-homeopathic approach has been adopted by some European farmers, particularly in Italy, and is generating interest, especially among organic farmers. Although initial observations are positive, further scientific research is needed to validate these results. If its efficacy is confirmed, this approach could provide an agroecological production model with a low energy impact [16]. On the other hand, agro-homeopathic studies are conducted both academically and practically in countries such as Italy, Brazil, Mexico, India, and Germany [17].

To the best of our knowledge, there are no scientific reports evaluating the effect of nosodes on agronomic variables (e.g., yield), phytochemical and physical responses, or post-harvest aspects, which would allow for a comprehensive assessment of plant conditions.

In light of this, it is relevant to expand research in this area, particularly in the cultivation of beans, as they are globally consumed legumes known for their significant antioxidant, protein [18], mineral, and dietary fiber [19] contents. The importance of beans extends beyond their nutritional value; beans play a fundamental role in food security and the economy of millions of farmers in various regions worldwide [20]. However, bean production faces numerous challenges, including pests, diseases, climate change, and the low purchasing power of producers, which affect the entire process from field production to post-harvest storage, ultimately resulting in economic losses [21]. To address this situation, this study aimed to evaluate the agronomic, physical (membrane stability index [MSI]), phytochemical, and phytopathological responses of bean crops and seeds to the application of nosodes during development and storage to demonstrate their effectiveness in agricultural management and/or post-harvest practices.

2. Materials and Methods

2.1. Study Area

This study was conducted in the municipality of Villas de Zaachila (Oaxaca, Mexico; coordinates: 16°54′ N, 96°44′ W, 1518 m a.s.l.). The area has an average annual temperature of 21.5 °C [22]. The land use is agricultural, with clayey soils, and the farming practices include conventional methods (positive control), both irrigated and rain-fed.

2.1.1. Preparation of Nosodes

Nosodes are homeopathic preparations obtained from tissues or substances associated with the targeted disease or from cultures of the pathogenic agent. These nosodes include insects, mites, and fungi associated with plant pests and diseases, which modulate host resistance, alleviate symptoms, or promote healing [11]. Specimens including twospotted spider mite (Tetranychus urticae Koch), Thrips [Trips spp. (Thysanoptera)], anthracnose [Colletotrichum lindemuthianum (Sacc. and Magnus) Briosi and Cavara], rust [Uromyces phaseoli (Pers.) Wint.], sweetpotato whitefly (Bemisia tabaci Genn), greenbug aphid (Schizaphis graminum Rondani), banded cucumber beetle (D. balteata), Mexican bean beetle (E. varivestis), and edible or Mexican grasshopper (Sphenarium purpurascens Ch.) were collected from bean crops as hosts in the field. Additionally, for Mexican bean weevil (Z. subfasciatus), beans infested with weevil larvae were purchased from a local market in Villa de Zaachila, Oaxaca, Mexico. The organisms were placed separately in polyethylene bags and then transported to the laboratory. In the laboratory, each bag was processed immediately, following the method described by Hahnemann [5].

Initially, approximately 10 fresh specimens of insects of each species, 2 g of fungi (and/or damage caused by fungi), or 2 g of mites were ground (Figure 1). To clarify, each type of specimen from each species was ground separately. Then, 0.05 g of the finely ground biological material from each specimen was then mixed with 1.67 g of refined sugar and triturated for 6 min. Subsequently, the mortar and pestle were scraped for 4 min to gather all the residues of the triturated sugar from the organism in the center of the mortar. The resulting mixture was triturated again for 6 min and scraped for 4 min. This procedure was repeated two times in the same mortar, with 1.67 g of sugar added in each iteration, over 1 h (approximately 5 g in total). Then, 1CH nosode was obtained. To obtain the 2CH nosode, 0.05 g of the 1CH nosode was placed in a mortar, and the above-mentioned procedure was repeated to obtain the 3CH nosode. Then, to obtain the 4CH nosode, 0.05 g of the 3CH nosode was placed in an amber glass vial containing 50 drops of distilled water and 50 drops of 96% alcohol. The mixture was vigorously shaken (or succussed) for 2 min and allowed to rest for another 2 min. Similarly, the other nosodes up to 3CH were added to the same vial. This results in 4CH dynamization of all organisms in a single vial. For dynamization from 5CH onwards, only one drop of the previous dynamization was added to 99 drops of 96° cane alcohol. The final nosode was prepared at a potency of 200CH, as previously described [23].

2.1.2. Field Treatment Application

On 20 November 2022, Delgado bean seeds, a variety of black creole bean known as “Delgado” from Villa de Zaachila, provided by the producer who owned the site where the experiment was conducted, were sown mechanically with a spacing of 20 cm between plants and 35 cm between rows. Germination was achieved 7 days after sowing (DAS), and watering was performed weekly. Manual weeding was performed 25 days after germination, and harvesting took place at 107 DAS. For the preparation of the nosode solution, one drop of the 200C nosode was added to 1 L of water, succussed for 2 min, and then added to a backpack sprayer with 19 L of tap water [23] The treatments (

Table 1. Nosode and synthetic product treatment of beans (Phaseolus vulgaris L.) during cultivation.

Treatment	Description
Negative control (C−)	Application of well water weekly.
Positive control (C+)	Conventional management with insecticides and fertilizers during the pre-flowering stage [cypermethrin and urea, respectively; Bayer® (Mexico City, Mexico)].
Once	Nosode was applied only once during the pre-flowering stage.
Three times	Nosode was applied three times during the crop cycle (pre-flowering, flowering, and pod-formation stages).
Fortnightly	Nosode was applied every 2 weeks, up until 1 month before harvest.
Weekly	Nosode was applied weekly, up until 1 month before harvest.

According to Meier [24], the pre-flowering, flowering, and pod-formation stages correspond to codes 51, 62, and 71, respectively.

) were applied foliarly in the morning (7–8 am), starting from the pre-flowering stage, corresponding to international code 51 [24] at 27 DAS (or 20 days after germination). The field experiment was conducted using a completely randomized design within a single large plot. Each treatment was randomly assigned to at least five rows of 100 m within the 8000 m² plot. For evaluation, three replicates were used. Three central rows per replicate of each treatment were evaluated, with edge rows excluded to avoid edge effects.

2.1.3. Agronomic Variable Evaluation

The root length, pod length, number of pods per plant, and number of empty pods per plant were evaluated in 20 plants post-harvest at 107 DAS. The yield was evaluated and expressed as g/plant and t/ha. The yield per hectare was calculated based on 132,000 plants. For biomass evaluation and the biomass/fruit ratio, 3 g of leaf, stem, pod, and seed samples were dried in an oven at 80 °C until the triplicate samples reached a constant weight. The results are expressed in g of dry weight, as previously described [25,26,27].

2.1.4. Incidence of D. balteata Larvae in Bean Pods

After harvesting and during pod threshing, the incidence of D. balteata larvae in bean pods was assessed. Twenty plants from each treatment were selected for evaluation. The number of pods with D. balteata larvae was recorded. Subsequently, the incidence was calculated by dividing the number of infested pods by the total number of pods and multiplying the result by 100 to express the incidence as a percentage. The total number of pods includes both infested and non-infested pods on each plant. This method provides a percentage that reflects the proportion of pods with D. balteata larvae relative to the total number of pods observed per plant.

2.1.5. Membrane Stability Index (MSI)

Central leaflets from young but fully developed trifoliolate leaves were collected from the plants 1 month before harvest, after the application of treatments was completed. MSI was evaluated as described by Khalil and collaborators [28] with slight modifications. Using a conductivity meter (OAKTON, CON 400 series, Singapore), the electrical conductivity (EC) of two leaf discs with a diameter of 2 cm (corresponding to approximately 300 mg) submerged in 10 mL of distilled water was measured after immersing in a water bath at 40 °C for 30 min (EC1). The same procedure was performed for another sample, but at 92 °C for 10 min (EC2). Finally, MSI was calculated with Equation (1), and each treatment was evaluated four times.

MSI (%) = 1 − (EC1/EC2) × 100

(1)

2.1.6. Total Phenolic Compound Content

After the completion of treatment applications (1 month before harvest), central leaflets of young but fully developed trifoliolate leaves were collected. The bean pods were exposed to natural sun drying for 15 days before proceeding with threshing.

Before the preparation of the extract, the samples of bean leaves and seeds were kept in a conventional oven at 50 °C until they reached a constant weight. They were ground in a coffee grinder to obtain a fine powder, which was sieved through a 1-mm mesh opening. Then, 5 g of the powder was mixed with 4 mL of 80% methanol (99.8%; J.T. Baker, Phillipsburg, NJ, USA). The mixture was kept in the dark for 48 h and filtered to obtain the methanolic extract. Phenolic compounds were determined following the methodology described by Parmar and collaborators [29], with certain modifications. Leaf or bean seed extract (1 mL) was diluted with 9 or 4 mL of 80% methanol, respectively. Then, 500 µL of Folin–Ciocalteu reagent (2N; Sigma-Aldrich, St. Louis, MO, USA) was mixed with 500 µL of the diluted extract, followed by the addition of 1 mL of 10% Na₂CO₃ (Fermont, Monterrey NL, Mexico) and 8 mL of deionized water. The mixture was kept in the dark for 60 min before measuring the absorbance at 765 nm using a spectrophotometer (model 1104; Zeigen. CDMX, México). Total phenolic compounds were assessed using a standard curve of gallic acid (GA; 0.016–0.099 mg/mL; Sigma-Aldrich, St. Louis, MO, USA), with the equation: y = 0.004x + 0.0038, and an R² value of 0.9967. The results are reported as milligrams of gallic acid equivalents (GAE)/g of dry weight, and each treatment was evaluated five times.

2.2. Mortality Bioassay on Z. subfasciatus

To collect Z. subfasciatus, beans containing weevil inoculum were purchased from a local market in Villa de Zaachila, Oaxaca, Mexico. They were then transported to the laboratory, where they were taxonomically identified using appropriate keys [30]. The insects were reproduced at room temperature (25 ± 2 °C) in plastic containers covered with fabric.

Twenty bean seeds from each treatment (C−, C+, once, three times, every fortnight, and weekly) were placed in 30 mL plastic containers. Five unsexed weevils (F1 progeny) were placed in each container. In addition, two treatments were evaluated. The first treatment involved submerging C-seeds in a nosode at 200C for 2 min (C-AN), followed by drying. The second treatment (C + P) served as a positive post-harvest control and involved the use of aluminum phosphide (3 g tablet per 50 kg of seeds, Phostoxin 333^®). Each treatment was replicated five times, and evaluations were conducted over 10 days. After this period, the percentage of mortality was determined as the ratio of the number of dead weevils to the total number in each container [31].

2.3. Statistical Analyses

A completely randomized design was used for the experiment.

To evaluate the differences between the means of agronomic variables, MSI, and phenolic compounds, an analysis of variance was conducted, followed by Tukey’s honest significant difference test for post hoc comparisons. The incidence of D. balteata larvae and the percentage mortality of Z. subfasciatus were analyzed using the non-parametric Kruskal–Wallis test because of the non-normal distribution of data. Post hoc comparisons were performed using Mann–Whitney U tests with Bonferroni correction. All statistical analyses were performed using R version 4.2.0.

3. Results

3.1. Agronomic Variables

The application of agro-homeopathic treatments to bean crops had a significant effect (p < 0.05) on bean yield (Figure 2a). Specifically, a single application of nosode during the pre-flowering stage promoted a 108% higher yield than the negative control and a 32% higher yield than the conventionally treated bean crop (positive control). With the increased frequency of nosode application, the yield decreased (p < 0.05) but was still 27% higher than that of the negative control. The biomass of plants treated with a single application of the nosode was statistically higher (p < 0.05) than that of all other treatments, being 26% higher than that of the negative control and 58% higher than that of the positive control (Figure 2b). However, the plant efficiency (fruit-to-biomass ratio) was lower, with the positive control being 27% higher and 53% more efficient than the negative control. For this variable, it was observed that increasing the frequency of nosode application reduced biomass production but kept the fruit-to-biomass ratio elevated, only 7% below the positive control. In addition to the response in yield variables, the application of nosodes had a significant effect (p < 0.05) on the root length of bean plants; regardless of the application frequency, the roots were up to 32% and 48% longer than those of the negative and positive controls, respectively (

Table 2. Root and pod length, and numbers of pods, damaged pods, and empty pods in bean plants during cultivation under different treatments, including no treatment (negative control), conventional management (positive control), and nosode application once, three times, fortnightly, and weekly. Significant differences (p < 0.05) within columns are denoted by different letters.

Treatment	Root Length (cm)	Pods/Plant	Pod Length (cm)	Damaged Pods/Plant	Empty Pods/Plant
Negative control	14.6 ± 3.7 b	27.3 ± 2.4 c	8.4 ± 1.2 ab	3.6 ± 1.1 a	1.2 ± 1.2 bc
Positive control	13.1 ± 6.3 c	31.4 ± 5.8 b	8.2 ± 1.0 ab	3.5 ± 1.1 a	0.3 ± 0.5 c
Once	16.9 ± 3.3 a	41.4 ± 4.8 a	8.6 ± 1.3 a	1.8 ± 0.9 b	1.4 ± 1.2 b
Three times	19.4 ± 5.0 a	31.0 ± 5.1 b	8.2 ± 1.3 ab	1.1 ± 0.5 b	1.7 ± 1.0 ab
Fortnightly	17.8 ± 3.2 a	33.5 ± 4.6 b	8.1 ± 1.3 b	1.5 ± 1.1 b	0.8 ± 0.6 c
Weekly	17.3 ± 4.5 a	27.6 ± 2.1 c	8.2 ± 1.2 ab	1.8 ± 1.1 b	2.4 ± 0.5 a

Mean ± standard deviation.

). Homeopathic plants exhibited the lowest number of damaged pods, with up to 227 and 218% fewer damaged pods than the negative and positive controls, respectively.

Plants treated with a single application of nosodes produced the largest number and size of pods. However, the number of empty pods was statistically similar (p < 0.05) to that of the negative control and 79% higher than that of the positive control, which had the lowest number of empty pods, similar (p < 0.05) to that observed in plants treated with homeopathy every 15 days. Plants treated with a single application of the nosode exhibited the highest pod counts and sizes. However, the number of empty pods was statistically similar (p < 0.05) to that of the negative control and 79% higher than that of the positive control. The positive control, which represented the lowest occurrence of empty pods, showed a similar (p > 0.05) effect to that observed in plants treated with homeopathy every 15 days.

3.2. Incidence of D. balteata Larvae in Bean Pods

Within the bean pods, D. balteata larvae were observed. When evaluating their incidence, it was observed that both plants corresponding to C+ and those treated with agro-homeopathy represented the lowest values (p < 0.05), with less than 1.3% (median incidence), whereas the negative control exhibited the highest (p < 0.05) incidence (up to 7.4%; Figure 3).

3.3. Membrane Stability Index

MSI reflects cellular membrane damage and its function [28]. A single application of the nosode induced the highest (p < 0.05) MSI in bean leaves, which was up to 37% higher than all other treatments and controls (Figure 4).

3.4. Total Phenolic Compound Content

The content of total phenolic compounds in bean seeds was up to 27% higher than that in C− when plants were sprayed with nosodes three times (during pre-flowering, flowering, and pod formation) or every eight days. In contrast, seeds obtained from plants treated with either single or fortnightly applications showed a statistically similar phenolic content to that of the positive control (Figure 5a). However, a different pattern was observed in the leaves, as both the C+ and weekly applications of nosodes induced the highest synthesis of phenolic compounds (up to 22%) compared to the other treatments (Figure 5b).

3.5. Mortality Percentage of Z. subfasciatus

Application of agro-homeopathy only once or conventional management (C+) during the post-harvest period and post-harvest treatment with aluminum phosphide (C + P) significantly increased the mortality of Z. subfasciatus inoculated on bean seeds (Figure 6), with up to 80% higher effectiveness than that in the untreated seeds (C−).

4. Discussion

The highest performance observed in the nosodes could be related to the greater induction of resistance to both biotic and abiotic stress conditions [32,33], which allowed the plant to maintain its energy and photosynthetic capacity [34] to promote high crop productivity [35,36]. In addition, homeopathic preparations induce changes in the physiological mechanisms of photosynthesis by promoting the efficiency of specific photosynthetic compounds [36]. This efficiency is favored by the reduction of stress in plants [37,38]. The yield in the homeopathic plants of this study ranged from 1.5 to 2.5 t·ha⁻¹, which is comparable to the yields reported by Mekbib [39] for high-yielding P. vulgaris species (between 1.511 and 2.216 t·ha⁻¹). These yields are comparable to the average yield in important varieties (Comapa, Flor de Mayo, and Negro Otomí) for the studied area, which has been reported at 1.87 t·ha⁻¹ [40]. Based on the premise of the positive effect of agro-homeopathy on yield, the authors expected that yield would increase as the application frequency increased. However, the opposite was observed, and the highest yield, attributed to a greater number and size of pods, was observed in plants treated with a single application of the nosode 200CH during the pre-flowering stage. In this regard, Jütte and Riley [41] mentioned that neither the low nor high potencies of homeopathic remedies suggest the superiority of low potency over high potency, or vice versa. Similarly, Ferreira and colleagues [11] indicated that the effect and intensity of the effect of a nosode depend on the potency of the dynamized nosode. They observed that lettuce growth was negatively affected as the potency of the applied nosodes increased. In addition to the higher yield of plants treated with a single application of nosode, they produced the highest biomass and the least membrane damage. This confirms what was mentioned earlier regarding the plant being in suitable conditions for efficient photosynthesis, given the greater membrane integrity, which could reflect increased stress tolerance [28], suggesting a robust cellular structure that effectively maintains electrolyte balance [42]. However, despite the low fruit-to-biomass ratio in plants exposed only once to nosodes, this result is not contradictory, as various studies have shown that high biomass does not necessarily guarantee a higher yield. The efficiency of light energy absorption and conversion is determined by the thermodynamic properties of the crop, its environment [43], and the stress conditions [34]. The agronosode had the longest root length and the lowest number of damaged pods.

Pod damage was caused by D. balteata larvae, and the incidence of this insect was lower in homeopathic plants than in those treated with the synthetic insecticide cypermethrin. This suggests that the nosodes had an effect similar to that of a synthetic insecticide. This effect could have been due to the root conditions favoring nutrient uptake and the active ingredient of the nosode potentially activating cellular mechanisms [11,44] through pattern recognition receptors associated with damage, which are present in the plasma membrane [45]. These receptors can send resistance signals to plants [11,44], allowing them to maintain their health. When a plant detects a signal, certain biochemical pathways are triggered, leading to the production of secondary metabolites for pathogen intoxication [16] and/or halting its migration through plant tissues [11]. In this regard, the current study observed that both plants treated with conventional (chemical) management and those exposed to the highest application frequency of nosodes had the highest phenolic content in the leaves. However, only plants treated every eight days produced fruits with high phenolic content. This suggests that homeopathic treatments induce plant defense through the production of phenolic compounds, which are commonly produced and accumulate in the subepidermal layers of plant tissues exposed to physical damage, wounds, drought, and pathogen attack [46] as a form of natural defense. Another way phenolic compounds act is by mitigating oxidative stress in plants through the formation of stable complexes (metal and protein-polyphenol) with their hydroxyl and carbonyl groups. Their synergistic capacity with other antioxidants enhances the radical scavenging ability [47].

In addition, the findings during storage were consistent in the sense that the mortality of Z. subfasciatus, aside from being induced in the presence of aluminum phosphide, was induced when these weevils were exposed to homeopathized seeds. Unlike aluminum phosphide, which acts as a toxic insecticide, the observed effect in treatments with homeopathized seeds, due to the absence of toxicity [48], may be related to the production of phenolic compounds or the composition of the seed, which, being unpalatable to weevils, leads to their death by starvation. This anti-feeding effect was not achieved with a brief exposure of the negative control seeds to the agro-homeopathic solution post-harvest. This suggests that to observe seed protection during storage, the agro-nosode must be present systemically in the seed as a consequence of an epigenetic effect [16] associated with EMIT (ElectroMagnetic Information Transfer). In this context, it has been proposed that water aggregates with an electric dipole moment could act as mediators of specific weak bioelectromagnetic signals, affecting target cells and modifying their proliferation, differentiation, apoptosis, or adaptive responses [49].

This study provides scientific evidence of the effects of agro-homeopathic application on the yield and seed quality of bean crops, particularly when a single application is made at the flowering stage. However, the frequency of application of agro-homeopathic remedies may yield variable responses, warranting further investigations. Our results suggest a potential limit to the frequency of nosode application, as frequent applications can direct the plant energy either towards defense or nutraceutical quality, as indicated by the production of phenolic compounds, whereas fewer applications may induce greater stability of the leaf area membrane and increase the seed yield. The synthesis of phenolic compounds is a plant defense response activated against certain pests, such as D. balteata and Z. subfacsiatus. Therefore, further studies should assess the response to the application frequency of nosodes in various crops, including bean crops, using different concentrations at different phenological stages. Furthermore, the proximal composition of homeopathic seeds and their association with the anti-feeding effects should be evaluated.