Study Site

Research was performed in the Experimental Agricultural Field of Centro de Investigaciones Biológicas del Noroeste (CIBNOR) within a metallic structure totally covered with a stabilized antiaphid polyethylene monophilament mesh, model 1.610 PME CR, thread 16 × 10 cm^-2 and opening 0.4 × 0.8 mm, crystal color with 40% shade. The site is located at 24° 08’ N and 110° 24’ W, 7 m altitude in EL Comitan, Baja California Sur (BCS), Mexico on coastal land in the southern portion of the Baja California Peninsula and 17 km westward from the port of La Paz, capital of the State. The experimental site has a climate type Bw (h’) hw (e) considered as semiarid with xerophilous vegetation (^{García, 2004}).

Genetic Material

Chiltepin chili (Capsicum annuum L. var. glabriusculum) seedlings were obtained from germinated seeds from the germplasm bank of CIBNOR Plant Biotechnology Laboratory, which came from a second generation of plants cultivated from seeds of wild fruit collected in Sierra San Antonio, BCS, Mexico. The seedlings were placed in plastic trays with commercial (Sunshine^®, Canada), substrate for the homeopathic treatments.

Homeopathic treatments

The homeopathic medicines are produced under governmental supervision in authorized laboratories. This procedure consists of serial dilution/succussion of a concentrated solution or mother tincture [(TM), All the acronyms are in Spanish] that contains what is known as the “active principle” in conventional medicine (allopathy). The dilution/succussion [decimal (D), centesimal (C), millesimal (M)] process is known as “potentiation” or “dynamization”, which is why it is common to identify the resulting product by adding a letter H in honor of Dr. Hahnemann (DH, CH, MH). For the development of this study, two centesimal homeopathic dynamizations (7CH and 13CH) were used from the medicine NaM of the Laboratorios Similia^® (CDMX, MX), authorized for human use and registered in the Health Ministry of Mexico (^{SSA, 2015}). The TM of NaM is a concentrated sea salt solution, whose composition is mainly NaCl, but it includes trace amounts of the minerals known in seawater, which include bioavailable magnesium, potassium chloride, iron, and calcium, among others (^{Mazón-Suástegui et al., 2018}). The stock NaM-6CH and NaM-12CH dynamizations in alcoholic dilution (ethanol 87°) were acquired in Farmacia Homeopática Nacional (CDMX, MX). The experimental dynamizations NaM-7CH and NaM-13CH (treatments) were prepared at CIBNOR Plant Biotechnology Laboratory by diluting them in distilled water (to avoid collateral effects of ethanol) and agitating in vortex for two minutes (^{Mazón-Suástegui et al., 2018}; ²⁰¹⁹).

Experimental Design

A completely randomized design with factorial arrangement (2A × 3B) was performed with four replicates of nine plants each, using as factor A two concentrations of NaCl (0 and 200 mM) and as factor B two dynamizations of the homeopathic medicine (NaM-7CH and NaM-13CH); a control treatment without medicine (distilled water) shall be referred in this study as NaM-0 for practical purposes because no homeopathic medicine was included.

Management of the Experiment

Two-month seedlings were transplanted individually in hydroponic baskets, placing a circular piece of synthetic sponge inside to support the radicle system of each plant. After transplanting, the pots were placed in an experimental hydroponic system, which consisted of plastic boxes containing nutritive medium, as well as polystyrene as floating support of the hydroponic baskets. The covered plastic boxes used were Merida green color model and 50 L capacity gauged at 30 L with water without salts and high density polystyrene 2.54-cm thick plates (Foamular^® 250; Mod. CAN24P3/0E, México) with openings ad hoc to insert (approximately ¼ of its total height) nine hydroponic baskets per box. The previous arrangement was performed to guarantee that each plant root could have contact with water, initially with the help of the support sponge and subsequently by direct water immersion. The water used was obtained from a desalination plant located at CIBNOR experimental field with an electrical conductivity of 0.22 dS m⁻¹. As nutrient formula, a solution adapted for chiltepin chili (Samperio, 1997) was applied, adding 666 mL to each 100 L of water with a pH in optimum (6.0 ± 0.4) range. To have a better dilution of each of one of the components, this nutrient solution was concentrated in 6 L of working nutrient solution. The medium of the hydroponic cultivation was prepared adding 200 mL of the working solution in each one of the boxes of the experimental hydroponic system, containing 30 L of water. The hydroponic cultivation pH was measured every other day, and when necessary, it was adjusted with potassium hydroxide (KOH) or sulfuric acid (H₂SO₄) according to that required to maintain a pH of 6.0 ± 0.4, and with equal frequency, electrical conductivity, total dissolved solids, salinity, and temperature of the hydroponic cultivation medium, using multiparametric equipment (Thermo Scientific^®, USA).

Eight days after transplanting (adaptation period), the seedlings were cut at a height of 10 cm. The homeopathic treatments NaM-7CH and NaM-13CH, the control (distilled water) group, and salinity treatments (0 and 200 mM of NaCl) were applied once the plants were adapted to the hydroponic system (18 days after transplant), showing new buds as a result of the initial cutting. Before applying the treatments, a total change of cultivation medium was performed in all the boxes of the experimental hydroponic system, adjusting the pH (6.0 ± 0.4) and determining electrical conductivity (CE), totals dissolved solids (STD). The application of the homeopathic treatments was made prophylactically, and thus a pre-conditioning stage of the plants started five days before applying the salinity treatments. With the purpose of avoiding an osmotic shock, the salinity treatments based on the addition of NaCl were applied gradually, starting with 25 mM of NaCl until a final concentration of 200 mM was reached (^{Murillo-Amador et al., 2007}).

Morphometric Variables Assessed

A week after the salinity treatments were applied at 100% (200 mM) of their concentration, the first sampling was performed randomly selecting two plants per replicate for a total of eight experimental units per treatment. The plants sampled were placed in labeled transparent plastic bags and transferred to CIBNOR Plant Physiology-technology Laboratory where the aerial part (stem and leaves) and root were separated to quantify fresh and dry weight (g) and length (cm) of both parts. Subsequently, leaf area was measured (cm²) using an integrator (Li-Cor^®, model-LI-3000A, series PAM 1701, USA) equipment. To obtain dry biomass of the aerial part and root of each plant, the corresponding material was placed in paper bags and then in a drying oven (Shel-Lab^®, model FX-5, series-1000203, USA) at 70 °C, until total dehydration was completed (72 h). Fresh and dry biomass was determined in an analytical balance (Mettler^® Toledo, model AG204, USA). A week later, a second sampling was performed following the same methodology.

Physiological Variables Assessed

Relative water content (CRA) was determined following the method of ^{Yamasaki and Dillenburg (1999)}, randomly selecting one plant per replicate, four per treatment. Leaves were taken from the middle section to minimize the effect that age could increase variability in the results. Leaf disks (1.3 cm) in diameter were extracted with a hole puncher obtaining their fresh weight (PF) and placing them in distilled water in closed Petri boxes; 24 h later, the leaf disks were dried superficially and without pressure with absorbing paper to obtain their turgid weight (PT). Subsequently, the leaf disks were placed in paper envelopes in the drying oven at 70 °C to determine their dry weight (PS) after 72 h. All the weight values of the vegetal material were obtained using a 0.0001 g precision analytical balance. The CRA was calculated by the following formula:

CRA = (PF-PS) / (PT-PS) × 100

Statistical Analyses

Analyses of variance (ANOVA) and Tukey’s honestly significant difference (HSD, P = 0.05) multiple comparison of means were performed. For all the variables, the average values were considered significantly different when P ≤ 0.05. For fresh and dry weigh of the aerial part and root fresh weight data were normalized with the equation: N = √x for fresh weight and N = √x+1 for dry weight where “x” was the value of the variable measure of each replicate. The statistical analyses were performed using Statistica model version 10.0 for Windows (^{StatSoft Inc., 2011}).

Aerial Part Fresh Weight (PFPA)

The chiltepin chili plants subjected to NaCl stress decreased the aerial part fresh weight PFPA (Figure 1a), which indicated that a high NaCl concentration caused a decrease in their nutrient absorption capability, reflected in a nutritional imbalance that affected plant development and growth (^{Batista-Sánchez et al., 2019}). These results were similar to those obtained by ^{Mazón-Suástegui et al. (2018)}, who reported a decrease in fresh biomass of the seedling aerial part of two basil varieties (Napoletano and Emily), as NaCl concentrations increased. The results obtained in the chiltepin plants treated with NaM made evident that PFPA was greater for the NaM-13CH treatment even when they were subjected to 200 mM of NaCl (Figure 1b).

Figure 1: Effect of the NaCl stress agent (a) and the homeopathic medicine NaM (b) on the fresh weight of the aerial part (PFPA) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

According to the analysis of factor interaction (NaCl × NaM), the plants treated with NaM-13CH and not subjected to salinity stress showed a greater value in the PFPA variable, which indicated greater biological (biomass) productivity. The greatest value of this same variable was also recorded in plants treated with Natrum muriaticum in the highest centesimal dilution (NaM-13CH), including in those that were subjected to a high salinity stress degree (200 mM of NaCl) although in a lesser degree of significance compared with those not stressed with NaCl (Figure 2). These results indicated a beneficial effect per-se on C. annuum L. var. glabriusculum plants of the most diluted homeopathic medicine (NaM-13CH). It was interesting because the nanoparticle presence of sea salt active principle (sodium, magnesium, manganese, and others) should be less in the 13^th centesimal dilution (NaM-13CH) than in the 7^th serial dilution of the same medicine (NaM-7CH). On the other hand, the serial dilution degree of NaM-13CH goes beyond the limits (1 × 10^-23) established by Avogadro’s Number since it is equivalent to one 26^th decimal dilution (1 × 10^-26), and at this dilution level, supposedly no molecule of the initial concentrated solution or “Mother Tincture (MT) exists. Nonetheless, several physical-chemical studies of homeopathic medicines in high dilution have established unequivocally the presence of the “active principle” nanoparticles contained in MT, including in very high centesimal dilutions, such as 30-CH and 200-CH (^{Chikramane et al., 2010}). Based on the previous, NaM-13CH should still contain molecules or nanoparticles of the respective MT, including magnesium, which is essential in forming chlorophyll, a molecule of great importance for photosynthesis, the most relevant process in plant biomass production. Additionally, Mg has an important participation in enzymatic activities associated to plant metabolism (^{Xiao et al., 2014}).

Figure 2: Effect of the NaCl × NaM interaction on the fresh weight of the aerial part (PFPA) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

In a recent study with basil (Ocimum basilicum L.) subjected to NaCl stress and treated with NaM-7CH and NaM-13CH, ^{Mazón-Suástegui et al. (2018)} also observed greater fresh biomass (PFPA) values, but the best results were obtained when NaM-7CH was applied. On the one hand, greater values in the PFPA variable were evident for chiltepin chili when NaM‑13CH was applied. This result could be explained because both species have different tolerance and requirements, which could also show among the varieties of the same species that react differently to stimuli caused by biotic or abiotic factors. On the other hand, the results obtained in both species confirm the validity of the homeopathic Law of Similars (“Similia Similibus Curentur”) because NaM is a homeopathic dilution of sea salt composed greatly by NaCl, which was the chemical agent used as salinity stressor. Moreover, sea salt contains a lesser proportion of salts of magnesium, manganese and many other compounds that have an important role in different plant physiological processes and reactions.

Fresh Root Weight (PFR)

The results showed significant differences in NaCl concentration (Figure 3a). Fresh root weight (PFR) was lower in plants subjected to salinity stress, which could have been due to NaCl inhibitor in high concentration (200 mM) that affected cellular division processes causing different metabolic problems by sodium toxicity and nutritional imbalance associated per-se to osmotic stress (^{Batista-Sánchez et al., 2019}). When the factor NaM was analyzed with its two dynamizations (7 and 13CH) and the control treatment (distilled water) without homeopathic medicine, the PFR values were greater in plants treated with NaM-13CH including those highly stressed with 200 mM of NaCl (Figure 3b).

Figure 3 Effect of the NaCl stressor (a) and the homeopathic medicine NaM (b) on the fresh root weight (PFR) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

The analysis of factor interaction showed a greater value in the PFR variable when the plants were treated with NaM-13CH even though they were subjected to NaCl stress conditions (Figure 4). The result can be explained in function of a compensatory increase associated to greater root length in response to NaCl stress and the interaction with original oligo-elements of NaM active principle since at greater cell division and elongation of the radicle system, a greater biomass of this organ is possible (^{Mazón Suástegui et al., 2018}).

Figure 4: Effect of NaCl × NaM interaction on fresh root weight (PFR) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum) subjected to NaCl stress. Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

Aerial Part Length (LPA)

The analysis of variance (ANOVA) showed significant differences in the aerial part length (LPA) for NaCl concentrations, which made a reduction of 80% evident in plants subjected to 200 mM of NaCl (Figure 5). Plant height is the result of nutrient assimilation and its cell division and elongation processes. A favorable response by salinity stress shows plant tolerance facing an interference in mineral, ionic toxicity, and in its photosynthetic process (^{Paellob, 2010}).

Figure 5: Effect of NaCl on the variable length of the aerial part (LPA) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum) subjected to saline stress. Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

The results obtained suggested that NaCl caused generalized nutritional disequilibrium and disorder in chiltepin chili plants, affecting physiological functions mainly due to the general imbalance derived from plant exposure to a high and stressful concentration of NaCl (200 Mm) in the nutritive medium.

Radicle Length (LR)

In both samplings, when the analysis of variance was performed independently, the radicle length variable showed significant differences for the NaCl treatments (Figure 6a). This result could be attributed to the direct affectation caused by Cl and Na ions at cellular level decreasing available water and increasing phytotoxicity, and consequently affecting cell division and elongation processes (^{Batista-Sánchez et al., 2017}). For the NaM treatments, the plants that received the dilutions 7 CH and 13 CH did not show differences (Figure 6b), but they did with respect to control (water, 0 NaM); this response may be caused by the oligo-elements contained in NaM active ingredients, which act favorably so the plants can develop physiological processes (^{Mazón-Suástegui et al., 2018}).

Figure 6: Effect of the NaCl stressor (a) and the homeopathic medicine NaM (b) on the root length (LR) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

Leaf Area (AF)

The leaf area showed significant differences among NaCl concentration with the lowest average AF values in the plants subjected to 200 mM of NaCl (Figure 7). One of the deleterious effects of salinity stress is the decrease in water absorption capacity, which affects the plant mechanism directly, reducing leaf expansion and causing turgidity loss in stem and leaf (Rosales-Nieblas, 2018¹). This same author worked with Chenopodium ambrosioides L., commonly known as “epazote” or wormseed plant subjected to NaCl stress in a hydroponic system and reported affectation in the leaf area at 100 mM of NaCl. In this study, chiltepin chile plants showed a reduction in leaf number and a degree of general plant withering when subjected to a concentration twice greater (200 mM) of NaCl in the hydroponic cultivation. This result indicates that C. annuum is a more tolerant species to salinity than Ch. ambrosioides L.

Figure 7: Effect of sodium chloride (NaCl) on the AF of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

The leaf area (AF) showed significant differences in NaCl × NaM interaction with average values signif icantly greater in plants that were not stressed with NaCl but were treated with NaM-13CH (Figure 8). On the contrary, the average value of AF in the plants subjected at 200 mM of NaCl was much lower than those of the plants not subjected to salinity stress. In the plants stressed with NaCl, no significant differences were recorded among treatments; however, the average value of AF was slightly greater in those treated with NaM-13CH with respect to NaM-7CH and the control group (Figure 8). These results suggested that NaM homeopathic medicine, developed by serial dilution/succussion of a sea salt MT, whose main component is NaCl, acted precisely to attenuate salinity stress (200 mM of NaCl) in chiltepin chili (Capsicum annuum L. var. glabriusculum) plants. These results corroborate and validate a philosophical and conceptual basis (“similia similibus curentur”), of homeopathic medicine (^{Mazón-Suástegui et al., 2019}).

Figure 8: Effect of NaCl × NaM interaction on the leaf area (AF) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

Aerial Part Dry Weight (PSPA)

The chiltepin chili plants subjected to concentrations of 200 mM of NaCl recorded effects on aerial part dry weight (PSPA) with a decrease of 90% with respect to those that did not receive a salinity treatment by adding NaCl (Figure 9). These results agree with ^{Pio et al. (2001)}, who asserted that dry matter decreased proportionally as salinity levels increased in five vine rootstocks. ^{Taffouo et al. (2009)} also studied the salinity effect on Vigna unguiculata L. Walp. plants and reported that dry weight reduced notably starting from 50 mM of NaCl, due to osmotic stress and that of the specific Cl^- and Na⁺ ions that reduce the osmotic potential of the solution, and in consequence, water availability for the plant.

Figure 9: Effect of sodium chloride (NaCl) on dry weight of the aerial part (PSPA) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

Root Dry Weight (PSR)

The root dry weight (PSR) showed significant differences among NaCl concentrations. This variable was reduced in plants subjected to 200 mM of NaCl without the application of homeopathic medicine NaM (Figure 10a). The decrease of PSR is attributed to the presence of NaCl in the nutritive medium since in high concentrations this salt is toxic for the plant, causing a nutritional imbalance and metabolic disorder that reduce plant tissue growth (^{Batista-Sánchez et al., 2017}). Additionally, significant differences were found between NaM dynamizations applied (Figure 10b), finding greater value in this variable in plants that received 13CH of NaM dilution. In the interaction NaCl × NaM analysis (Figure 11), the PSR was greater in plants treated with 13CH of NaM and without salinity stress; in those subjected to 200 mM of NaCl no significant differences were observed, but the best result corresponded to NaM-7CH (Figure 11). The results suggest that NaM-7CH and NaM-13CH has anti-salinity stress properties that favor growth and cellular elongation in chiltepin chili C. annuum L. var. glabriusculum, confirmed with that reported for other important commercial species, such as basil and bean (^{Mazón-Suástegui et al., 2018}; ²⁰¹⁹).

Figure 10: Effect of the NaCl stressor (a) and the homeopathic medicine NaM (b) on the dry root weight (PSR) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

Figure 11: Effect of NaCl × NaM interaction on dry root weight (PSR) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

Relative Water Content (CRA)

Significant differences were found with respect to factor A, making a decrease in relative water content (CRA) evident in plants subjected to 200 mM of NaCl (Figure 12a). Rosales-Nieblas (2018¹) mentioned that CRA reduced in wormseed plants developed under salinity stress conditions when the plants were subjected to 100 and 150 mM of NaCl. This results confirmed that water mobility decreased in salinity conditions and the plants were compromised to live in physiological drought conditions, including death of plant tissue. When the effect of the homeopathic treatments was analyzed, the CRA increased significantly in plants treated with dilutions 7 and 13 CH, which was greater for 7CH (Figure 12b).

Figure 12: Effect of the NaCl stressor (a) and the homeopathic medicine NaM (b) on the relative water content (CRA) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05). 

The interaction analysis of CRA showed significant differences in factor B where plants treated with NaM-7CH had the highest values, including in stressful salinity (200 mM) conditions (Figure 13). The non-stressed plants with NaCl and treated with NaM-7CH showed greater CRA values, followed by those that received leaf application of NaM-13CH. The plants subjected to salinity stress and without homeopathic medicine showed lower values compared with the rest of the treatments. The results of this study in chiltepin chili plants suggested that NaM homeopathic medicine could have a positive effect in the plant favoring water retention in the plant tissue subjected to salinity conditions. These results are not sufficient to know the precise mechanism of action of these homeopathic medicines of human use in plant physiological functions. However, the evidence cause-effect derived from this research contributes to bases for new studies in depth to help understand the specific way of the possible metabolic routes involved with the action of NaM active ingredient in plants.

Figure 13: Effect of the NaCl × NaM interaction on the relative water content (CRA) of chiltepin chili plants (Capsicum annuum L. var. glabriusculum). Bars with equal letters do not differ statistically (Tukey HSD P ˂ 0.05).