Introduction

Beans are a greatly important cultivation worldwide both as grain legume and vegetable. The greatest part of their production takes place in developing countries, and they are one of the cheapest main sources of calories, proteins, dietary fiber, vitamins and minerals for human consumption in those countries (^{Hillocks et al., 2006}). Beans are supplements of cereals and other food rich in carbohydrate to provide an almost perfect nutrition for people of all ages besides decreasing cholesterol and the risk of cancer (^{Muhamba and Nchimbi, 2010}). Archeological studies have revealed that beans originated in the American continent since 500-8000 year-old evidence exists in some regions of Mexico, the United States and Peru. A relative agreement exists with respect to the fact that beans originated in Mexico and from there the first seeds spread out toward the southern part of the American continent because it is possible to find wild prototype species of the five most cultivated ones of the genus Phaseolus. Nevertheless, other sources estimate that this legume was domesticated and cultivated before the Pre-Columbian era (^{García, 2005}), which through natural selection different types of seeds generated (^{Debouck and Hidalgo, 1985}). Such was the importance acquired by beans at that time, that Aztec civilization included them in the list of articles that should be collected as tribute from other tribes less powerful for taking advantage of the natural resources or the sites where these tribes were established or inhabited within the limits of the Aztec Empire (^{Voysest, 2000}).

Beans are a key cultivation in the national Mexican diet, but despite their high consumption and having experimented a slight increase in 2017 with respect to the previous year (^{SIAP, 2018}), currently, its production does not supply the levels of demand in the country. The low productivity of this important cultivation is attributed to adverse environmental factors, but predominantly to the presence of pest-organisms and diverse diseases associated to bacterial and viral pathogens that do not allow them to reach their productive potential (^{Bolaños et al., 2009}). As a result of the biotic and abiotic factor synergy, currently, this millennial cultivation does not produce the expected yield to supply its high demand, which has increased the need of searching for alternatives to overcome these limitations and take advantage of its great potential.

To date, research aimed at this objective has focused on biofertilizers to make their vegetative cycle more efficient, reducing inorganic fertilization with agrochemicals and the negative impacts their use implies affecting the environment (^{Estrada-Prado et al., 2018}). Additionally, studies have been performed on the use of green fertilizer and associations with other cultivations to improve soil fertility, nutrient content and their effect in biomass production (^{Rivero-Herrada et al., 2016}). Another line of research with growing tendency in the last years is the application of natural bio-stimulants with the capacity of strengthening plants and achieving greater productions (^{Moreno-Lorenzo et al., 2018}; ^{Calero-Hurtado et al., 2019}). Finally, an eco-friendly alternative of recent application and a high possibility of success due to the innocuousness of its products and economic viability is the application of homeopathic medicines authorized for use in humans with registration in official institutions, such as the Health Ministry of México (^{SSA, 2015}). These highly diluted and low-cost medicines, have proven the capacity of inducing biological responses that favor growth and development of diverse plant species (^{Mazón-Suástegui et al., 2018}, ²⁰¹⁹), allowing to obtain more vigorous plants with greater yield in biomass and thus with greater potential and economic feasibility in their cultivation. Scientific evidence of using homeopathic medicines in plants and their beneficial effects on stimulating growth and biomass production has been based on the direct action of the active ingredients on the physiological processes of the treated plants (^{Meneses-Moreno, 2017}). Moreover, homeopathic medicines have demonstrated to strengthen plants allowing a better development, including in unfavorable environments, such as those producing abiotic and biotic stress (^{Oliveira et al., 2014}; ^{Mazón-Suástegui et al., 2018}). Therefore, the objective of this study was to assess the effect of homeopathic medicines in physiological markers in “Yorimon” cowpea bean (Vigna unguiculata L., Walp) variety Paceña.

Materials and Methods

Research was performed in the Campo Agrícola Experimental (CAE, Experimental Agricultural Field) of Centro de Investigaciones Biológicas del Noroeste (CIBNOR) located northwest of the city of La Paz, Baja California Sur, México, at 24° 08’ 10.03” N and 110° 25’ 35.31” W, at 7 m altitude. The experimental study was performed in a semi-controlled environment under a metallic structure completely covered with a white 30% shade anti-aphid mesh, with a minimum, average, and maximum temperatures of 14.25±3.83, 27.64±4.01, 45.17±2.94 ºC, respectively, with relative humidity of 34.8 ± 5.07%. These climatological data were recorded with portable climatologic station (Vantage Pro2® Davis Instruments, USA) within the area of study. A completely randomized design was applied with two homeopathic treatments, Manganum metallicum 31CH (MnM-31CH), Magnesium-manganum phosphoricum 3CH (MaMnP-3CH), and distilled water (AD) as control treatment with six replicates per treatment. Seeds were disinfected previous to sowing with a 1.5% sodium hypochlorite solution, and a germination assay was performed according to the methodology of the International Seed Testing Association (^{ISTA, 2010}).

Seeds were sown in plastic pots with 1 L capacity of inert commercial substrate (Sogemix PM^®); daily irrigation was applied until substrate saturation with water was reached and nutritional solution calculated and adjusted for the cultivation of V. unguiculata, based on the manual instructions for nutritional solutions of ^{Samperio (1997)}. Homeopathic dilutions (MnM-31CH, MaMnP-3CH) and the control (AD) treatments were sprayed on leaves every other day during all the experiment (total of 50 days). Measurements started at day14 of sowing; photosynthetic and transpiration rates were taken from leaves completely turgid and healthy in sunny days and at the time of greater solar radiation (two times a week) using an IRGA equipment (LCpro-SD Portable Photosynthesis System) that included a broad-leaf camera (ADC, Hoddesdon, Herts, UK). Chlorophyll determination was performed on each plant taking samples from fresh and totally expanded leaves, previously cleaned with deionized water to remove any environmental contamination on their surface. For the extraction of photosynthetic pigments, 1 g of fresh material per sample was taken, which were ground in a mortar with 90% acetone, and subsequently left to settle in darkness for 72 h before measuring absorbance, which was read at 663 and 645 nm in wavelength with an UV/Visible spectrophotometer (Pye Unicam SP6-550, UK).

At 50 days after sowing, foliar area was assessed (cm²) with a measuring equipment (Li-Cor^®, model-LI-3000A, series PAM 1701-USA). Fresh biomass (g) was determined by root (BFR), stem (BFT) and leaf (BFH) weight in an analytical balance (Mettler Toledo^®, model AG204 USA). To obtain dry biomass, all the fresh material was placed separately (root, stem, and leaf) in paper bags and introduced in a drying stove (Shel-Lab^®, model FX-5, series-1000203) at 70 °C for 72 h for total dehydration. At the end of this process, they were weighed in an analytical balance (Mettler Toledo^®, AG204) expressing values in grams. An analysis of variance (ANOVA) was performed, and Tukey’s HSD multiple comparison of means was applied when significant differences P ≤ 0.05 were found among treatments, using Statistica v. 10.0 program for Windows (^{StatSoft, Inc, 2011}).

Results and Discussion

The results of the total chlorophyll and photosynthetic rate analyses showed significant differences (P = 0.02) among treatments with increase in photosynthetic pigment concentration in plants that received homeopathic treatments. This difference was higher than 90% in plants that received Manganum metallicum 31 Centesimal Hahnemanian dynamization (MnM-31CH) and 65% in those treated with Magnesium-manganum phosphoricum 3 Centesimal Hahnemanian dynamization (MaMnP-3CH), compared to those that received distilled water (AD) as control treatment (Figure 1A). The photosynthetic rate of the plants treated with homeopathic medicines increased in 71.6% for MnM-31CH and in 42.85% for MaMnP-3CH, compared with the AD control treatment (Figure 1B).

Figure 1: Effect of homeopathic medicines on the germination percentage of Salicornia bigelovii. Different letters show significant statistical differences (P = 0.05). 

If a traditional criterium is applied, these results could be explained by the presence of nanoparticles of the active ingredient in the homeopathic medicine applied, which could have a bearing on plant physiological processes, stimulating their growth and development even in adverse conditions (^{Mazón et al., 2018}). In the case of the MnM-31CH treatment, its active ingredient in the initial concentration or “mother tincture” from which it originated, is metallic manganese. These element is, after iron, one of the most demanded micronutrients by plants because it contributes to the function of various biological processes, including photosynthesis, respiration and nitrogen assimilation besides having an important role in protein synthesis (^{Teixeira et al., 2004}; ^{Prato and Gómez, 2014}). On the other hand, the MaMnP-3CH includes manganese and also magnesium, which plays an important role in forming chlorophyll molecules and favor green pigment concentration (^{Cakmak and Yazici, 2010}). At a greater photosynthetic pigment concentration, a greater absorption of luminous energy will exist, which in turn translates into greater efficiency of the photosynthetic process since more chemical energy is generated guaranteeing greater biomass accumulation and facilitating more vigorous plant production (^{Guo et al., 2016}).

As Figure 2 shows, significant differences were recorded among treatments for the foliar area (P = 0.036) and transpiration (P = 0.04), noting an increase in foliar area when bean plants were sprayed with the homeopathic medicines (MnM-31CH and MaMnP-3CH). The transpiration in treated plants with MnM-31CH reached the greatest value, followed by those that received MaMnP-3CH and the control treatments with less transpiration. Such results suggested that the homeopathic dynamizations used stimulated cell division and elongation in leaves of the treated plants, achieving in this manner greater foliar area with less transpiration. This response could have been associated to the direct action of the active ingredients of the homeopathic medicines used, which contained Mn²⁺ and Mg⁺² that play an important role in protein synthesis and nitrogen assimilation (^{Guo et al., 2016}). We can assume that transpiration was greater in plants that received MnM-31CH and MaMnP-3CH because of a greater availability of liquids in the inner part of the tissues, which could be related to an increase in fresh root biomass (Table 1). According to ^{Niu et al. (2014)} manganese, included as homeopathic ingredient in MnM-31CH and MaMnP‑3CH, boosted lateral root formation, allowing plants to achieve greater water and nutrient absorption given it increased the possibility of exploring greater soil area, which benefitted its growth and development.

Figure 2: Effect of homeopathic medicines in the foliar area (A) and in the transpiration (B) of Vigna unguiculata L., Walp. Different letters show significant statistical differences (Tukey’s HSD, P = 0.05). 

The analysis of the results showed significant differences among treatments for fresh and dry root, stem and leaf biomass (Table 1), observing and increase of 111.2 and 96.36% respectively, in the BFR variable for plants treated with MnM-31CH and MaMnP-3CH, compared with those not treated homeopathically (AD). The increase for the BSR variable in plants treated with homeopathy was higher than in those not treated in the control group (AD) making a better response more evident when plants were treated with homeopathic metallic manganese (MnM-31CH). No significant differences were observed among the homeopathic treatments (MnM-31CH and MaMnP-3CH) with respect to BFT and BST. Nonetheless, both treatments showed significant differences (P ≤ 0.05) in these variables with respect to the control group (Table 1). Fresh leaf biomass was significantly higher in plants treated with homeopathic medicine, reaching an increase of 65.5% for MaMnP-3CH, with respect to the control AD treatment. For the BSH the response was similar but with an increase of 56% in plants that received MaMnP-3CH. The increase in fresh and dry biomass observed in plants treated with homeopathic metallic manganese (MnM-31CH) and homeopathic magnesium-manganese phosphate (MaMnP-3CH), confirmed a positive response to the treatments since magnesium and manganese were key elements during the development of the biological processes responsible for biomass productions, such as photosynthesis, respiration and protein synthesis (^{Ceppi et al., 2012}).

Other authors have obtained similar results in the common bean Phaseolus vulgaris cultivation in biomass production when they have applied homeopathic medicines whose active ingredients have shown similar action in the physiological markers of the plants treated (^{Ruiz et al., 1997}). In the cultivation of Allium fistolosum a significant increase was observed in fresh weight when plants were treated with homeopathic medicines (^{Sánchez and Meneses, 2011}). Similar results were observed by ^{Mazón-Suástegui et al. (2018)} when Natrum muriaticum 7CH and 13CH were applied to basil Ocimum basilicum cultivation obtaining and increase in biomass although plants were subjected to stress by NaCl. The results set out in this research demonstrated that agricultural homeopathy is a viable alternative with the potential of achieving more biological production, mainly in future organic agriculture.

Conclusions

The two homeopathic medicines applied increased photosynthetic rate, total chlorophyll, foliar area, transpiration and fresh and dry root, stem, and leaf biomass significantly in “Yorimon” cowpea bean (V. unguiculata) variety Paceña. The best response (increase of 111.2% in BFR) was achieved with the treatment based on a high homeopathic dynamization (31 Centesimal Hahnemanian dynamization) of metallic manganese (MnM-31CH). These results confirmed that agricultural homeopathy can be a low cost and viable alternative to increase eco-friendly biomass production and financial feasibility of V. unguiculate cultivation with a positive impact on Latin American agricultural sector.