Introduction

Legumes (family Fabaceae) are very important basic food for human population and an essential source of proteins, vitamins, minerals, and fiber (^{Bellucci et al., 2014}). The common bean Phaseolus vulgaris L. is cultivated worldwide; it is one of the most important grain legumes for human nutrition, constituting 50% of consumption at world level (^{Broughton et al., 2003}). Nonetheless, a decrease in production of this important cultivation has been currently reported due to the presence of unfavorable abiotic and biotic factors (^{Rodríguez et al., 2009}; ^{Barrios et al., 2011}).

Soil salinity is one of the most important global problems that affect agricultural productivity negatively. Salinity affects plant growth and development by different effects associated to hydric stress and cytotoxicity deriving from an excessive absorption of some ions, such as sodium (Na⁺) and chloride (Cl⁻), which cause a nutritional disequilibrium in the plant. These harmful salinity effects tend to go together with oxidative stress due to the generation of reactive oxygen species (ROS) (^{Tsugane et al., 1999}; ^{Hernández et al., 2001}; ^{Isayenkov, 2012}).

Approximately 800 million ha of land, equivalent to more than 6% of the total global area of the planet are affected by soil salinity (^{FAO, 2008}). The excess of salts limits plant growth and productivity (^{Khan and Panda, 2007}) and inhibits growth in a very important manner because it affects essential metabolic processes, such as photosynthesis (^{El-Hendawy et al., 2005}), protein synthesis and enzymatic activity (^{Liang et al., 2005}; ^{Sadak and Abdelhamid, 2015}). Additionally, salinity affects transpiration, cell growth and particularly seed germination, which is the most sensitive to this stressing agent that affects water absorption and hinders seed hydration, affecting initial plant development in this manner (^{Meloni et al., 2004}).

Different research studies have been performed searching for alternatives to attenuate negative effects of salinity (NaCl), among which those found are obtaining tolerant varieties by complex and costly genetic techniques (^{Pedroza-Sandoval et al., 2016}), and the use of natural products, such as biofertilizers and biostimulants that favor plant growth, development, and yield (Álvarez, 2014¹). In the last years, alternative and eco-friendly experiments have been performed, promoting the evolution of the agricultural sector toward a sustainable agriculture in the medium and long terms. To achieve this purpose implies developing new options that allow cultivating soil with acceptable levels of productivity and economic yield, reducing harm to the environment to its possible maximum (^{Singh et al., 2011}). The use of homeopathic medicine is feasible in comparison with other most costly and harmful alternatives for the environment since they are highly diluted, effective and required in a very small amount (^{Sen et al., 2018}). The use of agricultural homeopathy has been increasing because of its innocuousness and tested efficiency in plant growth promotion (^{Mazón-Suástegui et al., 2018}). Moreover, the application of homeopathic medicines with registry in the Health Ministry of Mexico (^{SSA, 2015}) for use in humans, have also proven to stimulate plant growth and development even in abiotic stress conditions (Pereira et al., 2012; ^{Mazón-Suástegui et al., 2019}). Thus, agricultural homeopathy could be an excellent alternative to mitigate salinity stress conditions (^{Sen et al., 2018}). Taking this background into account, the objective of this study was to assess the attenuating effect of the homeopathic medicine for human use Natrum muriaticum (NaM) on salinity stress (NaCl), photosynthetic rate and morphometric variables of the common bean (Phaseolus vulgaris L.) variety white testa Quivican during initial plant growth stage.

Materials and Methods

Results and Discussion

The results of the analyses revealed a direct action of NaCl in all the studied variables (Table 1) since with a concentration of 75 mM of NaCl, which is considered as moderate salinity (^{Batista-Sánchez et al., 2017}), P. vulgaris plants experimented a significant decrease in LT (P = 0.04), BFR (P = 0.02), BSH (P = 0.00002), BST (P = 0.00004), AF (P = 0.0000) and in the number of leaves (P = 0.0000).

Salinity reduces plant growth by affecting its physiological processes, including the interruption of ionic equilibrium, mineral nutrition, stomatal response, and photosynthetic efficiency (^{Garzón and García, 2011}). Disparity in osmotic potential leads to water deficit, a reduced foliar area expansion and stomatal closing, which carries photosynthesis reduction and plant growth (^{Roy et al., 2014}). Ionic disequilibrium provokes an excessive accumulation of Na⁺ and Cl⁻ in the oldest leaves; this situation leads to premature senescence (^{Roy et al., 2014}) that generates an ionic disequilibrium, reducing capture of beneficial and essential ions for the development of physiological processes, such as K⁺, Ca²⁺ and Mn²⁺ (^{Hasegawa et al., 2000}), decreasing photosynthesis and enzymatic activity (^{Muchate et al., 2016}). According to ^{Távora et al. (2001)} the most notable harmful salinity effect on plants, besides altering osmotic potential, is toxicity and lack of equilibrium in nutrient absorption, which causes a generalized decrease in growth in plants stressed by salinity.

The results obtained in this study agree with those reported by ^{Can-Chulim et al. (2017)} who demonstrated that fresh and dry shoot biomass decrease of the common bean P. vulgaris is attributed to salinity stress that causes a reduction in water absorption. In this study, salinity caused by NaCl (75 mM) reduced fresh root biomass in 8.2%. This tendency was very clear and has already been observed for the same species (P. vulgaris L.) in other recent studies (^{Radi et al., 2013}; ^{Habtamu et al., 2014}).

The number of leaves per plant reduced in bean plants subjected to salinity stress by addiction to NaCl, compared with the plants in the control group (untreated). This result could have been associated to a difference in potassium, which agrees with ^{Murillo-Amador and Troyo-Diéguez (2007)} who reported that salinity induced to a reduction in the number of leaves in bean Yorimon (Vigna unguilata (L.) Walp) plants subjected to salinity stress.

While analyzing the interaction of the homeopathic medicine NaM and the stressor agent NaCl (NaM × NaCl), P. vulgaris plants without induced salinity stress (0mM de NaCl) and with the homeopathic NaM-7CH treatment were observed experimenting a significant increase of LR (P = 0.00509), BFR (P = 0.00019), BFH (P = 0.0065), BFT (P = 0.00002), BSR (P = 0.0188), BSH (P = 0.032), BST (P = 0.003) and AF (P = 0.033). These results mean the medicine has a favorable effect per se in P. vulgaris plants, so efficiency and productivity of the common bean P. vulgaris could increase with its application.

On the other hand, when the plants were subjected to salinity stress by NaCl without NaM homeopathic medicine, the response markers assessed LR, BFR, BFH, BFT, BSH, BST and AF decreased 8.04, 7.87, 55.55, 38.25, 6.0, 41.66 and 49.68%, respectively. These results confirm the favorable effect per se that NaM-7CH homeopathic treatment has. An additional result that ratifies the previous information is that plants subjected to salinity stress by adding NaCl that were provided with NaM-7CH and NaM-7+13CH homeopathic treatments (Table 2), all the response variables (except for BST and AF), showed an increase with respect to the plants in the control group (AD). These results agree with those obtained by ^{Lensi et al. (2010)} who demonstrated the efficiency of NaM (NaM-6CH and NaM-30CH) homeopathic dynamizations in common bean (P. vulgaris L.) plants after showing no signs of toxicity during their growth stage. The mechanism of action of the homeopathic medicines can imply physiological changes that conduce to the formation of secondary metabolic products related with the defense mechanism of the treated plants (^{Lensi et al., 2010}). According to these authors, bean plants treated with NaM-6CH showed a greater growth when compared to plants of the control group; in general, a low dilution of NaM-6CH showed to be more effective than a high dilution of NaM-30CH since no significant results were obtained in these treated plants when compared to non-treated in the control group. In agreement with these results, ^{Siqueira et al. (2010)} proved that the use of NaM promoted a significant plant growth, especially in the bean plants treated with NaM-6CH, whose application could be associated to a greater Relative Growth Rate of this cultivation.

In a research study ^{Mondal et al. (2012)} obtained an increase in seed germination of Yorimon Vigna unguiculata bean plants treated prophylactically with NaM and then subjected to salinity stress with NaCl; the biochemical analysis of the plants showed an increase in total protein content, total chlorophyll and sugar. In this study, P. vulgaris L plants revealed different favorable responses to the prophylactic application of NaM, confirming its preventive-protector effect in cultivated plants under salinity stress by addition of NaCl. These results can be explained in function of the oligoelements contained in the active ingredient of NaM that could have been acting as stimulators of physiological processes of the plants treated even when they were under salinity stress conditions by NaCl (^{Mazón-Suástegui et al., 2018}).

Other researchers as ^{Jaski et al. (2016)} performed a study in bean plants applying essential oils of Eucalyptus citriodora and E. globulus and homeopathically prepared, demonstrating growth stimulation in hypocotyl and radicle length, confirming the potential of homeopathic preparations to stimulate initial development of bean plants. ^{Bonato et al. (2009)} applied the homeopathic medicines Sulphur and Arsenicum album (dynamizations 6, 12, 24 and 30CH), for human use, in mint (Mentha arvensis L.) plants and recorded increases in plant height, fresh and dry biomass, concluding that homeopathic preparation can promote physiological changes in plants, such as greater growth and disease resistance. In this study, with the common bean P. vulgaris variety Quivican plants, no significant differences were observed in the variable FRB among the plants that received the treatments NaM-7CH and NaM-7+13CH under salinity conditions (75 mM of NaCl); nonetheless, these treatments showed differences with respect to the plants that received NaM-13CH. These finding can be explained because NaM-13CH dynamization has a smaller amount of nanoparticles of the active ingredient which could decrease the capacity of this homeopathic dynamization to induce measurable responses in the treated plants.

In this study a decrease in TF was observed when P. vulgaris, variety Quivican plants were subjected to stress (Figure 1). Salinity is known to affect the photosynthesis process in land plants, causing a decrease in their growth parameters (^{Pérez et al., 2014}). Photosynthesis decrease under salinity conditions is not only attributed to stomatal closing that leads to a reduction of intercellular CO₂ concentration but also to non-stomatal factors since evidence exist that salinity affects photosynthetic enzymes, chlorophyll and carotenoids (^{Stepien and Klbus, 2006}). Growth reduction of plants under salinity stress is attributed to photosynthetic rate, modification of carbohydrate metabolism, and to its subsequent distribution in the organism (^{Argentel et al., 2009}). ^{Taffouo et al. (2009)} reported that salinity reduced the concentration of K+, the relationship K/Na, plant length, foliar chlorophyll content, and photosynthetic activity in different bean (Vigna unguiculata L. Walp.) cultivars.

Figure 1: Effect of Natrum muriaticum on the photosynthetic rate of Phaseolus vulgaris plants subjected to stress by NaCl. 

Among the groups of treated plants, the one with greater TF was that with the NaM-7CH treatment in salinity stress (75 mM of NaCl) conditions, whose plants experimented an increase higher than 50% in this variable (Figure 1), compared with the control group (AD). These results can be justified because NaM-7CH contains nanoparticles of the active ingredient (sea salt), which is formed by oligoelements, such as magnesium, essential for the chlorophyll molecule that plays a priority role in photosynthesis (^{Mazón-Suástegui et al., 2018}). In a similar research study in bean (Vigna unguiculata L. Walp.) plants subjected to salinity stress and treated with the homeopathic medicine Sepia sucus for human use, positive responses were obtained, such as a significant increase in growth, content of sugar, chlorophyll, proteins and relative content of water in comparison to the control group without homeopathy (^{Sukul et al., 2012}).

In general, the salinity condition to which bean plants were subjected revealed a decrease in photosynthesis, which was reflected on the morphological variables assessed. Nevertheless, P. vulgaris revealed tolerance to salinity since it generated morphological responses, avoiding a drastic decrease in its initial growth stage. These and other results previously described suggest that homeopathic medicine has a great potential as prophylactic treatment to prevent damage caused to the plant by salinity stress. However, the results obtained in the morphometric variables of the plants treated with NaM demonstrated that the benefits of its application are even greater in the absence of a stressing agent as NaCl. By means of autoregulation, plants are known to respond intensely to homeopathic medicine in normal or stress conditions (^{Casali, 2004}) and that sustainable agriculture and homeopathic agriculture are compatible (^{Mazón-Suástegui et al., 2019}), which allows at the same time maintaining or improving the quality of the environment and conserving natural resources (^{Lisboa et al., 2005}).

The selection of the homeopathic medicines used to increase tolerance to abiotic stress in plants (mainly toxicity by metals and salt stress) is relatively simple because the denominated “Principle of Similars” (Similia Similibus Curentur: ’Let Likes Be Treated By Likes’) has demonstrated that it is one of the philosophical and conceptual cornerstones of homeopathy with a positive application in agriculture. This principle confirms the “diseases can be cured with something that induces the same symptoms of the disease itself “ (^{Sen et al., 2018}). During the development of this study a medicine used for humans (NaM) was applied, which was made from marine salt, precisely to attenuate the stress caused by the addiction to sodium chloride, main sea salt component. The results allowed hypothesizing that plants treated prophylactically with NaM may develop tolerance to NaCl because the active principle of the medicine could activate cellular mechanisms (still needs further study), capable of sending resistance to salinity signals to the plant before being exposed to a stressing agent. Based on the experimental results obtained, the application of MaM in common bean (Phaseolus vulgaris L.) variety white testa Quivican plants could generate a physiological response to minimize the negative effects of salinity stress by NaCl.

Conclusions

The treatment of the common bean P. vulgaris plants with NaM homeopathic medicine treatment increased the morphometric variables and Photosynthetic rate (TF), favoring a reduction of the stress effects caused by salinity (NaCl). Greater values of all the response variables assessed were obtained with the applications of NaM-7CH and NaM-7+13CH treatments, with a greater impact in TF. Both treatments included the centesimal dilution 7CH and were more efficient than higher (13CH) dilution. Agricultural homeopathy has demonstrated its value as an eco-friendly alternative for agriculture by reducing the negative effects of salinity stress without the use of chemical treatments that leave harmful residues in air, soil, and water. In general, the results obtained in this study suggest that the use of NaM has a great potential in sustainable organic agriculture, and its application may contribute to increase the biological productivity of P. vulgaris.