Antimicrobial action against of Macrophomina phaseolina and control of the grey stem in soybean by homeopathic remedies Nosode and Sulphur

Antimicrobial action against of Macrophomina phaseolina and control of the grey stem in soybean by homeopathic remedies Nosode and Sulphur Eloisa Lorenzetti*, José Renato Stangarlin, Elizana Lorenzetti Treib, Anderson Luis Heling, Sidiane Coltro-Roncato, Jeferson Carlos Carvalho, Lívia Hoepers, Bruna Broti Rissato, Jéssica Caroline Coppo, Cristiani Belmonte, Odair José Kuhn and Itamar Ferreira da Silva


INTRODUCTION
Brazil is the second largest producer of soybean in the world after the United States, with a production of 98,981.600tons (Conab, 2016).Over the years and with the increase in cultivated soybean area, is the emergence of diseases that impair productivity.Botrytis stem caused by the fungus Macrophomina phaseolina is one of the most important disease, It affects the roots and lower part of the stem; it first causes discoloration and then a dark brown or black coloration.When the weather is favorable, that is, periods of drought, heat and very high temperature, the disease can attack young plants.In mature plants, the infection usually occurs from the middle of the cycle (Almeida et al., 2014).Due to the degree of dangerousness of the disease, it is necessary to control it, which is based on the practice of direct planting and vegetation cover, in order to avoid water stress; chemical and physical soil management; rigorousness at the time of planting; use of recommended varieties for the region; promoting unsuitable environment for the fungus (Almeida et al., 2014), as well as alternative methods of control, such as homeopathic treatment.
Homeopathic medicine is all pharmaceutical form intended to be administered according to the principle of similarity, with preventive and therapeutic purposes, obtained by the method of dilutions followed by succussions and/or successive grindings.The succussion consists of vigorous and rhythmical agitation of dissolved drugs in an appropriate inert ingredient and the boosting results from the process of dilutions followed by succussions and/or successive grindings of the drug, with the purpose of medical power development (Farmacopéia homeopática brasileira, 2011).According to Carneiro et al. (2011), alternative methods of agriculture are modern methods, developed in sophisticated and complex system of agricultural techniques, whose main objective is not the economic exploitation short sighted and inconsequential, but the economic exploitation in the long term, maintaining stable and self-sustaining the agricultural ecosystem.Therefore, this study aimed to develop an alternative method through homeopathy to control M. phaseolina in soybean; check the antimicrobial activity of the homeopathic remedies Nosode of M. phaseolina and Sepia against M. phaseolina; and control stem botrytis in soybean plants by using these drugs.

MATERIALS AND METHODS
For this study, two experiments were performed in vivo and in vitro.For the in vivo test, the isolated M. phaseolina was put in rice medium and liquid medium PD (potato dextrose); and three discs of the M. phaseolina colony grown in Petri dish with 1 cm diameter.Containing what media was added to PDA medium (potato, dextrose and agar).For the in vivo test of the second experiment, the isolated M. phaseolina was put in petri dishes (0,1 cm diameter) with PDA medium and after the fungi present micro-sclerotia, medium with mycelial disks and micro-sclerotia were used for inoculation of the soybean plants in the pots.In both trials, the soybean seeds used in the in vivo experiment were from the cultivar NK 412113 (V-MAX convencional).In both trials the following homeopathic medicines were used: Sulphur and Nosode of M. phaseolina, the first being a homeopathic polychrest and the second starting the healing principle by the similar.The Sulphur Lorenzetti et al. 3413 was acquired in homeopathic pharmacy in the 6CH boosting and manipulated to 12, 24, 36 and 48CH as described by the Brazilian Homeopathic Pharmacopoeia (2011), diluted 1: 100 and sucussioning 100 times.The Nosode of M. phaseolina was obtained from mycelium grown on potato dextrose liquid medium (PD), which was allowed to stir in orbital shaker at 150 rpm for 7days, after filtration was carried out of the the fungus to separate all the culture medium.Three grammes (3 g) of the fungus (M.phaseolina) was placed in amber glass together with 27 ml of 70% grain alcohol with the glass encased by aluminum foil and left at rest for 21 days, stirring only once a day in a soft way, producing the mother tincture (Bonato, 2007) and through this, were manipulated as the Brazilian Homeopathic Pharmacopoeia (2011).
The dynamizations used were the same of Sulphur, that is to say 6, 12, 24, 36 and 48CH.
As additional treatments, we used distilled water and hydroalcoholic solution of 30% ethanol.Water and ethanol were used because they are solvents in the preparation of homeopathic drugs.
To determine the antifungal activity, we used medicines in their due boosting, being incorporated into the culture medium (PDA) still liquid and hot in the Erlenmeyer flask.0.005% and 5 uL of treatment were used to each 100 ml of PDA culture medium (Bonato, 2007).After, the PDA medium was poured and complete solidification was expected.It was added in the center of each petri dish a disc, removed from the fungal colony, to grow.The dishes were still incubated at room temperature in the dark.Daily measurements were initiated 24 h after the beginning of the experiment and ended when the fungal colonies reached the edges of the petri dish.Such measurements were performed according to the methodology described by Stangarlin et al. (1999) through the method of diametrically opposed measures to evaluate the diameter of the colony.At the end of the daily measurements for determining the area under the mycelial growth curve (AUMGC), we pulled out a known amount of the fungus, standardizing the pickup location and made to count the number of micro-sclerotia in magnifying glass.Through simple rule of three, the total number of micro-sclerotia in each petri dish was obtained.
For both tests in vivo tests, 1 liter pots containing the soil mixture were used, along wish sand and organic matter in the proportion 2: 1: 1, autoclaved at 120°C for 1 h.We used the drugs Sulphur and Nosode of the fungus M. phaseolina.The inoculum of M. phaseolina for the first test was produced using a rice medium (rice 25 g to 30 ml of water) and in each pot were added two grains of rice colonized with micro-sclerotia of the fungus M. phaseolina, in the center of the pot at 1 cm depth.Soybean seeds were sown around the inoculum, at a distance of 2 cm from it and subsequently held the thinning, maintaining three soybean plants per pot.In the first trial, the treatments were administered three days before sowing, in the sowing day, 3, 10 and 17 days after sowing.The application was on the soil in a proportion of 0.1% (100 uL of treatment to 100 ml of distilled water) or 1 uL per pot.We evaluated the drought of the stem giving values from one to four, and admitted one when there was an absence of drought, two in the presence of drought up to 1/3 of the stem, three for the presence of drought up to 2/3 of the stem, and four for the presence of drought in the whole stem.The values were transformed and calculated, the area under the disease progress curve (AUDPC).As for the second test, inoculum of M. phaseolina was produced on PDA and was added to each pot three discs containing the fungal mycelium, each of the disks placed in contact with the soybean plant root.This procedure *Corresponding author.E-mail: eloisa-lorenzetti@hotmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License was performed when the plants issued the first trifoliate leaf.In the same day, after the inoculation the soil of the pots was covered with hay.
The treatments were administered in the second test in the soil using spray three days before inoculation, on the day of inoculation, 3, 10 and 17 days after inoculation.Treatments were applied to the soil at a proportion of 0.1% or 1 uL per pot.Growth measurements were taken of the injury on the plant stem, every two days, using graduated scale.With the data obtained, we calculated the area under the disease progress curve (AUDPC).In both tests, the experimental design was a randomized block design with five repetitions, and the Tukey test at 5% probability of error was used to compare means.The factor was 2 x 5 + 2, being 2, the number of drugs (Sulphur and Nosode), 5 the number of dynamizations that were used, which were: 6, 12, 24, 36 and 48CH, and 2 corresponds to additional treatments, namely water and 30% ethanol.In the absence of a significant difference for the factorial and for the additional treatments, the data were shown in a table, otherwise, graphs were prepared.

RESULTS AND DISCUSSION
As shown in Table 1, for the area under the mycelial growth curve of the first test, there was no significant effect for Sulphur and Nosode on M. phaseolina.Through Figure 1, it is seen that in the second test for the area below the mycelial growth curve of M. phaseolina on PDA medium, both drugs showed the same behavior represented by quadratic equations, demonstrating the significance of Sulphur and Nosode of M. phaseolina.For dynamizations 12 and 48CH, Sulphur differed from Nosode of M. phaseolina showing greater reduction in AUMGC.Comparing Sulphur with the control there is a reduction of 14 and 15% in the dynamizations 12 and 48CH, respectively.In a work carried out by Toledo et al. (2015), it was found that the mycelial growth of Alternaria solani in the presence of Sulphur homeopathic preparation in 100 CH, was inhibited by 16.97% compared to the control.In another work, where Sinha and Singh (1983) observed the toxic effect of Sulphur on Aspergillus parasiticus, had 100% inhibition in mycelial growth using the boosting 200 CH, showing that Sulphur, subject to the boosting and on what the fungus will act, can be as effective as chemicals.Since there was no normal data for the amount of micro-sclerotia of M. phaseolina from the first test, it was carried out the transformation of the data into root of x+0.5.Through Figure 2, it can be observed that Sulphur showed a reduction of 50% in the number of micro-sclerotia, this effect being represented by a second degree equation.As for Nosode of M. phaseolina, it showed no significant reduction of micro-sclerotia.
For the amount of micro-sclerotia of the second test, as the data did not show any normality, it was carried out the transformation of that data into root of x+0.5.In Figure 3, it can be observed that Nosode of M. phaseolina did not show significance, while Sulphur showed a behavior represented by quadratic equation and provided a reduction of up to 33%, a very important number since the micro-sclerotia are the form of survival of the fungus on the ground, and it contributes to the spread and survival of this for long periods.The reduction of sporulation was also observed with Sulphur in the work conducted by Toledo et al., (2015) where it had 53% of reduction for the boosting 30 CH to 63% for boosting 6 CH.In work done by Toledo et al. (2013) seeking to evaluate the inhibition of sporulation of A. solani by Ferrum sulphuricum, it was found that the dynamizations 6,12,18,21,24,27,30,48,66,69,72,75,78,81 and 96 CH decreased sporulation and were statistically different from the controls distilled water and hydroalcoholic solution 70%, having the inhibition of 71.6% in the dynamizations 24 and 78 CH and variation of 88.42% in the dynamizations 21 and 96 CH of spore inhibition.Toledo et al. (2015) also found little effect of using isopathy, in the case of A. solani both for mycelial growth and also for the number of micro-sclerotia.With regard to the first test, according to Table 2, there was no statistical difference among the treatments and, bearing this in mind, none of the remedies showed themselves   efficient to reduce the area under the progress curve for botrytis of the soybean stem in the dynamizations tested.
In Table 3, for the area under the disease progress curve (AUDPC) caused by M. phaseolina in soybean plants, in the second test there was also not significance of the treatments.In a study conducted by Toledo et al. (2015), with tomato plants that had black spot, there was a reduction of 34.97% of AUDPC for Sulphur medicine in boosting 12CH and reduction of 16.79% for the same drug in boosting 30 CH, this compared to the control of distilled water.

Figure 1 .
Figure 1.Area under the mycelial growth curve (AUMGC) of M. phaseolina in PDA medium with dynamizations of the remedies Nosode of M. phaseolina and Sulphur.HS: Hydroalcoholic solution (30% ethanol).Data referring to the second test.

Figure 2 .Figure 3 .
Figure 2. Quantity of micro-sclerotia of M. phaseolina in PDA medium with dynamizations of the remedies Nosode of M. phaseolina and Sulphur.Data were transformed into root of x+0,5.ns : Absence of significance between dynamizations of the same remedy or among remedies.HS: Hydroalcoholic solution (30% ethanol).Data referring to the first test.

Table 1 .
Area under the mycelial growth curve (AUMGC) of M. phaseolina in PDA medium with different dynamizations of the remedies Sulphur and Nosode of M. phaseolina.Data referring to the first test.Absence of significance between dynamizations of the same remedy or among remedies.Additional treatment: distilled water.

Table 2 .
Area under the disease progress curve (AUDPG) of M. phaseolina in soy plants cultivated in a greenhouse and being treated with different dynamizations of the remedies Nosode of M. phaseolina and Sulphur.Data referring to the first test.

Boosting (CH) Sulphur Average Nosode of M. phaseolina
Absence of significance between dynamizations of the same remedies or among remedies.Additional treatment: distilled water.