Localization of experiments

Experiments were carried out at the Experimental Station of Lages/Agricultural Research and Rural Extension Company of Santa Catarina State - EPAGRI (Area 1), and in a farm located in the Pedras Brancas Community (Area 2), Lages (27°48' S and 50°19' W; 931 m a.s.l.), Santa Catarina State, Brazil, during the crop cycles of 2011 and 2012. The predominant climate in the region consists of an average temperature of 22°C in the warmest month, and in winter from 6 to 8°C. The total annual rainfall varies from 1,360 to 1,600 mm and the soils are classified as "cambissolo humico alico" (Embrapa, 2013).

High dilution preparations

High dilution preparations were obtained from the Laboratory of Homeopathy and Plant Health, Lages Experimental Station of EPAGRI, following the methodology described in the Brazilian Homeopathic Pharmacopoeia (2011). High dilutions of Arnica montana, Carbo vegetabilis, Silicea terra, and Sulphur were made from a matrix of 5CH dynamization, using a part of the matrix in 99 parts of 5% alcohol and followed by 100 times succussion with the help of mechanical arm (Autic^®, Denise Model 10-50), to achieve a 6CH dynamization; and so forward until reaching the desired 30CH dynamization.

Seedling production in greenhouse experiments

Three experiments were conducted in a greenhouse using a completely randomized design with four replicates from 30/09/2011 to 01/10/2012. The treatments were Arnica montana, Silicea terra, Carbo vegetabilis, and Sulphur at 6CH and 30CH, and water as the control plot. The experimental plot consisted of 20 plants in the middle of 64 seedlings in expanded polystyrene trays with capacity of 128 cells. Piracicaba precocious hybrid of broccoli was used for all experiments. Organic substrate was composed of cattle manure (composted for six months), soil, vermiculite, and rock phosphate, at volume rate of 3:1:1:0.2, respectively. The density of substrate material was 0.7 kg L^-1 of cattle manure; 1.0 of soil; 0.08 of vermiculite and 2.8 of rock phosphate. The materials were mixed and dried. The substrate was then transferred into tray cells. Two seeds per cell were placed at 0.5 cm depth. Thinning was done a week after germination, leaving one plant per cell. The irrigation of the seedlings was done daily by micro sprinklers during three minutes. Whenever necessary the irrigation was repeated. The spray treatments were applied every four days on the seedlings until transplantation, with total of seven applications. Trays were isolated from each other at the time of spraying. The applications were performed using manual sprayer Vonder^® with capacity for 1.5 L. Each homeopathic preparation was used at a rate of 10 mL L^-1 of water and homogenized with ten shakes. Double-blind technique was used for conducting experiments, where neither the assessor nor the applicator knew the nature of each treatment. Homeopathic preparations only revealed after the statistical analysis.

The evaluations were performed after 70% of seedlings reach the phenological stage V₀, characterized by seedlings with three to four leaves and 10 to 12 cm height (^{Jaramillo and Diaz, 2006}). Then the seedlings were harvested and the plant height (a), determined by the distance between the stem base to the apex of the newest leaf using a standard ruler; the root length (b) was measured with a standard ruler, and the stem diameter (c) was measured with a digital caliper. The fresh and dry mass of shoot and root were evaluated with the assistance of a digital scale. For the evaluation of dry mass of shoot and root, the fractions were wrapped separately in Kraft paper bags, and taken for drying in an oven with forced air at 65°C, until the weight was stabilized.

Broccoli production under field conditions

Two experiments were conducted in the field from 13/12/2011 to 17/04/2012 at the Experimental Station of EPAGRI (Area 1) and from 14/05/2012 to 17/09/2012 at a family farm (Area 2). Both experiments were located in Lages (27°48' S and 50°19' W; 931 m a.s.l.), Santa Catarina State, Brazil (Tab. 1). Piracicaba hybrid of broccoli was the variety used for all experiments.

TABLE 1 Chemical composition of soil for the experimental areas in Lages-SC, Brazil. 

The seedling production was done in expanded polystyrene trays with 128 cell capacity. The trays were washed with water and disinfected with sodium hypochlorite at 2.5% of active chlorine, in the proportion of 5 mL L^-1 of water. The substrate was composed of cattle manure, soil, vermiculite and phosphate, at rate of 3:1:1:0.2 in volume. One year before transplanting, soil tillage was performed with plowing and disking, followed by fertilization with green manure (oats + vetch). The fertilization was performed with 2.5 L m^-2 of compost [cattle manure: C (%) = 48; N (% dry matter) = 2.3; P₂O₅ (kg m^-2) = 4.1; K₂O (kg m^-2) = 3.8; Ca (kg m^-2) = 3.0], and phosphate in the proportion of 0.15 kg m^-2. Transplantation was done when seedlings reached the phenological stage V₀ (^{Jaramillo and Diaz, 2006}), where seedlings had three to four leaves and a height of 10 to 12 cm.

Treatments were of high dilution preparations of Arnica montana, Silicea terra, Carbo vegetabilis, and Sulphur at 6CH and 30CH. Pure water was used as control. The experimental design was organized in completely randomized blocks with four replicates. Each plot consisted of 10 plants transplanted in double line.

The irrigation was conducted according to the requirements of the broccoli crop and weeding was done by manual methods. One seedling was planted per hole with the spacing of 0.4 m between plants and 0.6 m between rows, resulting in a density of 41.500 plants per hectare.

The application of spray treatments was applied using backpack sprayer Guarany^® with capacity for 5 L. The frequency of applications was every fifteen days, starting seven days after the transplant until harvest (for a total of four applications). Each homeopathic preparation was dosed in a proportion of 10 mL per liter of water, followed by ten shakes. Double-blind technique was used for spray the treatments, where neither the appraiser nor the applicator knew the nature of each treatment, until after the statistical analysis was done.

At harvest the evaluated variables were: (a) plant height, determined with a measuring tape, from ground level up to the apical part and (b) stem diameter, measured at one cm from the ground with the assistance of digital caliper. External leaves of broccoli plants were removed and the fresh mass of the inflorescences was later determined with a digital scale, measured in grams. The dry mass of the inflorescences was obtained by packaging in Kraft paper bags and placing in a drying oven with forced air at a temperature of 65°C, until the weight stabilized.

Statistical analysis

Data were statistically analyzed with R version 3.1.2 (R Core Team, 2014), using analysis of variance (ANOVA). Means significantly were compared by Tukey test (P≤0.05).

Effect of high dilution preparations on seedling production

High dilution preparations of Silicea terra at 30CH and Arnica montana at 6CH increase of dry mass of shoot in the broccoli seedlings, when compared to the water control in at least one experiment (Tab. 2). ^{Bonfim et al. (2008)} also using of Arnica montana at 3CH, 6CH and 12CH found an increase of dry mass in rosemary (Rosmarinus officinalis L.) and royal sage shoots (Lippia alba (Mill.) N.E. Br.). Also ^{Grisa et al. (2007)} found an increase of dry mass in lettuce (Lactuca sativa L.) shoots with Arnica montana dynamizations at 6CH and 12CH. Further, ^{Almeida (2002)} observed an increase of 40% of fresh mass of basil inflorescences (Ocimum basilicum L.) when exposed to Silicea terra at 30CH.

Dry mass of root in broccoli seedlings treated with Silicea terra at dynamization of 30CH was higher than the water control plots. The preparations of Sulphur 6CH, Arnica montana 6CH and Carbo vegetabilis 30CH increased the dry mass of root in broccoli, in at least one of the three experiments (Tab. 2).

TABLE 2 Dry mass of shoot and root in seedlings of Piracicaba precoce hybrid of broccoli treated with high dilution preparations. 

Means ± S.E. followed by the same letter in the column did not differ significantly from each other by Tukey test (P≤0.05). Exp1 = Sep-Oct (2011); Exp2 = Feb-Mar (2012); Exp3 =Aug-0ct (2012).

Root length of broccoli seedlings was affected by the use of high dilution preparations, being that Sulphur 6CH and Silicea terra 30CH promoted greater root length compared to the water control in at least two of the three experiments (Tab. 3). The root system is a determining factor in the quality of the seedlings, and seedlings with poor root systems have growth difficulty in field conditions (^{Krasowski and Owens, 2000}; ^{Davis and Jacobs, 2005}).

Preparations of Silicea terra at a 30CH dynamization and Arnica montana at 6CH increased the stem diameter of broccoli seedlings when compared to the water control in two of three experiments. Sulphur at 6CH also increased this characteristic in one of three experiments (Tab. 3).

TABLE 3 Plant height, root length and stem diameter in seedlings of Piracicaba precoce hybrid of broccoli treated with high dilution preparations. 

Means ± S.E. followed by the same letter in the column did not differ significantly from each other by Tukey test (P≤0.05). Exp1 = Sep-Oct(2011); Exp2 = Feb-Mar(2012); Exp3 = Aug-0ct (2012). I56 Agron. Colomb. 35(1) 2017

There were no significant differences between the treatments in the three experiments concerning the height of broccoli seedlings (Tab. 3). These results suggest that the 6CH and 30CH dynamics are more appropriate for promoting the root strengthening, but not manifested in the height of the seedling. If the root system is strong, it is possible that this characteristic is observed in the post-transplant, as it can be measured in field experiments. According to ^{Bonato and Silva (2003)}, who observed different effects of high dilutions in different organs of plants. The same authors suggest that the action of high dilutions in plants should always be considered in its full cycle.

Effect of high dilution preparations on the production of broccoli in field

Piracicaba hybrid of broccoli plants conducted at area 2, when treated with Sulphur 6CH increased the fresh and dry mass of the inflorescences, compared to water control. Though this result was not confirmed in the conditions of area 1 (Tab. 4). Preparations of Sulphur at 6CH increased the production and the dry mass of cabbage (^{Pulido et al., 2014}), and the fresh and dry mass of mint plants (^{Bonato et al., 2009}). ^{Toledo et al. (2015)} also observed that Sulphur (6CH, 12CH, 30CH, and 60CH) increased the fresh mass of shoot and (30CH) root of tomato plants.

TABLE 4 Fresh and dry mass of inflorescences, plant height and stem diameter of Piracicaba precoce hybrid of broccoli treated with high dilution preparations. 

Means ± S.E. followed by the same letter in the column did not differ significantly from each other by Tukey's test (P≤0.05). Area 1 = Experimental Stationof Lages-Epagri, Dec-Apr (2011/2012): Area 2 = Farm in Peclras Brancas, Lages, May-Sep (2012).

Preparations of Silicea terra at 6CH, Carbo vegetabilis, and Sulphur at 30CH influenced the increasing of plant height when compared to water control, in area 2. However, no significant differences were observed for stem diameter in broccoli among treatments in both experimental areas (Tab. 4).

Fresh and dry mass of the inflorescences, plant height and stem diameter in broccoli did not differ among treatments applied in area 1 (Tab. 4). ^{Andrade et al. (2012}) also found no significant differences between the treatments in high dilutions in the characteristics of fresh and dry mass in plants of Justicia pectoralis. However, preparation of Arnica montana at 3CH, 30CH, 60CH, 100CH and 200CH dynamizations directly influenced the secondary metabolism of plants, causing changes in the coumarin content.