Homeopathy in the Rust Severity and Growth of Malva sylvestris L

The production of medicinal plants which have an association with biotrophic fungi requires non-residual and favorable methods to the host with tolerance to the presence of phytopathogens. The objective of this work was to evaluate the effect of homeopathic preparations on the rust severity and the growth of Malva sylvestris plants. M. sylvestris seedlings were prepared in 600 ml containers with commercial substrate. The seedlings were arranged in pots at 26 days of age and outlined in two experiments. The treatments consisted of Amonnium carbonicum (Am. carb.), Atropa belladonna (Bell.), Calcarea carbonica (Calc. carb.), Silicea terra (Sil.) and Sulfur (Sulf.), all at 30CH (centesimal Hahnemannian dilution order). The last two dynamizations (29 and 30CH) were prepared in distilled water for all treatments. Control plants were treated with water. Natural inoculation of the plants with Puccinia malvacearum occurred in the first experiment, and the applications of homeopathic preparations were carried out every seven days for five weeks. Four evaluations of rust severity, diameter, height and number of leaves were conducted. Next, M. sylvestris seedlings were transplanted into pots with 5 liters of substrate in the second experiment and the growth curve of the plant was observed in relation to the diameter and height variables. Am. Carb. reduced 18.29% of the rust severity in relation to the control plants. Sil. 30CH contributed to an increase in stem diameter. There was no interference in the plants’ height by homeopathic preparations. The application of homeopathies in M. sylvestris can contribute to their production, reducing the rust intensity considered in the crop cycle and can assist in the plant growth without leaving residues which can harm pollinators and hyperparasites.


Introduction
Malva spp. species evolutionarily developed in close interaction with the Puccinia malvacearum Bertero ex Mont. biotrophic fungus. This relationship is demonstrated by the increase in the number of cells with mucilage in the area with pustules and the passage of P. malvacearum through these cells without hindrance or degeneration (Classen, Amelunxen, & Blaschek, 2001). The bioactivity of Malva spp. is related to mucilage production (Tomoda et al., 1989;Classen & Blaschek, 1998). According to Mitchell and Roberts (1973), polysaccharides such as arabitol and trehalose can be stored in leaves infected with P. malvacearum in Althea rosea at pustule sites and trehalose in teliospores, which shows the evolutionary advantages of the intimate host and pathogen relationship.
The supply of M. sylvestris leaves and mericarps on the market is restricted by rust symptoms so that they cannot have more than one brown teliospore web of the P. malvacearum fungus per cm 2 (Brasil, 2000). On the other hand, in accordance with IN No. 17/2014 of the Ministry of Agriculture and Livestock (MAPA) and the National Policy on medicinal and phytotherapic plants of the Ministry of Health, medicinal plants cannot be handled with pesticides (Brasil, 2014(Brasil, , 2016. The risk of residues of active ingredients and heavy pesticide metals is related to the concentration of these ingredients in the plant's drying process and alteration of the chemical composition of the plants in processing and extracting the herbal medicine (Carvalho, Costa, & Carnelossi, 2010). In view of this problem, high dynamized dilutions, or homeopathic preparations, have been studied as an adequate measure in treating medicinal plants, since they do not leave chemical residues in fresh or processed products (Casali et al., 2010). not exclusive to these systems (Brasil, 2014). Pulido et al. (2017) demonstrated an increase in broccoli (Brassica oleraceae L. var. itálica) biomass through the use of Sulfur 6CH, Silicia terra 6CH, Carbo vegetalis 30CH and Sulfur 30CH. Leonel and Barros (2013) reported using homeopathy to manage coffee rust (Hemileia vastatrix), in which they used four leaf sprays with a complex of basic pharmaceutical preparations (Silicea 6CH, Sulfur 6CH, and Arsenicum album 6CH) and isotherapic rust pustules (30CH) during a period of cultivation (December-may), with a positive effect on stains and defoliation. Homeopathic preparations of propolis, Sulfur and Ferrum sulphuricum applied to tomato plants were effective in reducing the intensity of the Alternaria solani phytoparasite and interfering with tomato (Solanum lycopersicum L.) (sin.: Lycopersicon esculentum Mill.) growth (Toledo, Stangarlim, & Bonato, 2015). The mode of action of homeopathies in plants is still being investigated, but there are studies where the activation of mechanisms of resistance of plants to diseases has been observed (Carneiro & Teixeira, 2018).
Keeping in mind that the application of high dynamized dilutions can alter pathological processes established in plants and/or prevent them, the objective of this work was to evaluate the effect of homeopathic preparations on the rust severity and the growth of M. sylvestris plants.

Plant Location and Production
The study was conducted in semi-controlled conditions (greenhouse) in a residential-urban area in the municipality of Caçador, SC, in the period 2018, with support from the Epagri-Lages Homeopathy and Plant Health Laboratory and the UNIARP-Caçador Phytopathology Laboratory.
Malva seedlings were produced from Malva sylvestris seeds introduced in the garden of the Municipality of Caçador. The seedlings were kept in containers of expanded polystyrene (EPS) pots with 600 mL of Agrinobre ® TMX substrate. The plants were kept until the 26 th day in a greenhouse with sprinkler irrigation for 5 minutes, three times a day. The plants were then divided from that date into two experiments: (a) evaluation of the Area Under the Disease Progress Curve (AUDPC) and growth of M. sylvestris; and (b) evaluation of the height and diameter growth curve estimate of M. sylvestris.

Exp. 1 Evaluation of the Area Under the Disease Progress Curve (AUDPC) and Growth of M. sylvestris
The plants at 26 days remained in the EPS pots and in the greenhouse with sprinkler irrigation 10 minutes three times a day and were submitted to natural inoculation. The greenhouse was maintained with natural ventilation at the top so that the P. malvacearum spores from the M. sylvestris plants kept outside the greenhouse could be carried by the wind inwards to proceed with natural inoculation, being performed on a day with heavy rain and wind. The sprinkling time increased to 15 minutes and the number of irrigations was changed to four times a day for a period of 24 days. Rust symptoms appeared on the leaves one week after the natural inoculation, and all leaves were evaluated according to grades assigned as follows: grade 0 = 0%; grade 1 ≥ 0 < 5%; grade 2 ≥ 5 < 15%; grade 3 ≥ 15 < 25%; grade 4 ≥ 25%. The experimental design was a randomized block with five replications and a double-blind procedure, with the treatments for both the applicator of the preparations and the evaluator of the treatments being unknown. Each repetition consisted of one container.
The weekly treatments started one week after being considered a natural infection by applying the homeopathic preparations of Amonnium carbonicum (Am. Carb.), Atropa belladonna (Bell.), Calcarea carbonica (Calc. Carb.), Silicea terra (Sil.) and Sulfur (Sulf.) in the thirtieth centesimal Hahnemannian dilution (30CH). The choice of homeopathic preparations was made according to the characteristics of plant and rust symptoms, with analogy between the symptoms presented in the homeopathic matter medical. The symptoms used for repertorization were: eruptions, red; eruptions, pustules; thrist, large quantities, for; food and drinks, sugar desire; inflamation; light-desire for. The control was carried out with distilled water. The dynamizations were prepared according to FHB (Brasil, 2011a). The last two dilutions for preparing homeopathies for each potency/treatment were done in distilled water. The adopted homeopathic nomenclature, name and abbreviation were based on that described by Soares (2005). The treatments were applied once a week with 2±0.2 mL per plant, so that at least one drop touched each leaf and the rest was applied to the plant's stem for a period of five weeks. Evaluations were performed by measuring the growth in height and diameter by the difference between the first and last evaluation divided by the number of days. The evaluation of the area affected by rust of all leaves was performed weekly by the diagrammatic scale in the period of 4 weeks. Afterwards, the disease severity index per plant, McKinney index (McKinney, 1923), Equation 1 was calculated which was subsequently used to calculate AUDPC (Campbell & Madden, 1990), using four evaluations for the construction of the curve.
(1) jas.ccsenet.org Journal of Agricultural Science Vol. 13, No. 5;2021 where, y i represents the degree of the scale, f represents the number of plants with infection level y i , n represents the number of plants evaluated, and y k represents the maximum degree of infection.

Exp. 2 Evaluation of the Height and Diameter Growth Curve Estimate of M. sylvestris
Seedlings at 26 days were standardized and transplanted to pots with 5 liters of Agrinobre ® TMX substrate, kept in a shelter with a white screen and drip irrigated four times a day for five minutes. After 13 days, the plants received the same homeopathic treatments described above for a total of six applications. Plants were evaluated for diameter and height growth variables at 7, 14, 21, 28, 49 and 56 days after treatments. The experimental design was a randomized block with five replications and double blind, with the repetition being formed by a vase.

Statistical Analyses
Data analyses of the diameter, height and AUDPC variables of the treatments were performed using the classic analysis of variance, with the presupposition of normality being verified through the Shapiro-Wilk test and the homoscedasticity through the Bartlett test. Tukey's contrasts and the procedure for testing linear hypotheses described by Hothorn, Bretz, and Westfall (2008) were used to compare treatment levels.
The number of leaves variable was analyzed using the Poisson model with a superdispersion parameter (quasipoisson) as it is counting, and the deviance analysis was performed using the F test.
The growth curve for the height and diameter variables of the M. sylvestris treated with Am. Carb., Bell., Calc. carb., Sil., and Sulf. homeopathic preparations and control were adjusted by linear and logistic models, considering the treatments and the different evaluation dates (days) as descriptive variables. The Akaike Information Criterion (AIC) was used for selection among them. The comparison between treatments was performed using the confidence intervals of the parameters of the selected model.
All analyzes were performed using the R software program (R Core Team, 2017) considering 5% significance.

Results and Discussion
The use of Am. Carb in the 30CH provided a reduction of 18.29% in the AUDPC of rust compared to the control (Table 1). Am. carb is associated with rust symptoms on the leaves, as in the case of Bell., by analogy to the presence of dark red spore mass. According to Tyler (1992), Calc. carb. is Bell's chronic state and may also present weakness, late development and worsens in cold and wet weather (Tyler, 1992;Vannier & Poirier, 1987). By analogy to medical matter, Am. Carb. is indicated in cases of weakness when skin rashes worsen due to wet and rainy weather. It is a suitable remedy for the female organism, fainting, vertigo and considered antipsoric by Hahnemann (Hahnemann, 2018;Tyler, 1992). Therefore, there was a certain similarity correspondence in M. sylvestris with the difficulty of the plant to remain upright many times, marked weakness after flowering and fruiting, with signs of P. malvacearum on the leaves resembling eruptions, with humid and rainy weather favoring infection, and showy flowers with feminine characteristics. to that of the s represented i the control.
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Conclus
It  Vol. 13, No. 5;2021 sylvestris growth was affected by the use of Sil. 30CH due to the increase in diameter compared to the control with water.
Am. Carb 30CH is recommended for managing the Malva sylvestris and Puccinia malvacearum interaction.
Considering that the plant's resistance is overcome as the fungus develops specialized structures in the host forming a matrix of nutrient exchange, the use of Sil. 30CH can assist in assimilating nutrients and favor plant growth.