Revista Mexicana Ciencias Agrícolas   volume 11   number 3   April 01 - May 15, 2020

DOI: https://doi.org/10.29312/remexca.v11i3.2077

Article

In vitro evaluation of methods against Botrytis cinerea

Omar Essaú Larios-Palacios1

Érida Yasmín López-Vázquez2

Arturo Curiel Rodríguez1

Felipe de Jesús Ruíz-Espinoza3

Roney Solano-Vidal2

Miguel Ángel Serrato-Cruz1§

1, 2, 3Departments of Phytotechnics, Agricultural Parasitology and Regional Center of Anahuac-Chapingo Autonomous University. Mexico-Texcoco highway km 38.5, Chapingo, State of Mexico, Mexico. CP. 56230. (oma-ago95@gmail.com; jasmin.lov@outlook.com; acuriel@correo.chapingo.mx; agrohomeopatia@hotmail.com; roneysv@hotmail.com).

§Corresponding author: serratocruz@gmail.com.

Abstract

Although experimental methods are available for the control of fungal diseases in strawberry cultivation, including chemical, organic and biological, other low-cost methods such as homeopathic substances are not ruled out, which have not been evaluated against Botrytis cinerea. The objective of the present study was to compare conventional treatments and homeopathic substances, under in vitro conditions, in the mycelial growth of B. cinerea as a previous reference to in vivo evaluation. Treatments: PDA medium, synthetic fungicide Switch® (Fludioxonil, Ciprodinil) 1 g L-1, essential oil of Tagetes lemmonii (1%), commercial extract of Larrea tridentata (2 mL L-1), homeopathic substances, arsenic 6 CH and nosode of Botrytis 7 CH and dual test with Trichoderma harzianum. Registered variables: mycelial diameter, growth rate and percentage of mycelial inhibition; in the dual test, days at first hyphal contact and type of antagonism were measured. The data were subjected to analysis of variance and multiple means comparison test (Tukey, p≤ 0.05). Switch® and Tagetes oil inhibited mycelial growth by 100%, with Larrea extract the inhibition was 65.8%, T. harzianum inhibited 36.2% and hyphal contact occurred on the sixth day (type II antagonism), controlling the pathogen, the homeopathic substances nosode 7 CH and arsenic 6 CH inhibited the fungus in 31.2 and 11.8%, respectively. The effect of nosode 7 CH was different (p≤ 0.05) from the control, but homeopathic arsenic was not.

Keywords: antifungal effect, fungal diseases, homeopathic substances, organic and biological methods, strawberry.

Reception date: January 2020

Acceptance date: March 2020

Introduction

Strawberry (Fragaria vesca L.) is grown in Mexico in 12 states, but only Baja California, Guanajuato, and Michoacán generate 91.55% of total national production (Santoyo, 2009). Gray mold (Botrytis cinerea Pers. ex Fr.) is one of the main strawberry diseases; this plant pathogen has the capacity to act as a saprophyte and has been considered of high importance, since it affects a large number of crops around the world. The losses in the strawberry crop can be severe, up to 25% in the main harvest and around 37% in the second productive peak, so that without effective pre and post-harvest control it can cause large economic losses, affecting productivity in harvest quantity and quality (Ceredi et al., 2009).

For the control of B. cinerea, the use of modern fungicides that act on specific metabolic sites of the fungus has resulted in the formation of races resistant to certain chemicals, in this sense, the need has arisen to seek other resources to the integrated management of the disease (Cano, 2013).

Some methods explored in plants for fungal control consist of using microbial antagonists that regulate populations of phytopathogens (Chávez, 2004), the use of plant extracts has also been implemented to control fungal diseases, since they are a rich source of bioactive chemical products (Balandrin et al., 1985). These methods can currently be used as part of integrated disease management, and recently, the use of homeopathic substances is another promising resource for the biocontrol of phytopathogenic fungi and which, compared to previous methods, is attractive due to its low cost (Meneses, 2007 ).

The in vitro study model of substances or organisms to control fungal diseases is a useful methodological resource to select what is promising and to outline its evaluation in vivo. In this sense, in vitro tests of fungal sensitivity to fungicides help determine whether or not their biological activity is expected, either because the fungus develops resistance or due to aspects related to the quality and quantity of the synthetic product used (Mercier et al., 2010); therefore, the inclusion of control treatments and the verification of the fungicidal or fungistatic effect are necessary methodological aspects.

Regarding works on the use of plant extracts against fungi in vitro conditions, there are numerous investigations, but Botrytis highlights those related to extracts of Larrea tridentata (Lira, 2003) and Lippia origanoide (Taborda, 2015). In the case of extracts of governor plants (L. tridentata) its effect is positive for the control not only of Botrytis but also of Fusarium, Pythium and Rhizoctonia, this plant source of metabolites is distributed in arid regions of Durango and Coahuila (Lira, 2003).

In the case of essential oils against Botrytis, there are few studies on Tagetes that highlight the effect of total inhibition (Romagnoli et al., 2005), although in other works a positive effect against other fungus species is reported (Barajas et al., 2011 ; López et al., 2018). In general, there are few species of Tagetes explored for their effect against fungi and in the case of Tagetes lemmonii, a species distributed regionally in Sonora and Durango (Serrato, 2014), research is scarce.

The methanolic extract of T. lemmonii is antifungal in Botrytis (Bojorquez, 2017) and the essential oil causes inhibition of Fusarium oxysporun f. sp. ricini (Jarquin, 2016), the latter has not been explored against B. cinerea. With respect to Trichoderma harzianum, it is referred to as Botrytis antagonist (Merchan, 2014) and in works carried out under in vitro conditions, the total inhibition of the growth of the fungus is evident (Calvo, 2012).

In relation to the effect of homeopathic substances against fungal diseases in plants, there are several studies carried out under in vivo conditions (Oliveira et al., 2013; Hanif and Dewar, 2015; Lorenzetti et al., 2016; Rissato et al., 2016; Oliveira et al., 2017) and only six in vitro (Carneiro and Bueno, 2010; Gama et al., 2015; Lorenzetti et al., 2016; Rissato et al., 2016; Toledo et al., 2016; Serrato et al., 2018) among the latter, two of them refer to nosodes and four to various substances, showing direct antifungal effect and none of them evaluated against B. cinerea.

From the results of the application of homeopathic substances in vitro against fungi, the homeopathic principle stands out similia similibus curentur related to substances such as arsenic, phosphorus, sulfur, Arnica montana, etc. (Tichavsky, 2007), which inhibit fungi such as: Aspergillus niger, Alternaria solani and Sclerotinia scleroturum (Gama et al., 2015; Toledo et al., 2016; Rissato et al., 2016) also highlights another homeopathic principle, the most similar one , which corresponds to the elaboration of nosode, a homeopathic substance derived from the organism that causes disease or damage, as in the cases of Alternaria solani, Fusarium solani and Sclerotinia scleroturum evaluated in vitro (Carneiro and Bueno, 2010; Rissato et al., 2016).

Under in vivo conditions, the application of homeopathic substances against fungi presupposes signals that the plant receives preparing it to face the establishment of the pathogen; however, the incorporation of the homeopathic substance (dynamized substance or nosode) into the in vitro culture medium can directly influence the pathogen; that is, influencing it by contact, a biological response that has been little argued in the agrohomeopathic phenomenon (Carneiro and Bueno, 2010; Lorenzetti et al., 2016; Rissato et al., 2016).

Although comparative studies of chemical, organic and biological treatments under in vitro conditions against phytopathogenic fungi are reported (Jarquin, 2016), there are relatively few that include homeopathic substances (Oliveira et al., 2013; Gama et al., 2015; Lorenzetti et al., 2016; Serrato et al., 2018). Considering the limited exploration of T. lemmonni oil as an antifungal, the limited information on homeopathic substances with a direct effect on fungi and its limited inclusion in comparative studies of antifungal efficiency in the in vitro model, as well as the importance of reducing Costs in the control of strawberry B. cinerea. In this study, conventional treatments and homeopathic substances were compared in vitro against B. cinerea in order to generate useful information for verification in vivo.

Materials and methods

The present investigation consisted of carrying out an in vitro evaluation of a chemical fungicide, two vegetable substances, an antagonistic biological agent and two homeopathic solutions (Table 1). The experiment was carried out in the Laboratory of the Master in Plant Protection at the Autonomous University Chapingo (UACH) in 2018.

Table 1. Treatments for evaluation in the bioassay with B. cinerea.

Isolation of phytopathogenic fungi

The phytopathogenic fungus B. cinerea was isolated from strawberry plants in a greenhouse in the UACH Experimental Field. Plants with damaged fruits, showing a brown cover with a spore carpet texture, a peculiar symptom of Botrytis presence in that berry, were selected as a source of inoculum. Fragments of infected tissue were established under aseptic conditions in Petri dishes with Papa-Dextrose agar culture medium (PDA) and incubated in a growth chamber at 17 °C and 80% relative humidity for eight days and 1 hr of light daily.

Biological evaluation substances and organisms

The commercial chemical Switch® (Fludioxonil, Ciprodinil) was obtained from an agrochemical store in the municipality of Texcoco, State of Mexico. The commercial organic product Progranic® Mega (Larrea tridentata extract) was also purchased. The antagonistic organism (Trichoderma harzianum), in mycelium, was provided by Dr. Roney Vidal Solano, Department of Agricultural Parasitology, UACH.

From T. lemmonii A. Grey plants. (voucher 2012-199; Herbarium of the University of Sonora) Var. Limon (SAGARPA-SNICS, file 1423, record 1629) from a plantation established in the UACH Experimental Field in 2015 (19° 29.547’ north latitude, 98° 52.470’ west longitude and 2 267 masl, Cw1 climate type) were harvested stems in bloom in September 2018. This fresh biomass in quantity of 100 kg was subjected to a process of hydro-distillation by means of a stainless-steel distiller with a distillation capacity of 200 kg, the oil obtained was stored at 18 °C in the dark.

The homeopathic substances were prepared by Dr. Felipe de Jesús Ruiz Espinoza from the Regional University Center of Anahuac of the Autonomous University Chapingo (UACH), for the preparation of the mushroom nosode, the latter was isolated from a strawberry fruit and prepared by grinding with sugar. For the preparation, 0.05 g of the fungus sample was taken, which was placed in a 4-inch porcelain mortar. In three plastic bags of 6 x 10 cm, 5 g of sugar was added to each one, labeled with the name of Botritis cinerea T1C (bag 1), writing down the date. To bag 2, B. cinerea T2C and to bag 3, B. cinerea T3C.

Bag 1 was divided into three thirds, adding the first third and starting the crushing as follows: it was ground or crushed for 6 min, then, with a stainless steel coffee spoon, the pestle and mortar were scraped for 4 min. This was repeated by grinding 6 min and scraping that third for 4 min. When finished, the second third of the first bag was added. Grinding and scraping was repeated for 6 and 4 min, respectively. At the end, the last third was added, which was ground for 6 min and scraped for 4 min, repeating this sequence until completing 1 h.

From there the crushed was stored in the T1C bag. From the T1C crushing, 0.05 g was taken to start bag 2, which was prepared according to the times of the first bag and when the second bag was finished, the third bag was saved and started, when finished, it was saved and from there it was taken 0.05 g of the 3C trituration, which was added to a bottle containing 50 drops of distilled water and 50 drops of alcohol, stirred for 2 min and allowed to stand for 2 min.

At the end, this preparation constituted the 4C dilution or potency, hence the 5C, 6C and 7C were elaborated, according to the Hanamanianna centesimal scale, as mentioned by Sandoval (1961). This sequence of operations was done for arsenic, according to rule 7 of homeopathy (Zepeda, 2002), arsenic was obtained from the National Homeopathic Pharmacy located in the plinth of Mexico City and the characteristics of this substance are described in the work de García (1984).

Preparation and evaluation in Petri dishes

For the bioassay, the poisoned agar method was used, which consists of integrating the treatment substances into the culture medium. PDA culture medium was prepared in seven 250 mL Erlenmeyer flasks. For sterilization, they were autoclaved for 20 min at 120 °C and 15 lb pressure. When the autoclave temperature and pressure dropped to around 40 °C, the flasks were removed. The PDA medium plus the substances corresponding to the treatments were stirred before they were poured into the Petri dishes in order to homogenize the mixture.

The preparation was emptied into sterile 90 mm Petri dishes under aseptic conditions and allowed to cool and solidify at room temperature for 24 h. The next day, a PDA disc with the B. cinerea inoculum obtained with a sterile 3.5 mm diameter punch was placed and placed inverted in the center of the box. Petri dishes were incubated at 18 ± 2 °C in an oven. From the seeding of the fungus in the Petri dish and for 8 days, the diameter of the mycelium was measured with a digital vernier.

Preparation of treatments

The dual preparation with T. harzianum was carried out on sterile culture medium and placing a 3.5 mm diameter disk at one end of the Petri dish previously divided in half and then placing the mycelium of B. cinerea at the other end. The pure T. lemmonii essential oil was prepared at 1%, for which 0.1 mL of Tween 20 surfactant was added, shaking circularly to obtain an emulsion, and then it was homogeneously dissolved in 100 mL of PDA, then it was poured into a 250-ml flask mL. The same procedure was used to prepare the governor extract, where the 100 mL solution of culture medium was prepared with 2 mL of the organic product.

For the synthetic product Switch®, 1 g of the product was added in one liter of water and waited until it dissolved, stirring, transferring 100 mL of mixture to the flask to be sterilized. In the case of homeopathic substances, one drop of each homeopathic solution, Botrytis 7 CH and arsenic 6 CH, was added to one liter of distilled water, and then vigorously succussed the bottle for 2 min. Subsequently, the required quantity (100 mL) was deposited in the 250 mL Erlenmeyer flasks.

Experimental design

The experiment was analyzed by a completely randomized experimental design with six treatments each with five replications and an absolute control, the experimental unit consisted of a 90 mm diameter Petri dish with PDA culture medium incorporating the treatments to be evaluated.

Registered variables

The radial growth of the fungus was measured with a frequency of 24 h, ending until the Petri dish without treatment (control) was completely covered with mycelium (8 days), based on this time, evaluations of the dual growth Trichoderma-Botrytis were also performed. Measurements were made with a digital ernier. The growth rate of the mycelium (VC) was calculated with the formula of Sinclair and Cantero (1989).

Where: VC = growth rate (mm day-1); Df = final growth diameter (mm); Di= initial growth diameter (mm); Ti = initial growth time (days); Tf = final growth time (days).

The percentage of inhibition (% I) was determined by applying the formula of Fokkema (1973), cited by Kagezi et al. (2015).

Where: % I= percentage of inhibition of mycelial growth; D1= diameter of the mycelial growth of the control (mm); D2= diameter of mycelial growth of the influenced (mm).

To calculate the percentage of growth the formula was used.

Where: %C= percentage of mycelial growth; CI= influenced growth (mm); CT= control growth (mm).

To determine the class of antagonism under dual growth conditions, the scale proposed by Bell et al. (1982): 1. The antagonist overgrows the plant pathogen and covers 100% of the Petri dish; 2. The antagonist covers 75% of the Petri dish, stops the plant pathogen and can overgrow and sporulate on it; 3. No organism is dominant, each one covers 50% of the surface (antagonist and pathogen); 4. The phytopathogen covers 75% of the Petri dish and stops the growth of the antagonist and can overgrow and sporulate it; and 5. The plant pathogen overgrows the antagonist and covers 100% of the Petri dish.

Statistical analysis

For the statistical evaluation of the experiment, the data were subjected to a combined analysis of variance and Tukey's mean comparison test (p≤ 0.05) corresponding to a completely randomized experimental design with five replications. This analysis was carried out using the Statistical Analysis System software (SAS 9.0) with the Proc Anova procedure.

Results

The antagonistic agent T. harzianum controlled the growth of the mycelium of B. cinerea by inhibiting it in 36%, although its growth rate was lower (7.44 mm day-1) than that of the pathogen in the control treatment (9.8 mm day-1) (Table 2), in the Botrytis-Trichoderma treatment, Trichoderma managed to stop the development of Botrytis at the moment of contact (Figure 1). The coefficients of variation were 6 to 17%.

Table 2. Comparison of growth responses of T. harzianum vs. B. cinerea.

VCD= diametral growth rate (mm day-1); t(VCD)= VCD data transformation; INH= growth inhibition percentage; CREC= diametral growth percentage; DMS= minimal significant difference (Tukey, p≤ 0.05); CV= coefficient of variation; Z= averages with the same letter within the column are statistically the same. Values are means of five repetitions at 8 days after sowing.

Figure 1. T. harzianum antagonism vs Botrytis cinerea. a) absolute control with B. cinerea; b) and c) T. harzianum (right) and B. cinerea (left).

Hyphal contact between the two pathogens occurred on the sixth day. Trichoderma had exponential growth from the second day, exceeding half of the Petri dish before Botrytis, stopping its growth and at the same time causing the death of hyphae in the zone of interaction with B. cinerea (Figures 1 B and C). The dual test with these fungi highlighted a type two antagonism, which is characterized because T. harzianum invaded 75% of the Petri dish, in addition to overgrowing and sporulating on the plant pathogen, T. harzianum gradually invaded the growth of Botrytis until it was completely covered.

Treatments with substances mixed in the culture medium influenced the response of B. cinerea and coefficients of variation of 2 to 31% were recorded (Table 3). Botrytis mycelium had 100% growth in PDA, the highest growth rate (VCD) (9.8 mm day-1) and without mycelial inhibition; however, with the chemical fungicide the growth and the VCD were null, and the mycelial inhibition 100%. With T. lemmonii oil, from 24 h after inoculation of the fungus until the end of the experiment, the diameter of the mycelial inoculum was not modified, therefore, no VCD and 100% inhibition of the pathogen, a response similar to obtained with the fungicide product.

Table 3. Multiple comparison of means for the response variables of the bioassay.

VCD= diametral growth rate (mm day-1); t(VCD)= VCD data transformation; INH= growth inhibition percentage; CREC= diametral growth percentage; DMS= minimal significant difference (Tukey, p≤ 0.05); CV= coefficient of variation; Z= averages with the same letter within the column are statistically equal.

With governor extract (L. tridentata) the fungus grew 34%, VCD of 2.82 mm day-1 and mycelial inhibition of 65.8%. With homeopathic arsenic, the VCD was 8.56 mm day-1 and 11.8% inhibition, response statistically the same as the absolute control. With Botrytis nosode, the VCD was 6.76 mm day-1 and 31.24% inhibition, statistically different from the control treatment. Both in the homeopathic substances and in the PDA control, Botrytis had the highest growth percentage (68 to 100%).

In general, the fungicide Switch® and the essential oil of T. lemmonii were inhibitory of the growth of the mycelium and in the case of the biological agent T. harzianum there was antagonism against B. cinerea, these treatments were of high antifungal effect compared to those of inhibition mean of the governor extract (65%) and those with low inhibition (11 and 31%) of homeopathic substances (Table 3; Figure 2).

Figure 2. End of the 8-day bioassay when the absolute control completely covered the Petri dish, a) absolute control; b) T. lemmonii; c) homeopathic arsenic; d) Botrytis homeopathic; e) Switch®; f) governor; and g) antagonic Trichoderma harzianum.

Considering the diametral growth rate (using the transformation) on the eighth day, the high growth rate of the mycelium in PDA (9.8 mm day-1) and in homeopathic arsenic (8.56 mm day-1) stood out; the response was intermediate with Botrytis nosode (6.76 mm day-1), low speed with the governor extract (2.82 mm day-1) and null with T. lemmonii or Switch® (Table 3).

The dynamics of the growth rate of the mycelium in contact with the organic and homeopathic substances evaluated during the eight days (Figure 3) presented particular trends. Until the second day, all treatments had a similar response (10 mm day-1), but on the third day the growth rate of the fungus in the homeopathic and PDA control treatments increased from 19 to 31 mm day-1 on the 5th day. With the L. tridentata extract the growth of the fungus remained stable from day 2 to 4 (10 mm day-1), but on the 5th day it increased slightly (14 mm day-1) and continued to increase with a slow trend during the three subsequent days (19, 21 and 26 mm day-1).

In the control and in homeopathic arsenic and nosode, on day five it was observed that the mycelium acquired a different growth rate (35, 33 and 31 mm day-1, respectively), a trend more defined on days 6 (46, 42 and 38 mm day-1, respectively), 7 (59, 53 and 45 mm day-1, respectively) and 8 (76, 66, 54 mm day-1, respectively), separating the Botrytis nosode further in those last four days, by reducing the growth rate of the mycelium with respect to the control (decrease in VCD of 4, 8, 14 and 22 points, respectively) and homeopathic arsenic (decrease in VCD of 2, 4, 8 and 12 points, respectively).

Figure 3. Diametrical growth of the mycelium (mm∙day-1) of Botrytis cinerea during the in vitro evaluation period of the treatments: control (PDA), governor extract, homeopathic arsenic and B. cinerea nosode. The substances T. lemmonii and Switch® do not appear because the fungus did not show growth.

Discussion

The antagonistic effect of T. harzianum against strawberry gray mold in the in vitro study model confirms other antecedents in this regard (Calvo, 2012). Probably the temperature of 18 °C did not favor a higher growth rate of Trichoderma in the first days in relation to that reached by Botrytis in this regard, it is indicated that the optimal temperature for Trichoderma is 25 °C (Merchan, 2014). The result of the Trichoderma-Botrytis antagonism confirms the direct use of the biological agent in the fruiting and post-harvest stages of strawberry (Merchan, 2014).

However, since this result is favorable to decrease or replace fungicide products, the production or purchase of Trichoderma represents an investment cost, perhaps less than the purchase of the fungicide itself. For its part, the agrochemical had an immediate effect, reported both in vitro and in vivo in strawberry (Mercier et al., 2010); however, the negative implication to the human and the environment are food for thought. As for organic substances, the inhibitory effect of the governor extract coincides with that observed in other studies where this plant substance is attributed the property of fungistasis in Botrytis (Lira, 2003).

Regarding the outstanding antifungal effect of T. lemmonni oil, similar to that produced by the product Switch (Table 3), it had already been reported against Fusarium oxyosporum (Jarquin, 2016), but not with the same intensity with which it occurred against Botrytis, a result that is reported for the first time; the presence of dihydrotagetone, (E) tagetone and (E) ocimenone in the essential oil (Tucker and Marciarello, 1996) are possibly the substances responsible for the described biological activity. Due to the similarity of the response of the mycelium submitted to Switch® and to T. lemmonni oil, it is suggested that the essential oil of this species of Tagetes has a biocidal property. An ongoing investigation aims to explore other concentrations and establish the inhibition concentration, in addition to its toxicological evaluation in vivo.

The result obtained on the effect of mycelial inhibition of B. cinerea caused by the nosode of the same pathogen (Table 3; Figure 1) is of special importance because it would be the seventh experience that shows direct effect of homeopathic substances on the growth of phytopathogenic fungi (Gama et al., 2015; Rissato et al., 2016; Toledo et al., 2016; Hanif and Dewar, 2017; Oliveira et al., 2017; Serrato et al., 2018).

In vitro bioassays of application by fumigation with nosode directly on mycelium or spores could solidly confirm some published results and also those of the present work. On the other hand, Botrytis 7 CH nosode was better than 6 CH homeopathic arsenic (Figure 1), when the latter in that same potency, in vitro conditions inhibits the germination of Alternaria spores (Trebbi et al., 2016) This comparison suggests specificity of the homeopathic according to the dynamisation or specificity by biological origin of the homeopathic substance.

The successful results related to the application of homeopathic substances against fungal problems in plants indicate that the fungus does not establish in the plant (Hanif and Dewar, 2015) or, it grows in a restricted way without damage to the plant (Rissato et al., 2016 ; Hanif and Dewar, 2017; Oliveira et al., 2017) furthermore, it is not clear whether nosode or other homeopathic substances have the same inhibitory effect, nor is it established whether their action is direct against the pathogenic organism. Therefore, the in vitro model has favored partially answering the previous questions.

In this case, it was evident that the fungus is influenced in its growth by the homeopathic product. Taking into account published works on the action of homeopathic substances applied to the foliage of plants to control fungal diseases, it follows that the homeopathic stimulus, either by substances derived from the pathogenic organism or from any other origin, trigger plant response mechanisms to your protection. It has recently been shown that nanoparticles with greater biological activity than with macromolecules are generated with the dynamization and succussion process of substances (Rajendran, 2017).

From an ecological perspective, organic, biological and homeopathic treatments offer advantages compared to synthetic fungicide. The possibility of reducing the concentration of essential oil of T. lemmonii is considered important to strengthen the threshold of use of this substance, especially in a preventive strategy, additionally its economic viability would be valued. Rudilla oil is remedial in action, while Governor's extract, Trichoderma, and homeopathic substances are slow and may be preventative in their best performance.

Especially the low cost of Botrytis nosode represents a promising advantage that stimulates research to continue exploring different dynamizations from those tested or to test other homeopathic substances, whenever there is accompanying in vivo work, that in general they are few in the subject of agrohomeopathy.

Conclusions

In vitro conditions in which responses of B. cinerea mycelium were evaluated, antagonistic effect of T. harzianum, biocidal effect of the agrochemical and essential oil of T. lemmonii, and fungistatic effect of L. tridentata extract and nosode 7 CH were evidenced.

Cited literature

Balandrin, M. F.; Klocke, J. A.; Wurtele, E. S. and Bollinger, W. H. 1985. Natural plant chemicals: sources of industrial and medicinal materials. Science Magazine. 228(4704):1154-1159.

Barajas, P. J. S., Montes, B. R.; Castrejón, A. F.; Flores, M. H. E. y Serrato, C. M. A. 2011. Propiedades antifúngicas en especies del género Tagetes. Rev. Mex. Micol. 34:85-91.

Bell, D. K.; Well, H. D. and Markham, C. R. 1982. In vitro antagonism of Trichoderma species against six fungal plant pathogens. Phytopathology. 72(4):379-382.

Bojórquez, V. J. J. 2017. Actividad fúngica in vitro de extractos orgánicos contra patógenos de tomate (Solanum lycopersicum). Tesis de Maestría. Universidad Autónoma Chapingo (UACH). México. 102 p.

Calvo, A. J. A. 2012. Aislamiento y evaluación in vitro de antagonistas de Botrytis cinerea en mora. Rev. Agron.  Mesoam. 23(2):225-231.

Cano, M. 2013. Estrategias biológicas para el manejo de enfermedades en el cultivo de fresa (Fragaria spp.). Rev. Colomb. Cienc. Hortíc. 7(2):263-276.

Carneiro, S. and Bueno, E. 2010. Effect of biotherapic of Alternaria solani on the early blight of tomato plant and the in vitro development of the fungus. Inter. J. High Dilution Res. 9(33):147-155.

Ceredi, G.; Antoniacci, C.; Montuschi, E.; De Paoli, G. and Gengotti, S. 2009. Ten years of field trial on grey mold control on strawberries. Acta Hortic. 842(60):327-330.

Chávez, N. 2004. Combate del moho gris (Botrytis cinerea) de la fresa mediante Gliocladium rosenum. Rev. Agron. Costarric. 28(2):73-85.

Gama, E.; Silva, F.; Santos, I.; Malheiro, R.; Soares, A.; Pereira, J. and Armond, C. 2015. Homeopathic drugs to control red rot disease in sisal plants. Brazilian J. Agron. Sustainable Development. 35(2):649-656.

García, T. E. 1984. Compendio de la materia médica homeopática. Ed. Propulsora de Homeopatía. 4ta (Ed.). México, DF. 17-21 pp.

Hanif, A. and S. Dawar. 2015. Use of homeopathic drugs in combination with fertilizers for the control of root rot fungi. Pakistan J. Bot. 47(6):2455-2462.

Hanif A. and Dewar S. 2017. Antifungal activity of homeopathic drugs treated seeds against root decay pathogens and improvement of growth on crop plants. Pakistan J. Bot. 49(SI):355-362.

Jarquín, C. S. 2016. Evaluación in vitro de fungicidas químicos, orgánicos y biológicos contra Fusarium oxysporum sp. ricini en higuerilla. Tesis de Licenciatura. Universidad Autónoma Chapingo (UACH). México, DF. 110 p.

Kagezi, G.; Kucel, P.; Olal, S.; Pinard, F.; Seruyange, J.; Musoli, P. and Kangire, A. 2015. In vitro Inhibitory effect of selected fungicides on mycelial growth of ambrosia fungus associated with the black coffee twig borer, Xylosandrus compactus Eichhoff (Coleoptera: Curculionidae) in Uganda. African J. Agric. Res. 10(23):2322-2328.

Lira, S. R. H. 2003. Estado actual del conocimiento sobre las propiedades biocidas de la gobernadora [Larrea tridentata (D.C.) Coville]. Rev. Mex. Fitopatol. 21(2):214-222.

López, L. E.; Peña, O. M. G.; Colinas, L. T. B.; Díaz, C. F. y Serrato, C. M. A. 2018. Fungistasis del aceite esencial extraído de una población de Tagetes lucida de Hidalgo, México. Rev. Mex. Cienc. Agríc. 9(2):329-341.

Lorenzetti, E.; Stangarlin, J. R.; Treib, E. L.; Heling, A. L.; Coltro-Roncato, S.; Carvalho, J. C.; Hoepers, L.; Rissato, B. B.; Coppo, J. C.; Belmonte, C.; Kuhn, O. J. and Silva, I. F. 2016. Antimicrobial action against of Macrophomina phaseolina and control of the grey stem in soybean by homeopathic remedies Nosode and Sulphur. African J. Agric. Res. 11(36):3412-3417.

Meneses, M. N. 2007. Agrohomeopatía una opción para la agricultura. Boletín Informativo de Homeopatía Agrícola. 1(6):1-25.

Merchán, G. J. B. 2014. Efecto de dos cepas de Trichoderma en el control de Botrytis cinerea y la calidad del fruto en fresa (Fragaria sp.). Rev. Colomb. Cienc. Hortíc. 8(1):44-56.

Mercier, J.; Kong, M. and Cook, F. 2010. Fungicide resistance among Botrytis cinerea isolates from California strawberry fields. https://www.plantmanagementnetwork.org/pub/php/ research/2010/strawberry/.

Oliveira, J. S. B.; Gomes, S. M. T. P.; Schwan-Estrada, K. R. F.; Mesquini, R. M.; Bonato, C. M. y Romano, E. D. B. 2013. Patogenesia do óleo essencial e homeopatias de Eucalyptus citriodora em plantas de feijão (Phaseolus vulgaris). Brazilian J. Medicinal Plants. 15(4):734-741.

Oliveira, J. S. B.; Schwan-Estrada, K. R. F.; Bonato, C. M. and Gomes, S. M. T. 2017. Homeopathy with essential oils in the germination of spores and induction of phytoalexins. Rev. Cienc. Agron. 48(1):208-215.

Rajendran, E. S. 2017. Nano pharmacological aspect of homeopathic drugs -a comparative study of different scales of ultra-high dilutions based on HRTEM analysis and np characterization of homeopathic drug natrum muriaticum 6C-CM and LM1 - LM30. Saudi J. Medical Pharmaceutical Sci. http://scholarsmepub.com/sjmps/.

Rissato, B.; Stangarlin, J.; Coltro, S.; Dildey, O.; Goncales, E. and Lorenzetti, E. 2016. In vitro activity of homeopathic drugs against Sclerotinia sclerotiorum. J. Sci. Agrar. Paranaensis. 15(3):320-323.

Romagnoli, C.; Bruni, R.; Andreotti, E.; Rai, M. K.; Vicentini, C. B. and Mares, D. 2005. Chemical characterization and antifungal activity of essential oil of capitula from wild Indian Tagetes patula L. Protoplasma. 225(1-2):57-65.

Sandoval, L. G. 1961. Farmacopea homeopática mexicana. Ed. Propulsora de Homeopatía. México. 36-37 pp.

Santoyo, J. J. A. 2009. Paquete tecnológico para la producción de fresa. Fundación Produce Sinaloa, AC. SAGARPA. México. 21-35 pp.

Serrato, C. M. A. 2014. El recurso genético Cempoalxóchitl (Tagetes spp.) de México (diagnóstico). Universidad Autónoma Chapingo (UACH)- SINAREFI-SNICS-SAGARPA. 182 p.

Serrato, C. M. A.; López V. E. Y.; Ruiz E., F.; Solano, V. R. y Hernández, H. I. 2018. Evaluación in vitro de aceites esenciales y preparados homeopáticos en el crecimiento de algunos hongos fitopatógenos. In: homeopatía hoy. Ruíz, E. F. de J. y Durán, C. V. (Coords.). Universidad Autónoma Chapingo (UACH). Texcoco, Estado de México. 83-89 pp.

Sinclair, C. G. and Cantero, D. 1989. Fermentation modelling. In: fermentation a practical approach. McNeil, B. L. and Harvey, M. (Eds.). IRL Press, New York. 65-112 pp.

Taborda, A. L. A. 2015. Efecto fungistático de extractos y aceites esenciales de Lippia origanoides HBK y Thymus vulgaris L. como alternativas de manejo de Botrytis cinerea en fresa. Rev. Acta Agron. 11 (39):3824-3838.

Tichavsky, R. 2007. Manual de agrohomeopatía. Instituto Comenius en colaboración con la Secretaría de Desarrollo Social. México. 30-71 pp.

Toledo, M. V.; Stangarlin, J. R.; Bonato, C. M.; Mioranza, T. M.; Müller, M. A.; Rissato, B. B.; Lorenzetti, E.; Coltro-Roncato, S.; Kosmann, C. R. and Assi, L. 2016. Fungitoxicity activity of homeopathic medicines on Alternaria solani. African J. Agric. Res. 11(39):3824-3838.

Trebbi, G.; Nipoti, P.; Bregola, V.; Brizzi, M.; Dinelli, G. and Betti, L. 2016. Ultra-high diluted arsenic reduces spore germination of Alternaria brassicicola and dark leaf spot in cauliflower. Hortic. Bras. 34(3):318-325.

Tucker, A. O. and Maciarello, M. 1996. Volatile leaf oil of Tagetes lemmonii Gray. J. Essential Oil Res. 8(4):417-418.

Zepeda, L. G. 2002. Diccionario médico homeopático ilustrado. Ed. Porrrúa. México. 33 p.