Introduction

The use of synthetic pesticides have caused problems due to their irrational use, such as: pest resistance to pesticides, presence of residues in food chains and physical environment (air, water, and soil), destruction of beneficial fauna that is responsible for natural control of potential pests, pest resurgence after treatment with pesticides. Therefore, the unintelligent use of pesticides has led to the reduction of trophic interaction between organisms, biodiversity loss, ecological imbalance and decrease of environmental stability (^{López et al., 2006}).

The need to reduce the use of synthetic chemicals in agriculture has increased the interest by possible application of plant extracts and essential oils for the control of plant pathogens. Plant extracts and essential oils of each plant can have over sixty components and of them can be several with antifungal properties; generally are present as compounds mixtures and pathogens may be affected differentially by individual compounds or by mixtures in certain concentrations and ratios (Montes, 2009).

Several authors around the world have reported the antifungal activity of plant extracts, essential oils and their compounds performing in vitro, in greenhouse and field trials; using different extraction methods for metabolites and showing varying responses depending on the species, parts used, concentration, strains of pathogens and assessment technique used (^{Wilson et al., 1997}; ^{Gogoi et al., 1997}; ^{Pitarokili et al., 1999}; Montes et al., 2000; ^{Meepagala et al., 2002}; ^{Hernández et al., 2007}; ^{Ramírez, 2013}).

However traditional extraction methods to obtain extracts have limited applicability because they require high processing times, toxic solvents, high temperatures that affect the quality of the final extract and not economically competitive with other alternatives of control (^{Ramírez, 2006}).

As alternative have been developed new extraction techniques environmentally friendly, the product and the environment, low risk levels to human health and superior quality in bioactive products. Among these alternatives are: the use of supercritical fluids that selectively extract certain families of compounds with high performance and temperatures below 50 °C (^{Martínez et al., 2011}); microwave assisted extraction (MAE) which requires less time, less use of solvents with higher extraction rate, saving energy and better products at lower cost and ultrasound-assisted (UAE) which significantly reduces processing times thanks to the action of ultrasound higher than 16 KHz inside the extractor (^{Hayat et al., 2009}).

Crops such as tomatoes and papaya are of great economic importance for many countries, and specifically for the state of Chiapas are considered as an alternative for many producers, according to the Agro food and Fishery information service for 2014, the state ranked fourth nationally in papaya growing area with a yield of 80 t ha^-1, while for red tomato ranked twelfth and with a yield of 35.72 t ha^-1 considered low compared to other producing states. A common factor in the production of these two crops is high consumption of pesticides that allow them to control plant health problems, which leave residues in fruits, increased production costs and contribute to environmental pollution. So tomato and papaya product systems have included in their master plans the need to implement alternative management for major diseases that are a limiting factor for their production (^{SAGARPA, 2012}).

Thus diseases in tomato such as Alternaria (Alternaria solani) and fusarium (Fusarium oxysporum) and anthracnose (Colletotrichum gloeosporioides) in papaya are primarily treated with chemical pesticide, plus some producers to maintain a longer shelf life of their products without the presence of pathogens apply products a few days before taking them for sale, so pesticides are unable to degrade, thereby reducing the biological quality of products to market.

This work studied in vitro effectiveness of plant extracts from Pimienta dioica, Cinnamomum zeylanicum Nees, Syzygium aromaticum L., obtained by distillation and microwave on Colletotrichum gloeosporioides isolated from Carica papaya, as well as pathogens Alternaria solani and Fusarium oxysporum isolated from Lycopersicum esculentum with the purpose to generate viable alternatives to manage these pathogens.

Methodology

In the Laboratory of agro-technologies from the Autonomous University of Chiapas were isolated and characterized the fungi Alternaria solani and Fusarium oxysporum from tomato and Colletotrichum gloeosporioides from papaya collected in the Frailesca region, of the state of Chiapas, Mexico; which they were incubated on PDA medium at 23° +/- 2 °C. The plants of which extracts were obtained to determine their effectiveness to control fungi were Pimienta dioica, dried bark of Cinnamomum zeylanicum Nees and dried flower buds of Syzygium aromaticum L., with which were prepared the extracts using two extraction methods:

Hydrodistillation: a distiller adapted to obtain extracts was used. The dried plant material (250 g L^-1) was placed inside the pot distiller along with water, hermetically closed for the continuous extraction process through the application of heat and constant pressure, the steam was taken to a condenser and, by cooling with tap water the hidrodistillate was obtained.

Microwave assisted hydrodistillation extraction: a microwave radio frequency adapted to extract metabolites, under the following conditions was used: 220 Kw power, period of 1 800 s and 2 450 MHz. To perform the extraction was placed in the output of the equipment a reflux condenser with cold water. The sample was placed in gauze retained by a cotton mesh, the vapor generated at the bottom of the flask, to this end 300 ml of purified water were placed and the steam generated crossed the samples pulling the essential oil with the steam. Water began to boil after 590 s reaching a temperature of 89 °C. A final temperature of 91 °C was obtained. It worked with a sample of 250 g. Finally the essential oil of the hidrodestillate was separated, for this purpose a separatory funnel was used.

Trial 1. Screening to determine the effectiveness of extracts: poisoned medium with the extract was used. Initially, a screening test in which each extract was added individually to the medium potato-dextrose-agar (PDA) at a concentration of 30% (vol/vol) was performed for hidrodestillates, for oils it was 0.1%; once the medium is prepared with the extract, it proceeded to pour it in sterile petri dishes and inoculation of the respective fungus was performed. Cultures were maintained in culture room under controlled conditions of 23 °C +/- 2 °C and an absolute control in which the fungus was cultured in the original medium (PDA) without any control was included. A total of 10 treatments per pathogen and a total of 30 for this test were studied.

The inhibitory effect was quantified through growth every 24h of mycelia diameter from pathogen for 12 days. Spore production was also quantified by Neubauer chamber. The experimental unit consisted of a petri dish and the treatments were distributed in a completely randomized design with five replications. To determine the effects of the treatments under study, the data underwent an analysis of variance and Tukey at 0.05%.

Trial 2. Determination of minimum inhibitory concentration (MIC)

Extracts showing complete growth inhibition and pathogen development, were determined MIC; evaluating concentrations of 20, 10 and 5% (V/V) and for oils 0.05 oil and 0.01% (V/V). Similarly agar diffusion was employed and growth and conidia formation with the same procedure described for the above test was evaluated.

Results

Trial 1. The results for the two variables evaluated for each fungus are presented in Table 1. In the case of Alternaria solani it can be seen how clove (obtained by distillation and microwave) and oil did not allow growth and development of the pathogen, while for cinnamon this inhibitory effect was made by the microwave and oil treatments which recorded statistically significant differences with respect to absolute control. The analysis of variance conducted for the three pathogens recorded differences between treatments.

In the case of pepper, both Hydrolates and oils allowed the growth and formation of conidia of the pathogen; however, it is appreciated that treatments allowed mycelial growth inhibiting between 58.61% to 28.31% conidia formation compared to absolute control, showing statistically significant differences with it.

These results show higher inhibition percentages in growth and conidia formation to that reported by other authors; Such as ^{Pupo et al. (2011)} who evaluated 11 Cuban naturalized plants on Alternaria solani and found that flowers extracts of T. erecta and leaves of L. peltata stimulated the growth of A. solani. While flower extracts of L. camara and complete plant of C. viscosa with 2 mg mL^-1 inhibited more than 50% conidia germination and with 3 mg ml^-1 exceeded 80%. Flower extract of L. dulcis, at the same dose, reached 93.5% inhibition which was statistically similar to the activity shown by Zineb (91.2%). ^{Santander and Aquino (2009)}, report the regulating effect of the aqueous extract of Allium sativum at concentrations above 50%, however report fungistatic effect since showed growth after 7 days of incubation.

In the case of F. oxysporum, clove (both obtained by distillation and microwave) exerted total inhibitory effect, as cinnamon obtained by microwave, however in cinnamon and clove oils and pepper obtained by microwave, although registered growth, conidia formation was not present, and showed statistical differences compared with the control, which recorded the highest growth values and conidia formation. These results confirm those reported by López-Benítez et al. (2005), who showed that aqueous extracts of cinnamon and clove inhibited the growth of F. oxysporum, however showed how clove extract had lower activity on this pathogen than on Rhizoctonia solani and Verticullium danliae at 144 h of incubation.

Research by ^{Barrera and García (2008)} indicate that cinnamon and clove oils did not completely inhibit the growth of Fusarium sp. Isolated from papaya, being clove oil which showed the greatest inhibition (84 to 29%), while cinnamon recorded inhibition of 68 to 30% using 100 mg mL^-1 - 300 mg mL^-1, results that agrre with the ones from this investigation since neither of the two oils inhibited the growth of F. oxysporum; however, the highest inhibition percentage was recorded for cinnamon oil with 78%, while clove oil was 69%, difference that may be due to the origin of the material or from the extraction process.

The results of treatments effect on C. gloeosporioides, shows that only in oil pepper allowed the growth and conidia formation surpassing even absolute control, while cinnamon obtained by distillation, only allowed growth of the pathogen , registering difference with other treatments, which inhibited both growth and conidia formation, appreciating that the extraction process using microwaves allows to extract larger amount of active metabolites, since the same concentration completely inhibited growth and conidia formation.

The results obtained with clove and cinnamon oils do not match those reported by ^{Martínez et al., (2012)}, who evaluated different concentrations of these oils integrating them into edible films to coat papayas finding that at concentrations of 0.08 to 0.06% reduced the pathogens infection being more effective clove oil with inhibition of 88.9%, while the cinnamon had 78%.

However, ^{Guillén- Sánchez et al. (2014)}, also using chitosan films, beeswax / oleic acid mixed with thyme, cinnamon and clove oil report regulatory effect of the three oils but at concentrations over 0.5 and 1%, being the most sensitive fungus C. gloeosporioides to the oils effect in inhibiting its mycelial growth, while Penicillium digitatum, Rhizopus stolonifer, Fusarium oxysporum and Alternaria alternata only presented inhibition at higher concentrations.

Trial 2. Determination of minimum inhibitory concentration (MIC).

MIC was determined for each of the extracts that completely inhibited growth and development of the pathogen in the latter trial. For A. solani the results are presented in Table 2, analysis of variance performed recorded differences between treatments; for clove obtained both by distillation as microwave at 20% concentration recorded growth but did not allow conidia formation, effect that was also recorded by clove obtained by microwave at 10% concentration, while distillation at the same concentration had higher growth and conidia formation, as well as hidrodistillates at concentration of 5%, although all showed inhibition in conidia formation compared with the control with whom the statistical analysis detected differences but did not record statistical difference between them for this variable, similar to that reported by ^{Ramírez (2013)}, who reported that cinnamon and cloves obtained by distillation at concentrations of 10 and 20% (v/v), although allowed growth of Moniliophthora roreri, inhibited conidia formation.

Regarding cinnamon oil, totally inhibited growth and conidia formation at a concentration of 0.05%, while at 0.01% decreased by 50.23% the amount of conidia and 16.5% mycelial growth and recorded statistical differences with the absolute control; while clove oil showed growth inhibition and conidia formation at concentration 0.05%, and 0.01%, inhibiting 100% conidia formation and 27% mycelial growth.

These results indicate a greater effect of cinnamon extracts obtained either by distillation or microwave assisted on A. solani, since its MIC were lower than those presented by cloves and pepper extracts; and shows that the hidrodistillate obtained with microwave turns out to be more effective, since both extracts from clove and cinnamon at the same concentration had higher regulatory activity than those obtained by distillation. cinnamon obtained by microwave at 10%, and cinnamon and clove oil at 0.05%, were the treatments that had the best regulatory effect fir this pathogen of tomato, even with better results than those reported by other authors like ^{Cortina (2014)}, who evaluated cinnamon and clove oil on Alternaria alternata isolated from rice, since 300 µg /mL, did not record total growth inhibition, and ^{Ramirez (2013)} which recorded a MIC for M. roreri of cinnamon 30% (v/v) and clove 20% (v/v) hidrodistillate.

In the case of C. gloeosporioides, initially concentrations of 20 and 10% of pepper, cloves and cinnamon hidrodistillates and of 0.05% for of clove and cinnamon oil were assessed; analysis of variance indicated statistical differences. The results are presented in Table 3, showing complete growth and sporulation inhibition for clove both obtained by distillation and microwave, and hydrodistillates obtained by microwave of cinnamon at 20 and 10% and pepper at 20%, thus clove and cinnamon oils at 0.05%, which recorded difference to absolute control.

So it was necessary in the case of this pathogen, to perform a second triak and be able to determine MIC, evaluating concentrations below, 5% for cinnamon, clove and pepper obtained by microwave and cinnamon and clove by distillation and 0.01% for oils (Table 4), all treatments inhibited in different percentages growth and conidia formation, registering statistical differences with absolute control, being cinnamon and clove oil and clove obtained by microwave which inhibited to a greater degree (79- 55%), so their MIC would be 10% and 0.05% for hidrodistillates and oils respectively.

The above results allowed us to appreciate the regulatory effect of hydrodistillates and oils of cinnamon, clove and pepper for three fungi evaluated both on pathogen growth, as in conidia formation in different inhibition percentages, however the results indicate differences in activity of these extracts and oils on the three fungi, being C. gloeosporioides the pathogen that was more sensitive to hidrodestillates from the three plants and to cinnamon and clove oil, while for the F. oxysporum pepper hidrodistillate in the two forms (distillation and microwave) and cinnamon by distillation at 30% concentration did not inhibited the pathogen in its totality as well as cinnamon, clove and pepper oils to the assessed concentration, which although presented inhibition percentages in growth of 73.2 to 6.5% and in conidia formation of 100 to 22.4%, did not inhibited completely.

The high antifungal capacity found in essential oils of C. zeylanicum and S. aromaticum coincides with the results reported by ^{Montes and Carvajal (1998)} who observed that Aspergillus flavus was completely inhibited by the essential oils of these plants. ^{Wilson et al. (1997)} evaluated 49 essential oils and found that C. zeylanicum showed the highest antifungal activity against Botrytis cinerea. Bravo et al. (1998) found that essential oils of M. piperita, E. globulus, T. ambrosioides, C. zeylanicum, S. aromaticum and T. vulgaris at doses of 10 000 and 7 500 ppm inhibited the mycelial growth of Fusarium moniliforme. These authors also report inhibitory effect on sporulation of the same fungus at doses of 150 to 300 ppm of C. zeylanicum, S. aromaticum and T. vulgaris oils.

Fungicidal activity of C. zeylanicum and S. aromaticum against pathogens causing anthracnose in banana has been reported by ^{Ranasinghe et al. (2002)}. The fungitoxic properties of essential oils from six populations of Thymus zygis against Rhizoctonia solani, Fusarium oxysporum and Colletotrichum acutatum were reported by ^{Pérez et al. (2007)}. These authors also published the chemical composition of essential oils and the correlation between compounds concentration and antifungal activity reported by ^{Barrera and García (2008)}.

The results of this research indicate that both traditional method of hydrodistillation and microwave assisted using clove, cinnamon and pepper allows to extract metabolites capable of inhibit in different percentages both growth and conidia formation, however it is appreciated that microwave assisted allows to obtain higher concentration of them, which allowed to decrease MIC in which affect to the pathogens compared to traditional distillation method. Corroborating the report from Zharia (2011) cited by ^{Aguilar and López (2013)}, who used clove hidrodestillation coupled to microwave and was compared with traditional hidrodestillation where there were no major differences as for components obtained from essential oils recovered by both methods; however the extraction time and the sample size were lower for microwave. The results of this research provide alternatives using hidrodestillates and vegetable oils, which must be adjusted to management plans for each crop and establish its effectiveness in the field.

Conclusions

Hidrodestillates and C. zeylanicum, Z. aromaticum and P. dioica oils obtained by traditional distillation and microwave assisted possess metabolites capable to reduce growth and conidia formation of A. solani, F. oxysporum and C. gloeosporioides, which can be an alternative for handling these pathogens of tomato and papaya.

C. zeylanicum, Z. aromaticum and P. dioica hydrodistillates obtained using microwaves appear to have a greater amount of active compounds than those obtained with distillation, since MIC were lower or inhibitory activity on fungi at the same concentration was superior.

Pathogens C. gloesporioides and A. solani seem to be more sensitive to the effect of hydrodistillates and oil of C. zeylanicum, Z. aromaticum and P. dioica, than F. oxysporum.

Clove obtained by distillation completely inhibited the development of A. solani and F. oxysporum at a concentration of 30% (v/v) and at 10% C. gloeosporioides, while microwave-assisted of cinnamon inhibited A. solani and C. gloeosporioides at 10% concentration (v/v), and 30% to F. oxysporum; and clove at 30% inhibits F. oxysporum and A. solani and at 10% C. gloeosporioides and cinnamon and clove oils at 0.05% inhibited A. solani and C. gloeosporioides.