Introduction

Tomato is a vegetable that generates currency for Mexico (^{SIAP, 2014}), it is also one of the hosts most affected by nematodes, among them Nacobbus aberrans, which cause yield losses of 36 to 55% in the greenhouse (^{Manzanilla et al., 2002}) and from 50 to 100% in the field (^{Mendoza, 1999}). Chemical control is the most used method against nematodes, but it induces resistance, in addition to eliminating natural enemies (^{Gutiérrez et al., 2013}). On the other hand, nematicides are expensive and not easily accessible to small producers; consider that in the last decades the availability of efficient commercial products has been reduced (^{Sorribas and Ornat, 2011}).

Given the economic importance of this vegetable and the consequences of using nematicides, alternatives are being explored such as biological control, genetic resistance of tomatoes and the use of vegetable substances. The use of aqueous extracts and essential oils, due to their cytotoxic properties, are potential inputs for the control of pests and diseases (^{Isman, 2000}). In particular, essential oils have a promising use in the control of nematodes (^{Andrés et al., 2012}).

Although there is great plant diversity in Mexico, the biological properties of several plants that could be useful in the control of nematodes are unknown (^{Silva et al., 2005}). The most known botanical families for the presence of essential oils are: Asteraceae, Lamiaceae, Lauraceae, Labiateae, Myrtaceae, Poaceae, Rutaceae, Turaceae and Umbeliferae (^{Andrés et al., 2012}), but Asteraceae is the most studied and recognized as a source of compounds with pesticide properties (^{Choi et al., 2003}). Asteraceae represents 18.3% of the total species that make up the flora of Mexico (^{Villaseñor et al., 2005}), in this family the Tagetes (Tageteae) stands out for its allelopathic potential against plant parasitic nematodes, documented for more than 75 years (^{Steiner, 1941}).

In this regard, ^{Tyler (1938)} reports that 29 species of Tagetes are not attacked by Meloidogyne spp. Root-knot nematodes, because the excretions produced by the root, which reduce the incidence of nematodes in the soil, contain thiophenes, polyacetylene compounds that they are secondary metabolites responsible for the biological effect (^{Marotti et al., 2010}). Due to these chemical properties of Tagetes, plants of this genus are used as an intercropped, imbricate, covert or rotating crop with other species of economic importance for nematode control (^{Serrato and Argomedo, 1993}); On the other hand, the use of Tagetes essential oils as an applicable input against nematodes is being considered, since this natural resource is abundant in Mexico (^{Serrato, 2014}). In this regard, there are several references to the effect of Tagetes essential oil on nematodes; For example, it is reported that T. minuta oil controls eggs and juveniles of Meloidogyne incognita (^{Adekunle et al., 2007}), T. erecta has a toxic effect against populations of nematode eggs of Haemonchus contortus (^{Macedo et al., 2013}) and the nematicidal effect of T. zypaquirensis oil was demonstrated (^{Álvarez et al., 2016}).

In particular, T. lucida or pericon is a species that is widely distributed in Mexico, mainly in agricultural lands such as arvense or ruderal at elevations of 800 to 2 700 meters above sea level (^{Villareal, 2003}). The essential oil of the pericon can be used as an insecticide in larvae of Aedes aegypti L. (^{Vera et al., 2014}), coleoptera such as Sitophilus zemais (^{Nerio et al., 2009}) and adults of Tribolium castaneum (^{Olivero et al., 2013}), as an extract obtained with solvents, has an effect against bacterial agents that cause respiratory infections (^{Caceres et al., 1991}) and gastrointestinal infections (^{Céspedes et al., 2006}) and with effect on nematodes (^{Siddiqui and Alam, 1988}; ^{Omer et al., 2015}).

These last two references are the only ones on the effect of substances extracted from T. lucida against nematodes such as Meloidogyne incognita, Rotylenchulus reniformis, Tylenchorhynchus brassicae, Hoploimus indicus, Helicotylenchus indicus and Tylenchus filiformis, but T. lucida oil has not been evaluated against this type of organisms. The chemical composition of the essential oil of T. lucida is recorded since 1938, estragole is the compound first referred to in the oil of this species (Anonymous 1938, cited by ^{Visbal et al., 2010}).

This secondary metabolite, of the group of phenylpropanoids, has been identified as a high percentage compound (96.8% and 97.3%) for populations of Cuba (^{Regalado et al., 2011}) and Costa Rica (^{Cicció, 2004}), respectively; however, in addition to estragole, other major phenylpropanoids are reported in pericon samples from Guatemala, such as anethole and methyl eugenol (^{Bicchi et al., 1997}). The chemical characterization of this natural resource of Mexico has not been sufficiently explored (^{Serrato, 2014}).

In the State of Mexico T. lucida is present in 22 municipalities (^{Discover Life, 2014}), it is a ceremonial and useful plant in traditional medicine (^{García et al., 2012}). Considering that there is little background on the activity of the oil of this species against nematodes and taking into account the natural availability of the pericon in that entity, the objective was to evaluate the toxic activity of the essential oil of a natural population of pericon in the formation of galls by N. aberrans in the greenhouse, in order to assess whether this local resource, in specific doses, influences the process of galling in the roots of tomato plants, in addition to specifying the sequence in which this vegetable input could be applied , methodological aspect little referred in toxicological evaluations of this type.

Materials and methods

Tagetes lucida Cav. In the the Joya 3 of Mayo colony Linda Vista of the Ixtapan of the Sal municipality, State of Mexico, coordinates 18º 49’ 31’’ north latitude and 99º 41’ 18’’ E, altitude 1 853 m climate Cw₁(w)i’g, in October 2014, floral stems of ruderal plants were collected for the extraction of essential oil. Seeds of this plant population were entered into the National Plant Germplasm Bank (BANGEV) in the Department of Plant Technology of the Autonomous University of Chapingo (UACH) (JZE-Tateges-001) and specimens were also sent to the Herbarium-Hortorio “Jorge Espinosa Salas” of the Department of Agricultural Preparatory, UACH.

Tomato (Solanum lycopersicum L.). For the corresponding tests, the Rio Grande variety of the Edena^®, brand was used, a variety susceptible to nematodes. In unicel trays of 200 (2.5 x 2.5 x 6.5 cm) and as peat moss substrate; one seed was planted per cell on May 7, 2015, the transplant was made on June 3 of the same year in plastic pots with a capacity of 1 L. Potted plants were established in the greenhouse area of the Institute of Horticulture, UACH.

Results

Chemical composition of T. lucida essential oil

In the essential oil of the Ixtapan population of T. lucida, 19 compounds were identified, five of them major and the rest as traces. The major compounds were: geranyl acetate (40.83%), β-ocimeno (15.14%), nerolidol (8.19%), caryophyllene (5.29%) and β-cubebeno (5.17%) (Figure 1, Table 1).

Figure 1 Chromatogram showing the peaks of: β-myrcene (retention time, Rt, 3.93) β-ocimene (Rt, 4.62), geranyl acetate (Rt, 9.07), carifilene (Rt, 9.68), β-cubebeno (Rt, 10.44) and nerolidol (Rt, 11.36). 

Table 1 Chemical composition of the essential oil of floral stems of the Ixtapan population of T. lucida. 

Peak	Compound	Tr	Area	(%)
1	β-Pineno	3.87	1 225 630	0.55
2	β-Mirceno	3.93	11 923 332	4.79
3	Acetato de alcohol de hojas	4.07	556 487	0.29
4	3,6,6-2-Norpineno	4.47	2 270 961	0.88
5	β-Ocimeno	4.62	41 693 860	15.14
6	Linalool	5.3	2 222 315	1.23
7	Geranyl acetate	9.07	92 446 673	40.83
8	Humuleno-(v1)	9.28	3 692 376	1.57
9	†Biciclo [4.3.0]nonan-2-eno, 8-isopropylideno-	9.33	1 165 226	0.55
10	Caryophyllene	9.68	11 650 343	5.29
11	Copaeno	9.78	473 212	0.21
12	Farneseno	9.97	2 449 168	1.27
13	α-Caryophyllene	10.1	1 011 519	0.45
14	β-Cubebeno	10.44	10 046 965	5.17
15	γ-Elemeno	10.62	6 598 214	3.97
16	δ-Cadinne	10.89	2 055 984	0.86
17	Nerolidol	11.36	18 323 420	8.19
18	Caryophyllene oxide	11.71	6 250 778	1.68
19	tau-Cadinol	12.43	2 935 784	1.19

^†= no trivial name was found.

Discussion

The major compounds identified in the essential oil of the aerial part of flowering plants of T. lucida correspond to the chemical groups of monoterpenes (geranyl acetate and β-ocimeno) and sesquiterpenes (caryophyllene, nerolidol and β-cubebeno) (^{Lange and Ahkami, 2013}). Monoterpenes and sesquiterpenes, in vegetable extracts of Tagetes obtained with solvents, may have biological activity against nematodes (^{Marotti et al., 2010}) and it would be expected that their presence in the composition of the essential oil of T. lucida could have similar effect, as further on it is exposed (Table 2).

Geranyl acetate (40.8%) was the most abundant in the essential oil of the Ixtapan population of T. lucida (Table 1), this compound had not been reported in populations of Guatemala, Cuba, Costa Rica and Mexico (^{Bicchi et al., 1997}; ^{Ciccio, 2004}; ^{Serrato et al., 2007}; ^{Regalado et al., 2011}); this molecule and nerolidol are attributed toxic activity against Aedes aegypti (^{Muñoz et al., 2014}), but there was no reference to their effect against nematodes. The compound β-ocimeno (15.14%) has antibiotic, anti-inflammatory and anti-oxidant properties, it also highlights its effect against pathogens and protects plants against insect pests (^{Adorjan and Buchbauer, 2010}), but without a history against nematodes.

The caryophyllene (9.6%) has an insecticidal effect against mosquito larvae (^{Jaenson et al., 2006}) and β-cubebeno (5.17%), also has activity against Escherichia coli (^{Bezic et al., 2005}); none of these compounds is related to effects against nematodes. Most of these compounds, in trace amounts, had already been described in the composition of T. lucida essential oil (^{Bicchi et al., 1997}; ^{Ciccio, 2004}; ^{Serrato et al., 2007}; ^{Visbal et al., 2010}; ^{Regalado et al., 2011}), the distinctiveness of the oil composition of the Ixtapan population of T. lucida was the high abundance of some of them and the presence of the compound geranyl acetate.

Considering that the root galling of tomato plants is a consequence of the presence of female nematodes and their penetration into the roots (^{Manzanilla et al., 2002}), it was clear that the application of T. lucida oil reduced the process of galling by N. aberrans (Table 2), result that constitutes the first report for this nematode. Possibly the inhibition of galls was due to the fact that the essential oil affects the nervous system of the organism, consequently causes paralysis and finally death (^{Maffei, 2010}), effect of immobility of nematodes previously reported with the application of root and part extracts of T. lucida, obtained with solvents (^{Siddiqui and Alam, 1988}; ^{Marotti et al., 2010}; ^{Omer et al., 2015}); the effect of T. lucida essential oil against nematodes had not been evaluated before the present work.

The CL₅₀ values ​​were expected to be decreasing in proportion to the number of intervals, but the trend was not appreciated; when analyzing the number of plants with galls by factorial analysis, it was confirmed that there was no interval effect, so that applying once, or perhaps twice, is convenient. These results constitute a useful reference for the management of vegetable substances, since there is little information about them. From the practical point of view, the highest concentrations produced the best results, although the possible use of 0.35 to 1 mg mL^-1 stands out; without phytotoxicity.

With the control treatment, the inhibition was proportional to the concentrations at which the oil was diluted and applied; this did not happen with the preventive application, therefore only the CL₅₀ was obtained for the first two intervals. However, the low values ​​of CL₅₀ (0.06-0.13 mg mL^-1) in the control scheme, the slope was always lower than 0.7 in the three intervals, indicating that the population of individuals responded heterogeneously to the application of the oil , which could mean that oil molecules do not affect effectively the J2 females of N. aberrans, although it is also possible that a heterogeneous proportion of female individuals in the inoculum has dispersed to the root zone of the seedlings, even there have been differences in the parasitic ability of the populations of the nematode in the host, as well as the colonizing ability (^{Bourne and Kerry, 1999}).

The essential oil of species such as T. minuta has been evaluated against Anopheles gambiae by recording CL₅₀ of 1.49 mg mL^-1 (^{Kyarimpa et al., 2014}), of T. patula against Aedes aegypti with CL₅₀ of 0.13 mg mL^-1 (^{Dharmagadda et al., 2005}) and of T. lucida against Tetranychus urticae with an CL₅₀ of 0.016 mg mL^-1 (^{Caramillo et al., 2008}) such results and those achieved in this study in the case of control treatments with CL₅₀ of 0.06-0.13 mg mL^-1, suggest strong biological action of T. lucida oil against N. aberrans.

To apply essential oil at a concentration of 0.06 mg mL^-1 to one hectare would require 120 mL of oil in 199.8 L of water, while 260 mL would be required in 199.7 L of water for the 0.13 mg mL^-1 concentration. In lands not cultivated in Ixtapan, it is estimated that in 1 m² of surface there are nine plants of T. lucida and in 1 ha, 90 000 of them with an oil yield of approximately 0.68 mL m^-2 and production of 6.8 L ha^-1. The wide distribution of populations of T. lucida in the State of Mexico and its abundance in numerous localities such as Ixtapan, are favorable conditions for the use of this natural resource and low cost.

Conclusions

The essential oil obtained by hydrodistillation of flowering plants of the Ixtapan population of T. lucida presented the major compounds were geranyl acetate, β-ocimeno, nerolidol, β-cubebeno and caryophyllene, highlighting geranyl acetate that had not been reported as abundant. The essential oil of T. lucida influenced the process of galling of N. aberrans in tomato seedlings, the concentration effect being critical and the application interval little effective, in this way the best IC₅₀ results of 0.06-0.13 mg mL^-1 to inhibit galling were achieved by applying the oil as a control treatment.