¹Departamento de Fitotecnia, Universidad Autónoma de Chapingo, Carretera México-Texcoco, Chapingo, México; ^*Corresponding Author: serratocruz@gmail.com

²Laboratorio de Química Orgánica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala s/n, Casco de Santo Tomás, México D.F., México

Copyright © 2014 Miguel Ángel Serrato Cruz et al. This is an open access article distributed under the Creative Commons Attribu- tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accordance of the Creative Commons Attribution License all Copyrights © 2014 are reserved for SCIRP and the owner of the intellectual property Miguel Ángel Serrato Cruz et al. All Copyright © 2014 are guarded by law and by SCIRP as a guardian.

ABSTRACT

The genus Tagetes is a possible source of es- sential oils for the biorational control of pests and diseases in Mexico. The aim of the present study was to assess the distance between plants (PD; 15 × 80, 30 × 80 and 60 × 80 cm) and urea fertilization (0, 60 and 120 kg∙he⁻¹ N) for bio- mass production and essential oil in Tagetes terniflora HBK. Oil was obtained by the aerial part hydro-distillation and its chemical compo- sition was analyzed by gas chromatography coupled to mass spectrometry. Factors N, PD and their interaction did not change plant height or the number of branches per plant; however, PD factor modified fresh tissue (FW) and dry tissue (DW) weights and the amount of oil per plant (p ≤ 0.05) which represent a good biomass production (30 to 72.5 ton∙he⁻¹ FW, 11 to 27 ton∙he⁻¹ DW) and oil producing (168 to 383 L∙he⁻¹) potential in the field. Urea did not have effect on both biomass and oil response per plant but interaction of 15 x 80 DP and 120 N could favor higher biomass and oil production potential (78 ton∙he⁻¹ FW, 28 ton∙he⁻¹ DW and 608 L∙he⁻¹, re- spectively). Essential oil yield varied from 0.3 to 2.1% according to the management conditions. A total of 11 major compounds were identified in essential oil, the relative quantity was constant in different agronomic management factors: E- tagetone (22%), cis-tagetenone (20.4%), trans- tagetenone (20.4%), dihydrotagetone (13.4%) and cis-β-ocimene (10.3%), trans-β-ocimene (5.0%), propenyl anisole (4.3%), sphatulenole (1.1%), allyl anisole (0.7%), Z-tagetone (0.5%) and limonene (0.5%).

Keywords

Tagetes terniflora; Biomass; Essential oil; chemical composition; distance between plants; nitrogen

1. INTRODUCTION

Essential oils extracted from aromatic plants are natu- ral sources with broad-spectrum biological activity against insects, mites, nematodes, fungi and bacteria [1, 2]. The genus Tagetes (Asteraceae) consist of 58 species [3], most of them are aromatic species. The essential oil of T. terniflora HBK has a biological effect against lice Pediculus humanus capitis [4], weevils Sitophilus ory- ceae and Tribolium castaneum [5], flies Ceratitis capita- ta [6] and this essential oil also has a bactericidal effect [7]. The essential oil of T. terniflora has major com- pounds corresponding to monoterpenes, sesquiterpenes and phenylpropanoids [6,8,9] their relative content depends on the phenological stage of the plant and the characteristics of the habitat [8].

T. terniflora is distributed in Peru and Argentina, and it has recently been found in Chiapas, Mexico, perhaps as an introduced species [10]. In South America, collected and natural habitat populations for oil extraction may have yields from 0.4% to 1.5% (mL/100g fresh tissue) or from 1.1% to 4.1% (dry base) [8]; so far there is no available information on agronomic management aspects of T. terniflora such as distance between plants and ferti- lization, which are important agronomic management factors for other species to improve biomass and essen- tial oil [11,12].

The presence of T. terniflora plants in Chiapas gene- rates interest to grow and use them to control pest and diseases in Mexican agriculture. Therefore, the aims of the present study were to evaluate two agronomic man- agement factors (population density and a nitrogen ferti- lization source) to generate preliminary information on biomass and essential oil production of this species.

2. MATERIALS AND METHODS

2.1. Biological material

A total of 20 plants were grown under greenhouse conditions from seeds of T. terniflora collected in 2011 in San Cristóbal de las Casas, Chiapas, Mexico; seedl- ings were obtained from seeds of these plants to perform an experiment on agronomic management at Chapingo, Mexico in 2012 and to analyze the content of oil. “Eitzi Matuda” herbarium specimens provided by the Univer- sidad de Ciencias y Artes de Chiapas, Mexico were ana- lyzed for taxonomic identification of the species.

2.2. Agronomic management factors, Variables and statistical analysis

The present study was carried out at Chapingo, 19˚29' N and 98˚W, with an altitude of 2250 m; subhumid tem- perate climate (C(w0)(W)b(i)g); average annual rainfall of 654 mm and average temperature of 15˚C with early frosts at the end of September and late frosts in April [13].

Sowing in greenhouse was carried out on April using germination trays of 3 × 3 cm and 7 cm with peat moss (Cosmopeat) as substrate. Once the second pair of leaves was visible, the plant was transplanted (May), placing one plant per shrub between furrows with a spacing of 80 cm on moist soil. Before the field was plowed, the soil was left fallow and tilled. The field received heavy irrigation the same day after transplanting, two irrigations in May and then, the moisture supply was by temporary rain. The soil was mechanically tilled twice for weed control.

Three plant distances (15 × 80, 30 × 80 and 60 × 80 cm) and nitrogen fertilization with urea (46-00-00) at levels 0, 60 and 120 kg∙he⁻¹ of nitrogen (units of N) were tested. N was applied 30 days after transplanting. The experimental field was used for non-leguminous plants without using any fertilizer; no previous soil nutritional diagnosis was carried out.

Treatments were distributed in field according to a randomized block design with six replications in experi- mental plots of 12 m²; one plant corresponded to the ex- perimental unit. Plants in full competition, in 100% flo- wering stage (150 days after transplanting) were consi- dered for evaluation. The plants selected were cut 2 cm below the ground level, immediately they were weighed and measured, each sample was place inside a paper bag and dried in an oven at 60˚C for 3 days. The measured variables were: plant height, number of branches (from the first branch appearance at the base until the last visi- ble branch at the apex; branches outside the main stem were not considered), fresh weight and dry weight of the aerial part and amount of essential oil per plant.

Data analysis was carried out using the following two-factor (3 × 4) completely randomized bock design:, where, value of the dependent variable recorded in the experimental unit, in which the i-th dose of fertilization of the j-th dis- tance of the transplanting distance of the k-th block was applied, general average of all experimental plots for the conditions of the experiment, effect to apply the i-th dose of fertilizer, j-th effect per transplant- ing distance, combined effect of the levels of fac- tors involved, effect of the k-th block, and experimental error related to each. The components of the model, and, are orthogonal to each other. The Student’s test was used to compare means of each factor and of the combination of the factor levels for independent samples using a significant level of p ≤ 0.05. Statistical analyzes were performed using SAS [14].

Oil extraction

Oil was extracted from plants in full flowering stage using the methodology of hydro distillation reported by [15]. Three replications of each treatment were recorded to sample the production of oil; each sample corres- ponded to one plant cut into pieces of 1 to 3 cm and time of distillation was 45 min.

2.3. Determining compounds

The composition of essential oil was analyzed by gas chromatography coupled to mass spectrometry (CG-EM) using a Polaris Q Finnnigan Trace GC Ultra^® equipment (USA) with a Polaris Q mass detector, electronic impact (70 eV). A column RTX-5MX, diphenyl-dimethylpoly- siloxane (5:95), of 30 m × 0.25 mm Ø (diameter) × 0.25 µm was used. The injector and detector were set at 250 and 300˚C. The temperature of the oven started at 70˚C, and remained like this for 1 min and was programmed to reach 250˚C at a rate of 20˚C min⁻¹. Helium was used as carrying gas, at a flow rate of 1 mL min⁻¹. Diluted sam- ples were injected manually (1/100 in methylene chloride v/v) of 1 μL, in split mode. Quantitative data were ob- tained electronically from the area percentage of the chromatographic peak. Detected mass range was 35 - 500 m/z. N-alkanes are used as references in the calcula- tion of Kovats indices. Three processed samples were measured in the identification of the components by comparing the relative retention indices and mass spectra using the NIST database of the GC-MS system and Adams’s data published by Corp. Carol Stream, USA [16].

3. RESULTS AND DISCUSSION

3.1. Distance between plants and Fertilization

According to the F-test of the analysis of variance, plant height (160 to 188 cm) and the amount of branches per plant (23 to 47) of T. terniflora in 100% flowering did not change their expression due to the agronomic management factors evaluated (individual and interaction effects), an opposite result to that observed in other spe- cies of Tagetes subject to the application of nitrogen fer- tilizer and population densities [17,18]. However, bio- mass production and essential oil were strongly changed by plant distance (PD) (Table 1), underlining that by increasing the plant distance, increased fresh and dry tissue biomass (66% in 60 × 80 and from 32% to 37% in 30 × 80, with respect to that recorded with 15 × 80) and the amount of oil (76% in 60 × 80 and 10% in 30 × 80 with respect to that recorded with 15 × 80) (Table 1), a trend that has been recorded in previous studies, in which different species of Tagetes have been studied [12,17]. The urea as a nitrogen source did not cause significant effects on the amount of biomass and plant oil (Table 1), this was not expected considering that some researches [12,18] confirm that by increasing the units of this nu- trient, also increases the growth of the plant, as total biomass. Perhaps the rapid dissolution of urea in the soil, and if it was once used, contributed to a low availability of nitrogen during the 150 days that plants were kept on the field until harvest, time when the experiment data was recorded; there is also a strong suspicion that tilling the soil (fallowing, ploughing, furrowing, weeding), ra- ther than the application of urea in the doses tested, largely influenced the growth of T. terniflora, an agricul- tural practice that in the case of other wild species of Tagetes provokes a biomass increase up to 300% with respect to the non-tilling system [19]. The experimental verification of the cases referred is required, and also the testing of other nitrogen sources and dosages to evaluate their effect on biomass and oil production of T. terniflo- ra.

Although F-test indicated significant effects of PD × N there was not a clear tendency of interactions and only few ones favored the expression of the biomass as plants were further apart (Table 1), for example, applying 120 N, with a plant distance of 15 × 80 less fresh or dry tis- sue was produced (945 g/plant and 339 g/plant, respec- tively) than with a plant distance of 60 × 80 (1175 g/ plant and 445 g/plant, respectively) (Table 1), this trend

^†Means with different lower case letters are significantly different (p ≤ 0.05). ^¶Means with different capital letters are significantly different (p ≤ 0.05).

was also observed for the amount of oil per plant without applying N (Table 1). Other interactions showed that by increasing N with a PD of 15 × 80, increased the oil per plant (3.2 mL without N, 4.3 mL with 60 N and 7.3 mL with 120 N), this suggests the importance of taking into account the specific management conditions to obtain desired results. For example, in the case of PD of 15 × 80 without N (case 1), and 60 × 80 with 120 N (case 2), the lowest (3.2 mL, 918 g) and highest (9.3 mL, 1548 g) amount of oil and fresh biomass per plant obtained, re- spectively, suggests that, in a production per hectare (10 000 m²), the case (1) could favor the production of fresh biomass of 76 ton and a volume of oil of 266 L; however in case (2) 32 ton and 193 L could be produced (Table 2).

Oil yield from 0.3% to 0.8% on fresh base and from 0.9 to 2.1% on dry base (Table 3) obtained from differ- ent management conditions evaluated, is located within the yield limits allocated for South American populations of T. terniflora in 100% flowering stage without any management [8]; it is necessary to determine the yield in other phenological phases, such as fruiting where a high- er yield is possible [8].

3.2. Essential oil Composition

A total of 11 compounds in greater proportion with re- tention times were detected according to the fragmenta- tion pattern in the following sequence: limonene (3.94 min), cis-β-ocimene (3.95 min), dihydrotagetone (4.1 min), Z-tagetone (4.5 min), trans-β-ocimene (4.75 min), E- tagetone (5.01 min), alilanisole (5.55 min), cis-tagetenone

(5.72 min), trans-tagetenone (5.8 min), propenyl anisole (6.34 min) and spathulenol (7.18 min) (Figure 1). Four groups of compounds stood out according to the relative percentage: abundant 20% to 22% (E-tagetone, cis- and trans-tagetenone), moderately abundant 10% to 13% (di- hydrotagetone, cis-β-ocimene), less abundant 4% to 5% (propenyl anisole, trans-β-ocimene) and very less abun- dant 0.5% to 1% (Z-tagetone, limonene, allyl anisole, spathulenol). Secondary metabolites found in the present study had already been identified in previous work [6,8, 9,20] where it is stated that the group of monoterpenes (ocimene, tagetone and tagetenone) is more abundant that phenylpropanoids (propenyl anisole and alyl anisole) and sesquiterpenes (spathulenol and limonene) groups, as also found in this study.

The relative abundance of each compound in essential oil of T. terniflora population grown for the first time in the area of Chapingo, Mexico was different with respect to that recorded for the same species in populations of different geographic origin [6,20], which is possibly due to environmental or genetic causes.

The presence of these secondary metabolites and sim- ilar relative amount of them in essential oil in samples analyzed corresponding to the plants grown in the expe- riment with the various agronomic management factors were responses that had previously been found in other wild species of Tagetes introduced to cultivation sub- jected to different dates of establishment in the field [21], at population densities and nitrogen amounts [12].

This result is important because now there is informa- tion available of some management aspects in field con- ditions that can help to have a quality control of the oil produced, however, it is recommended to continue with the study of other management factors such as date of establishment.

The extrapolation of biomass yield and oil per hectare (Table 2), good estimated yields of oil (Table 3) and a relatively homogeneous composition of the essential oil of T. terniflora on management conditions established (Figure 1), form a basic reference for: experiences of commercial production to validate the important man- agement factors found in this study, tests of other ferti- lizers and nitrogen sources such as phosphorus and po- tassium that could improve the productivity of T. ternif- lora and toxicological studies against the various pests and diseases of important crops in the region of the upper valleys of Estado de México, state where Chapingo is located.

4. CONCLUSION

The distance between plants, but not the application of nitrogen in the form of urea, influences the production of biomass and essential oil per plant of T. terniflora, some interactions also influenced the production of biomass and oil. Oil yield varied according to the management conditions. A total of 11 compounds were identified in essential oil with abundance of the tagetone and tagete- none groups, followed by ocimene, phenylpropanoids, spathulenol and limonene; the relative amount of each chemical component did not showed changes under the