Use of plant essential oils in the management of phytonematodes

Meloidogyne It is among the main species of nematodes that affect agricultural crops of commercial importance, such as soybeans, cotton, sugar cane and corn. In the search for alternative measures, which, added to other integrated strategies, can help in the difficult management of these organisms, the use of essential plant oils is still hampered by their viscosity and low solubilization in water. Hence the importance of studies that evaluate the interference of solvents in relation to the survival/mortality of these phytonematodes.

There are several factors that limit plant production, such as pathogens, including fungi, phytoparasites, bacteria, viruses and nematodes. Phytonematodes stand out, which feed on intensely strong plants for economic exploitation and cause reductions in the productivity of these crops. Among the species in this group, which cause greater damage to the main crops, such as soybeans, cotton, sugar cane and corn, the Meloidogyne spp. These, in turn, affect plants through their negative effects on the root system, which hinders the absorption and translocation of nutrients (DAYS et al., 2009). Such pathogens are difficult to eradicate due to species polyphagy. Meloidogyne, physiological variability and the wide dissemination of the different producing regions, which leads to a limitation in the use of control measures (SILVA et al. , 2001).

Currently, among the most used strategies is the series of crop or plant rotation, the use of resistant or tolerant cultivars and chemical treatment (ARAÚJO AND MARCHESI, 2009). However, in all these procedures, many losses in agricultural production are observed. In this context, alternative measures become essential in the search for control of these phytonematodes. Knowledge of secondary metabolic compounds present in plant essential oils (EO) and their nematicidal or nematostatic activity is attractive for exploring biological activity in relation to disease control (SILVA et al., 2005).

Essential oils have been an alternative for controlling these pathogens, agricultural pests and weeds (OOTANI et al., 2013). In view of this, to use essential oil against nematodes, in most cases, prior solubilization is necessary, due to its viscosity and low solubilization in water. In this context, the objective of this study was to examine, for the first time, the interference of chemical solvents in relation to the survival/mortality of nematodes Meloidogyne javanica.

The inoculum was prepared from organisms obtained from individual monospecific eggs from the Meloidogyne sp. resulting from infected soybeans in the city of Santa Flora, state of Rio Grande do Sul, Brazil. Tomato seedlings (Lycopersicum sculetum L.) cv. Santa Clara were used for continuous multiplication of the pathogen in pots with a capacity of two liters kept in a growth room under ideal conditions for phytonematodes, with a temperature of 26 °C, humidity of 60% and a photo period of 12 hours.

To obtain juveniles from the second stage (J2), the methodology used was proposed by HUSSEY AND BARKER (1973), adapted by BONETI & FERRAZ (1981). Part of the suspension containing the eggs and (J2), obtained by this method, was subjected to the modified Baermann funnel technique (CHRISTIE & PERRY, 1951) and incubated at 26 °C for 36h in conditions stimulating the eggs to hatch and obtain of J2 nematode.

The solvents Ethyl acetate PA, manufacturer Synth, batch 168 668; Ethyl Alcohol 95% PA, manufacturer Fmais, lot 41823; n-Butyl-PA alcohol, manufacturer Synth, lot 149 013; Crodamol GTCC, manufacturer Alpha, chemical batch M2889/13; Dimethyl sulfoxide PA, manufacturer Nuclear, lot 10111414; Glycerin PA, manufacturer Vetec, batch 0908321; Propylene glycol,  manufacturer Alpha Química, batch M2096 / 13; PEG 400, manufacturer Synth, lot 141 959; Tween 20, manufacturer Synth, Lot 154.201, were obtained commercially.

The nematicidal activity of solvents in J2 of Meloidogyne spp. was carried out in 24-well microdilution plates, following the PÉREZ method et al. (2003), in concentrations of 5%, 10%, 25%, 50% of each solvent separately, in duplicate. For the test vitro a suspension of 1 mL of distilled water containing 120 nematodes was placed in individual wells of a microplate. Meloidogyne spp. from J2. A solvent solution was then added due to the corresponding concentration, calculated based on the percentage of desired solvent. As a control,  distilled water with the nematodes was used.

Then, the plates were sealed with plastic film, placed under circular motion with a shaker at 80 rpm and incubated in a growth room at 26 °C,  in the dark. Dead J2 was counted using an optical microscope (10x magnification) and occurred 24 hours after assay incubation. Mortality assessment was carried out 24 hours after the test was set up. All mobile and static J2 were taken into account. To confirm death, the samples were transferred to a polystyrene Petri dish (4 cm in diameter) and then viewed under a light microscope at 40X with the aim of verifying minimal movement (CAYROL et al., 1989). The mortality rate was calculated by the equation: J2 dead (%) = (J2 dead x 100) / (+ J2 J2 dead alive). Two replicates per concentration were performed. Nematode survival was assessed using the Kaplan-Meier test with post log-rank test (SPSS 18 software). The minimum level of significance was set at P ≤ 0,05.

The solvents, propylene glycol, n-butanol, glycerin, crodamol and ethanol and ethyl acetate induced the death of the entire population of nematodes present in the sample, differing significantly from the control (control) (P < 0,001). This mortality can be observed at all concentrations tested, and at higher concentrations of 25% and 50% of nematodes there was a notable change in the structure observed under a microscope. The PEG 400 and ethyl acetate solvents showed feasibility of use in concentrations below 5%. In this case, mortality was 40% of the total nematode population, thus triggering a statistical difference from the control (P < 0,001). In the case of Tween 20 at a concentration of 5%, no mortality occurred and nematode movement was normal. At a concentration of 10%, mortality occurred in 15% of the total nematode population and those that remained alive were slow moving. At a concentration of 25%, mortality was 20% of the tested population and those who remained alive showed minimal mobility. Thus, at concentrations of 5%, 10% and 25% there was no significant difference compared to the control. However, at a concentration of 50%  there was a statistical difference from the control group (P <0,001). The DMSO solvent proved to be toxic to nematodes in most of its concentrations, but at a concentration of 5%, mobility was observed in the total nematode population and there was no significant difference compared to the control, unlike the others, where the solvent altered the conformation of the cell membrane of the phytoparasites and showed statistical changes compared to the control group (P <0,001).

Essential oils are potentially useful in treating diseases of cultivated plants (SALGADO et al., 2003). The insolubility of essential oils and many of their constituents in aqueous media is likely to impair their performance in sensitivity tests. Attempts to overcome this problem have been made using solvents (KATIKI et al., 2011). Research into chemical components from natural products, such as essential oils, is of fundamental importance for the development of new anthelmintic drugs (ASSIS et al., 2003).

Given the results obtained, it is recommended to use Tween 20 to dilute essential oils when applied to nematodes. If the essential oil to be tested does not show complete miscibility in the Tween 20 solvent, other solvents must be selected, such as DMSO, PEG 400 and ethyl acetate in concentrations lower than 5%, so as not to interfere with the results of the experiments.

Jessyka A. Cunha, Federal University of Santa Maria; Rodrigo G. Madalosso, Centro Universitário Franciscano; Paulo S. Santos, Phytus Institute; Cecília de Á. Scheeren, Federal University of Santa Maria; Andrezza N. Lopes, Marcelo G. Madalosso, Instituto Phytus; Berta M. Heinzmann, Federal University of Santa Maria; Roberto C. V. Santos, Franciscan University Center

Article published in issue 201 of Cultivar Grandes Culturas.