Nematode control methods

Compounds derived from plants, such as oils and plant extracts, have been the target of studies in the search for the control of phytonematodes. This is the case with essential oil of Melaleuca alternifolia, in a test against the species Meloidogyne javanica.

Controlling plant parasitic nematodes is a difficult task, mainly due to the limitations that most methods present. Combat measures based on crop rotation for species of the genus Meloidogyne, shows some limitations, due to the great ability of this parasite to feed on a vast number of plants. The use of resistant varieties, although desirable, is still limited, due to the scarcity of resistant cultivars and chemical control that has not resulted in great effectiveness in the field.

For these reasons, the scientific community has sought alternative control methods. A clear example of this is the search for plant-derived compounds, such as vegetable oils and extracts. These substances have advantages over synthetic pesticides, as they are less concentrated and less toxic. Furthermore, they are quickly biodegradable, have a broad mode of action and are derived from renewable resources. In the literature there are already several studies that point to the nematicidal and/or nematostatic effect of these substances on phytonematodes. Some even serve as a basis for the development of bionematicides.

The oil from a plant native to Australia has been the target of studies on the control of phytonematodes. Known as the tea tree (Tea Tree), belongs to the family of myrtaceaeor a Melaleuca alternifolia and has approximately 100 active compounds. Among them are hydrocarbons, terpene, monoterpenes, sesquiterpenes and associated alcohols. Its biological and antimicrobial activity of the essential oil is mainly attributed to the Terpinen-4-ol component, the majority of the oil (30%-40%) (CARSON et al., 2006).

There are few reports in the literature that show the activity of compounds from essential oil of M. alternifolia in the control of plant parasitic microorganisms, only in the control of human and animal pathologies (MARTINS, et al., 2010). Because of this, the Phytus Institute in partnership with the Franciscan University UNIFRA (both Rio Grande do Sul) tested under laboratory conditions “vitro”, the nematicidal and/or nematostatic activity of the essential oil of M. alternifolia in control of Meloidogyne javanica.

The essential material was obtained commercially from the Manufacturer Evalar, Lot 2013003 and characterized in gas chromatography (CG) on the Agilent Technologies 6890N CG – FID system. 15 components present in the essential oil were identified, representing 95,86% of the total composition used in the study (Table 1). 

The inoculum was prepared from individual egg masses obtained from a monospecific population of the species Meloidogyne javanica coming from infected soybean plants in the municipality of Santa Flora, Rio Grande do Sul. Tomato Plants (Lycopersicum sculetum L.) cv. Ogata fukuju grown in 2-liter pots, containing soil and sand, were used for the continuous multiplication of the pathogen in growth room conditions, with controlled temperature and humidity.

To obtain J2 second stage juveniles, the methodologies proposed by Hussey & Barker (1973), adapted by Boneti & Ferraz (1981), were used. The suspension containing juveniles and eggs was subjected to the modified Baermann funnel technique (CHRISTIE & PERRY, 1951), and incubated in a hatching chamber at 26 ºC for 36 hours, to stimulate egg hatching and obtain juveniles.

The nematicidal activity of the essential oil on J2 of M. javanica was conducted in 24-well polystyrene plates, according to the methodology of Pérez et al. (2003) in alternating concentrations of 5 mg/ml, 10 mg/ml, 15 mg/ml (Figure 1). In each individual well of the microplate, a suspension of 1 mL of water containing 120 juveniles of Meloidogyne javanica, and then added to the solution, according to the corresponding concentrations that were calculated based on the density of the O.E.

Distilled water and Tween 20 (1%) were used as controls. Then, the plates were sealed with plastic film, placed under a shaker with a circular motion of approximately 80 rpm and incubated in a growth room at a temperature of 26 ºC in the dark. The counting of mobile and immobile J2 was carried out using an optical microscope after 24 hours of incubation. To better confirm the mortality or paralysis of juveniles, the specimens were transferred to a petri dish (4 cm in diameter) and then viewed under an optical microscope at 40x magnification, to verify the slightest movement. The youth of M. javanica they were considered dead when immobile (CAYROL et al., 1989) (Figure 2).

The percentage of mortality was calculated by the equation: J2 dead (%) = (J2 dead x 100) / (J2 dead + J2 alive). Two replications were performed per concentration. To confirm the nematicidal activity of the oil, juveniles were collected and transferred to Petri dishes containing distilled water and monitored for 24 hours.

The test result vitro showed that all concentrations tested presented nematicidal action on J2 juveniles of M. javanica when compared to the witness. In the mortality curve it was possible to refer to the essential oil of M. alternifolia an inversely proportional action between O.E. and time to mortality. Thus proving that the higher the concentration, the shorter the time for inducing mortality of the pathogen. Concentrations of 5 mg/mL, 10 mg/mL and 15 mg/mL provided 100% mortality in 20, 20 and 16 hours, respectively (Graph 1). 

The results obtained in this experiment are in line with other work already carried out by several researchers who have highlighted the potential effect of substances extracted from plants on the mortality of phytonematodes. According to Carson and Riley (1995), antimicrobial activity is dependent on the compound Terpinen-4-ol, the main and active compound of olive oil. M. alternifolia, ensuring that the greater the quantity found in the essential oil, the better its action. The same compound has anti-inflammatory action (HART et al, 2000). However, other constituents have the same antimicrobial properties, such as γ-terpinene, α-terpinene (BURT, 2004) and 1,8-cineole (ALBORNOZ, 1992).

The oil of M. alternifolia should cause interference in the metabolism of juveniles M. javanica inhibiting vital functions, acting synergistically through the membrane, which tends to cause its rupture, and/or affect the nervous system. These actions may explain the mortality of juveniles. However, future studies in similar cultivation conditions should be carried out to better understand the dynamics of oilseed oil. Melaleuca alternifolia in control of M. javanica. Furthermore, the combined use of biological products with chemicals can be an alternative to reduce the concentration of the latter, without losing effectiveness.

Maiquel Frigo, Phytus Institute; Rodrigo Gripa Madalosso, Centro Universitário Franciscano; Gracieli Rebelatto, Paulo Sergio dos Santos, Marcelo G. Madalosso, Instituto Phytus

Article published in issue 209 of Cultivar Grandes Culturas.