Root-knot nematodes (RKN;
The control of nematodes has become increasingly difficult due to many reasons. Many nematicides and soil fumigants have been removed from the market due to their toxicity and their harmful impact on the environment. An example is methyl bromide, a common fumigant that has been banned since 2005 (Karpouzas et al., 2007). The control against nematodes is further complicated by the lack of inherent resistant mechanism in most vegetable against nematode invasion or the cost-ineffective aspects of preventative approaches such as the long-term crop rotations or the scarcity of biological control agents in the market. As such, there is a great need for new alternatives (Sikora and Fernandez, 2005; Davies and Spiegel, 2011; Giannakou, 2011). In face of the recent European Union (EU) environmental restrictions, it is necessary to develop more ecologically rational alternative systems, like biological control and natural products (Faria et al., 2016).
In the actual context of research and development, a large number of essential oils and their constituents have been studied against a number of pests. In particular, terpenes, which are the primary volatile constituents of essential oils (EO), are among the most promising compounds for biorational pest control (Isman, 2000; Shaaya and Rafaeli, 2007; Sousa et al., 2015). One of these terpenes is eugenol (C10H12O2; 4-allyl-2-methoxyphenol) (Fig. 1), a volatile phenolic constituent and a flavoring agent of great economic importance in the food and cosmetic industries, mainly obtained from
Eugenol has shown interesting biological activities, such as antimicrobial (Marchese et al., 2017), anti-inflammatory (Daniel et al., 2009), antioxidant (Ogata et al., 2000), antibacterial (Da Silva et al., 2018), insecticidal (Park et al., 2000), and nematicidal (Tsao and Yu, 2000; Ntalli et al., 2010) among others. Previous lines of research have shown that eugenol at concentration of 660 µg/ml was nematotoxic to
The objectives of the present study were to determine (i) the nematicidal and nematostatic activity of eugenol on second-stage juveniles (J2) of
A population of
Solutions of eugenol were tested for J2 motility at the doses of 62.5, 125, 250, 500 and 1,000 ppm. Eugenol (Merck; Germany) was dissolved in ethanol (Sigma-Aldrich, Italy) and serially diluted in distilled water containing Tween-20 to produce test solutions of the above doses. In all cases, final terpene solutions were prepared containing double the test concentration. Eugenol solution (0.5 ml) was pipetted into each well of a Cellstar® 24-well plate together with J2 suspension (0.5 ml containing approximately 40 J2) at a ratio of 1:1 (v/v), to produce the desired concentration of J2-terpene suspension. Tween-20 is a polysorbate nonionic surfactant and is used as an emulsifying agent for the preparation of stable oil-in-water emulsions. Final concentrations of ethanol and Tween-20 never exceeded 1 and 0.3%, respectively. Second-stage juveniles exposed to these concentrations of ethanol and Tween-20 were not affected, as preliminary tests and previous work indicate (Ntalli et al., 2010). As controls we used either distilled water or water with ethanol and Tween-20 at concentrations identical to those used in the treatment wells. All treated and control plates were covered with a lid to diminish terpene volatilization and incubated at 26 ± 1°C. Juveniles were observed with the aid of an inverted microscope (Zeiss, Germany) at 100 × magnification after 12, 24, 48, and 96 hr and were ranked into two distinct categories: motile or dead. For a window of 10 sec, we checked the juveniles for motility by probing them with a needle. Lack of movement was considered as a strong indication of juveniles’ death. The experiment was conducted twice and all treatments were replicated five times.
Eugenol was dissolved in ethanol and diluted serially in distilled water containing Tween-20 resulting in solutions at the doses of 62.5, 125, 250, 500, and 1,000 ppm. In total, 50 ml of each test solution was placed in 250-ml Erlenmeyer flask and then newly hatched J2 were added. In all cases, working solutions were prepared containing double the test concentration and then mixed in flasks at a ratio of 1:1 (v/v) with a 50 ml suspension containing approximately 1,200 J2. A plastic tube connected to an air pump for oxygen supply was inserted into each flask. This was necessary to avoid lack of oxygen since each flask contained a high number of nematodes. Evaporation was avoided by covering the flasks with cotton plug and incubating at 26 ± 1°C temperature settings in dark. Solutions of water with ethanol (1%) and Tween-20 (0.3%) at the same concentrations as the ones in the treatment flasks as well as distilled water were used as controls.
After 12 hr, two solutions of 5 ml each were removed from every flask and used independently. The first one was divided into five aliquots of 1 ml each and placed into wells (containing approximately 40 J2 per ml/well). Second-stage juveniles were observed under an inverted microscope (100 ×) and ranked as motile or dead, as previously described. The second 5-ml solution was placed on a 38 μm sieve and J2 were rinsed with tap water to remove excess of eugenol. Then J2 were placed in a beaker and 5 aliquots of 1 ml each, containing approximately 35 J2, were placed in wells covered with a lid to avoid evaporation. Motile and dead J2 were counted under an inverted microscope (100 ×) after 12 hr to monitor recovery. The same procedure was repeated after 24, 48, and 96 hr. If any J2 regained motility, the effect was considered as nematostatic. Two solutions of 5 ml each was removed from flasks after 24, 48, and 96 hr and the same procedure, as described above, was followed. The experiment was conducted twice and all treatments were replicated five times.
Following the procedure described by Hussey and Barker (1973), we used sodium hypochlorite solution to extract
The effect of eugenol solutions on the development of eggs at the doses of 62.5, 125, 250, 500, and 1,000 ppm was tested. Eugenol was initially dissolved in ethanol and brought to the desired volume using Tween-20 in water, as previously described. In all cases, working solutions were prepared containing double the test concentration and then mixed in Cellstar® 24-well plates at a ratio of 1:1 (v/v) with suspension of eggs added to each well. Eggs suspension (0.5 ml containing approximately 50 eggs) was pipetted into each well and immediately eugenol solution (0.5 ml) was added. Distilled water and water with ethanol plus Tween-20 at concentrations equivalent to those in the treatment wells, served as the controls. All plates were covered with a lid to avoid evaporation and maintained at 26 ± 1°C. In total, 90% of eggs were undifferentiated at the beginning of the experiment. The number of either eggs having a fully developed juvenile or emerged J2 were counted in each well every 7 days (Tzortzakakis and Trudgill, 2005) using an inverted microscope (100 ×). For monitoring the egg development, eggs were observed on day 0 and were categorized either as differentiated (fully developed juvenile) or undifferentiated (eggs containing only cells). Undifferentiated eggs were considered those with cell division (one, two, or more cells).
The experiment was terminated after three weeks. It was conducted twice and each treatment was replicated four times.
Mature egg masses were handpicked using sterilized forceps, from roots previously rinsed thoroughly, and placed in small plastic extraction trays made by 6 cm Petri dishes (one mature egg mass per extracting tray). Eugenol solutions (62.5, 125, 250, 500, and 1,000 ppm), initially dissolved in ethanol and brought to volume using Tween-20 in water (as described previously), were added to each extracting tray to cover the egg mass (10 ml eugenol solution/extracting tray). Egg masses were maintained for seven days and then test solutions were discarded. Then each egg mass was carefully submerged twice in clean water to remove excess of eugenol and finally was placed in extracting tray filled with clean water. The extracting trays were covered to avoid loss of water and placed in incubator at 26 ± 1°C. Hatched J2 were counted every week, they were discarded and the water was renewed with fresh one. The experiment was terminated after 5 weeks. Then every egg mass was removed from the extracting tray to a drop of water on a glass microscope slide, gently squashed with a coverslip and the number of unhatched eggs per egg mass was counted under an inverted microscope. The experiment was conducted twice and all treatments were replicated five times.
Sandy soil was collected from a field in Gargalianoi village, Messinia, Southern Greece. It was sieved using a 2 mm sieve to separate soil from debris and it was sterilized in an autoclave for 20 min at 100°C. Subsamples were removed to determine the soil moisture (oven drying at 50°C for 24 hr) and maximum water holding capacity (MWHC) (gravimetrical measurement following saturation of the soil with water and allowing to drain for 24 hr) (Pantelelis et al., 2006).
In total, 48 plastic pots (7 cm depth and 5 cm diameter) were filled with 40 g soil each. Every pot was inoculated with a
The efficacy of eugenol against nematodes was tested at concentrations of 62.5, 125, 250, 500, and 1,000 ppm. Eugenol stock solution was prepared in ethanol and Tween-20 (0.6%) to overcome insolubility, while distilled water with Tween-20 (0.6%) was used for further dilutions according to the above method. Control pots were consisted of soil with J2 only. The above experimental procedure was performed at two levels of temperature (20-22°C and 30°C).
For both temperatures, the plastic pots remained in climate room for three days. For the experiment at the 30°C, the plastic pots were placed on a metal tray with dimensions 60 cm × 40 cm × 8 cm (L × W × H). At the bottom of the metal tray a flexible silicone resistance was placed, connected with an automatic thermostat. Throughout the experiment the tray was filled with wet sand and plastic pots were immersed in the sand. After three days soil from every pot was removed and nematodes were extracted following a modification of Cobb’s decanting and sieving methods (Flegg, 1967) as suggested by Brown and Boag (1988). Juveniles were collected after 2 days and counted with the aid of an inverted microscope at 100 × magnification. The experiment was conducted twice and every treatment was replicated four times. Before statistical analysis, all numbers were calculated according to the Abbott (1925) formula:
The efficacy of eugenol was evaluated using tomato seedlings cv. Belladona grown in plastic pots (10 cm depth and 6 cm diameter). All seedlings were 6 weeks old and at the four leaf stage. Initially, 15,000 newly hatched J2 were transferred from a graduate cylinder into four 250-ml Erlenmeyer flasks containing a total volume of 100 ml solutions of eugenol with concentration of 33.75, 67.5, 135, and 270 ppm and incubated at 26 ± 1°C. Eugenol was dissolved in ethanol (Sigma-Aldrich, Italy) and serially diluted in distilled water containing Tween-20 to produce test solutions of the above concentrations. Distilled water, as well as a solution of water with ethanol and Tween-20, at concentrations equivalent to those in the treatment flasks, served as control. After 24 hr, 1/3 of the suspension from each flask was placed in a 38 μm sieve and excess of eugenol was removed by washing with tap water. Then, nematodes were transferred to a beaker and four aliquots of 1 ml containing approximately 40 J2 were transferred to a 24-well plate. Second-stage juveniles were scored as motile or dead using an inverted microscope. The remaining J2 were maintained in the flasks for another 48 and 96 hr when the same procedure as previously described was repeated.
In total, 10 ml of a nematode suspension containing approximately 300 motile J2 was used to infect 6-weeks-old tomato plants. All plants were maintained for 30 days in a growth room at 26 ± 2°C. Plants were uprooted and roots were carefully washed free of soil and stained with acid fuchsin solution as described in Byrd et al. (1983). Roots were then washed in water and placed in vials containing equal volumes of glycerol and distilled water. The female nematodes were counted in the whole root system of each plant using a stereoscopic microscope at 12.5 × magnification. The experiment was conducted once in a randomized block design with five replicates per each treatment.
All experiments were conducted using the completely randomized design. Data were subjected to one-way analysis of variance (ANOVA) using the General Linear Model (GLM). Treatments means were compared using the LSD test. Statistical analysis in all cases was conducted using SAS statistical package (SAS University Edition). All experiments (except the pot experiment) were conducted twice and they were combined and analyzed together since no variation was revealed between data.
The effect of eugenol on J2 motility of
Effect of eugenol on the motility of
Exposure time (hr) | ||||
---|---|---|---|---|
12 | 24 | 48 | 96 | |
Dose (ppm) | Dead J2 (%) | Dead J2 (%) | Dead J2 (%) | Dead J2 (%) |
0 | 0 d | 0.8 d | 1.3 d | 4.7 e |
62.5 | 0.4 d | 1.6 d | 2.4 d | 10.7 d |
125 | 0.7 d | 2 d | 6.4 c | 19.8 c |
250 | 6.5 c | 6.9 c | 24.5 b | 80.8 b |
500 | 74.1 b | 92.2 b | 99.8 a | 100 a |
1000 | 99.5 a | 100 a | 100 a | 100 a |
No nematostatic effect was observed. The death of nematodes was further confirmed by maintaining all nematodes in wells with clean water (Cellstar® 24-well plates) and evaluated after 12, 24, 48, and 96 hr. The percentage of dead J2 was similar to the previous experimental result when the nematicidal activity was tested in bioassay experiment (data not shown).
The inhibitory effect of eugenol in different doses on eggs differentiation after exposure for 21 days is presented in Table 2. The lower percentage indicates higher efficiency in inhibiting the egg differentiation. Eugenol at the doses of 500 and 1,000 ppm significantly inhibited eggs differentiation (66.2 and 21.4%, respectively) compared to control (92.1%). Also, there was no significant difference between the treatments of 62.5, 125, and 250 ppm compared to the control treatment (Table 2). The experiment was terminated since no further egg differentiation was observed in the control treatment.
Effect of eugenol on the differentiation of
Exposure time (21 days) | |
---|---|
Dose (ppm) | Eggs differentiation (%) |
0 | 92.1 a |
62.5 | 90.6 a |
125 | 90.5 a |
250 | 82.1 a |
500 | 66.2 b |
1000 | 21.4 c |
The highest number of hatched J2 (83%) was observed in egg masses remained in clean water (control) throughout the experiment (Fig. 3). Significantly fewer nematodes were hatched from egg masses treated with 500 and 1,000 ppm resulting in more than 70 and 80% fewer J2 counted, respectively, compared to the control. Eugenol also reduced at doses of 62.5, 125, and 250 ppm resulting in about 40, 57, and 65% hatching reduction as compared to the control treatment (Fig. 3). The experiment was terminated since not any more J2 were hatching.
The toxicity of eugenol on J2 was evaluated by the contact-vapor mortality bioassay (Fig. 4). As doses increased from 62.5 to 1,000 ppm there was a corresponding increase in J2 mortality. The number of J2 was significantly lower in contact bioassay at both levels of temperature. Eugenol at doses of 500 and 1,000 ppm showed strong contact mortality of J2 resulting in about 80 and 90%, respectively, in both levels of temperature. At a dose of 250 ppm, a 68 and 56% decrease of J2 in the soil was recorded in the contact bioassay at 20 and 30°C, respectively. On the other hand, a lower mortality was recorded at doses of 62.5 and 125 ppm in both temperature levels. In contrast, no vapor toxicity to
The effect of sublethal doses activity of eugenol against
Terpenes constitute the largest class of secondary metabolites in the plant kingdom (Dudareva et al., 2006) and they possess nematicidal activity against
This is the first report of eugenol’s inhibition on eggs differentiation using
In more details, we observed a corresponding decrease in egg hatching of
We further tested the efficacy of eugenol which was found to exhibit strong contact mortality of
The invasion of nematodes in roots was affected by treating J2 with sublethal doses. Most juveniles treated with eugenol remained motile, although some of them were not able to infect roots. Eugenol at doses of 270 and 135 ppm significantly reduced the number of females per gram of root compared to the control, after exposure of 24 and 96 hr (Fig. 5). Previous investigations have shown that eugenol at concentration of 1,500 mg/kg soil reduced the number of galls caused by
The mechanism of action of EO and terpenes against nematodes remains unclear. Investigation on mode of action of EO and their constituents is important for nematode control, as useful information can be collected on the formulation and delivery means. According to Oka et al. (2000), there is a correlation between nematicidal and insecticidal activity and suggested the involvement of EO components in interrupting the nematode nervous system but also changing the permeability of the cell membrane.
In conclusion, phenolic monoterpenoid eugenol could be useful as a potential plant-based nematicide with contact action to control