Fig 1.
Effect of D. cf. concentrica culture plate on M. javanica J2 viability.
J2s were exposed to 0–3 culture plates of D. cf. concentrica for 48 h. The numbers on the x-axis represent the numbers of Petri plates (50 mm in diameter, containing 5 mL of growth medium, and the fungal culture) in each 1-L sealed box. The number 0 indicates control treatment in which the nematodes were not exposed to the fungal culture plate. The viable J2s were separated using 30 μm sieves, and the numbers on the y-axis represent the numbers (mean ± SE) of viable J2s counted following the incubation. There were 10 repetitions, each using 300 J2s. The results were subjected to analysis of variance followed by the Tukey-Kramer multiple comparison test; different letters above the bars indicate a significant difference between samples at P ≤ 0.05. The experiment was independently repeated three times, each time with similar results.
Fig 2.
Effect on M. javanica J2 viability of a SVM compared with that of D. cf. concentrica culture.
J2s were exposed for 48 h either to the SVM at 1 mL/L (V/V) [3-methyl-1-butanol (0.2 mL, 1.84 mmole), (±)-2-methyl-1-butanol (0.2 mL, 1.86 mmole), 4-heptanone (0.4 mL, 2.86 mmole), and isoamyl acetate (0.2 mL, 1.35 mmole)] or to three 50-mm-diameter fungal culture plates, each with 5 mL of growth medium, and then the viable J2s were separated using 30 μm sieves. The numbers on the y-axis represent the numbers (mean ± SE) of viable J2s counted following the incubation. Each treatment was composed of 10 technical repetitions using 300 J2s in each repetition. The results were subjected to analysis of variance followed by the Tukey-Kramer multiple comparison test; different letters above the bars indicate a significant difference between samples at P ≤ 0.05. The experiment was independently repeated three times, each time with similar results.
Fig 3.
Effects of individual compounds on M. javanica J2 viability.
J2s were separately exposed for 48 h to 3-methyl-1-butanol (0.2 mL, 1.84 mmole), (±)-2-methyl-1-butanol (0.2 mL, 1.86 mmole), 4-heptanone (0.4 mL, 2.86 mmole), isoamyl acetate (0.2 mL, 1.35 mmole), or to 1 mL combination thereof (SVM), before viable J2s were separated using 30 μm sieves. The numbers on the y-axis represent the numbers (mean ± SE) of viable J2s counted following the incubation. There were 7–10 repetitions, each using 300 J2s. The results were subjected to analysis of variance followed by the Tukey-Kramer multiple comparison test; different letters above the bars indicate a significant difference between samples at P ≤ 0.05. The experiment was independently repeated three times, each time with similar results.
Fig 4.
Nematicidal effect of the chemical compound 4-heptanone on M. javanica J2 viability.
In each treatment the nematodes were incubated for 24 or 48 h in the presence or absence of 4-heptanone (0.4 mL, 2.86 mmole), before viable J2s were separated using 30 μm sieves. The numbers on the y-axis represent the numbers (mean ± SE) of viable J2s counted following the incubation. There were 8–10 repetitions, each using 300 J2s. In the treatment designated as "4-heptanone 24 h + recovery 24 h" the nematodes were exposed to the compound for 24 h and then removed to a new container with no VOCs for recovery. The results were subjected to analysis of variance followed by the Tukey-Kramer multiple comparison test; different letters above the bars indicate a significant difference between samples at P ≤ 0.05. The experiment was independently repeated three times, each time with similar results.
Fig 5.
Phenotypes of Meloidogyne javanica J2 following exposure to D. cf. concentrica culture, SVM, and 4-heptanone.
The nematodes were exposed to each compound for 48 h at 25°C in the dark, before visualization. A. Control—untreated nematodes. B. Nematodes exposed to three 50-mm-diameter culture plates, each with 5 mL of growth medium, on which D. cf. concentrica had been pre-grown for 4 days. C. Nematodes exposed to SVM preloaded onto 125 mg of perlite particles at 0.25 mL/L (V/V) [3-methyl-1-butanol (0.05 mL, 0.46 mmole), (±)-2-methyl-1-butanol (0.05 mL, 0.46 mmole), 4-heptanone (0.1 mL, 0.72 mmole), and isoamyl acetate (0.05 mL, 0.34 mmole)]. D. Nematodes exposed to 4-heptanone (0.05 mL, 0.36 mmole) preloaded onto 25 mg of perlite particles. The bar represents 500 μm.
Fig 6.
Effects of D. cf. concentrica volatiles and of the SVM on M. javanica eggs.
The eggs were exposed for 48 h either to D. cf. concentrica grown in three 50-mm-diameter culture plates, each loaded with 5 mL of growth medium or to 0.25 mL/L (V/V) of the SVM [3-methyl-1-butanol (0.05 mL, 0.46 mmole), (±)-2-methyl-1-butanol (0.05 mL, 0.46 mmole), 4-heptanone (0.1 mL, 0.72 mmole), and isoamyl acetate (0.05 mL, 0.34 mmole)] preloaded onto 125 mg of perlite particles. Each treatment used 10 repetitions each using 800 eggs. The numbers on the y-axis represent the means (± SE) of percentage of egg hatching. The results were subjected to analysis of variance followed by the Tukey-Kramer multiple comparison test; different letters above the bars indicate a significant difference between samples at P ≤ 0.05. The experiment was independently repeated three times, each time with similar results.
Fig 7.
Activity of the SVM in loamy soil.
A 0.4 mL stock of SVM [3-methyl-1-butanol (0.08 mL, 0.73 mmole), (±)-2-methyl-1-butanol (0.08 mL, 0.74 mmole), 4-heptanone (0.16 mL, 1.14 mmole), and isoamyl acetate (0.08 mL, 0.54 mmole)] was aliquoted, loaded on perlite particles, and mixed with 60 g of soil in a sealed 50-mL cup, before the addition of 500 J2s. The cups (five repetitions) were sealed and incubated for 48 h, after which viable J2s were determined according to the Baermann funnel method. The numbers on the y-axis represent the means (± SE) of viable J2s extracted from the soil. The results were subjected to analysis of variance followed by the Tukey-Kramer multiple comparison test; different letters above the bars indicate a significant difference between samples at P ≤ 0.05. The experiment was independently repeated twice, each time with similar results.
Fig 8.
Effects of the SVM in greenhouse experiments.
Susceptible tomato seedlings were planted in inoculated soil, with or without pretreatment with the synthetic volatile mixture (SVM). Four treatments were designated: Control NIR—soil inoculated with nematode-infected roots (NIR) infested by M. javanica in amounts equivalent to 4,000 eggs in root suspension in each pot; SVM NIR—soil inoculated with nematode-infected roots infested by M. javanica in amounts equivalent to 4,000 eggs in root suspension in each pot and supplemented with the synthetic volatile mixture; Control J2 –soil inoculated by direct insertion of 4,000 M. javanica J2s into each pot; and SVM J2 –soil inoculated by direct insertion of 4,000 M. javanica J2s and supplemented with the synthetic volatile mixture. (A) Galling index (mean of five repetitions). The results were subjected to nonparametric comparison using Wilcoxon method; different letters above the bars indicate a significant difference between samples significant at P ≤ 0.05. (B) Mean numbers (± SE) of M. javanica eggs per gram of root. The results were subjected to analysis of variance followed by the Tukey-Kramer multiple comparison test; different letters above the bars indicate a significant difference between samples at P ≤ 0.05. (C) Mean weights (± SE) of tomatoes roots. The results were subjected to analysis of variance. No significant differences (P > 0.01) between treatments in root weight were found. The experiment was independently repeated twice, each time with similar results.