The Role of Ascorbate-Glutathione System and Volatiles Emitted by Insect-Damaged Lettuce Roots as Navigation Signals for Insect and Slug Parasitic Nematodes

Abstract

The effect of wireworm-damaged lettuce roots on the antioxidative defense system (ascorbate-glutathione cycle, photosynthetic pigments) and movement of insect/slug parasitic nematodes towards determined root exudates was studied in a glasshouse experiment. Lettuce seedlings were grown in a substrate soil in the absence/presence of wireworms (Elateridae). The ascorbate-glutathione system and photosynthetic pigments were analyzed by HPLC, while volatile organic compounds (VOC) emitted by lettuce roots were investigated by GC-MS. Herbivore-induced root compounds, namely 2,4-nonadienal, glutathione, and ascorbic acid, were selected for a chemotaxis assay with nematodes Steinernema feltiae, S. carpocapsae, Heterorhabditis bacteriophora, Phasmarhabditis papillosa, and Oscheius myriophilus. Root pests had a negative effect on the content of photosynthetic pigments in the leaves of infested plants, indicating that they reacted to the presence of reactive oxygen species (ROS). Using lettuce as a model plant, we recognized the ascorbate-glutathione system as a redox hub in defense response against wireworms and analyzed its role in root-exudate-mediated chemotaxis of nematodes. Infected plants also demonstrated increased levels of volatile 2,4-nonadienal. Entomopathogenic nematodes (EPNs, S. feltiae, S. carpocapsae, and H. bacteriophora) proved to be more mobile than parasitic nematodes O. myriophilus and P. papillosa towards chemotaxis compounds. Among them, 2,4-nonadienal repelled all tested nematodes. Most exudates that are involved in belowground tritrophic interactions remain unknown, but an increasing effort is being made in this field of research. Understanding more of these complex interactions would not only allow a better understanding of the rhizosphere but could also offer ecologically sound alternatives in the pest management of agricultural systems.

Keywords: ascorbate–glutathione system; entomopathogenic nematodes; lettuce; root volatile organic compounds; slug parasitic nematodes; wireworms.

Figure 1

To conduct the experiment, three circular marks with a diameter of 1 cm were created on the bottom of the Petri dish. The first mark was placed in the center, while the other two were positioned 1.5 cm from the edge on the right and left sides of the dish. A tested substance at a concentration of 0.03 μg mL⁻¹ was added to the right side of the agar surface using a pipette, creating the treated area. The left side of the agar surface was used as the control area and was treated with 10 μL of distilled water. Both sides of the agar surface were considered outer segments. The application of VOCs was performed immediately before the introduction of nematodes onto the agar plates. A 50 μL drop containing 100 IJs was placed in the center of the agar surface, which was considered the inner segment. In the control treatment, distilled water was applied to both the control and treated areas, and 50 μL of 100 IJs was placed in the center of the agar surface.

Figure 2

Concentrations of total ascorbate (nmol/g DW) and dehydroascorbate (% of total) in the roots and leaves of non-attacked plants and plants which had been attacked by wireworms. Different letters (a–d) indicate statistical differences (p < 0.05) between control and attacked plants.

Figure 3

Concentrations of total glutathione (nmol/g DW) and oxidized glutathione (% of total) in the roots and leaves of non-attacked plants and plants which had been attacked by wireworms. Different letters (a, b) indicate statistically differences (p < 0.05).

Figure 4

Concentrations of violaxanthin, antheraxanthin and zeaxanthin (mg/g DW) in the roots and leaves of non-attacked plants and plants which had been attacked by wireworms. Different letters (a, b) indicate statistical differences (p < 0.05) between control and attacked plants.

Figure 5

Concentrations of chlorophyll a + b (µg/g DW) and the ratio of chlorophyll a/b in leaves of non-attacked plants and plants which had been attacked by wireworms. Different letters (a, b) indicate statistical differences (p < 0.05) between control and attacked plants.

Figure 6

Examples of extracted ion chromatograms (XIC) from HS–SPME–GC-MS analysis of lettuce roots for the molecular mass of 2,4-nonadienal (m/z 138), with indicated peak of interest (at 12.19 min). (A) Lettuce roots control group; (B) lettuce roots infected with wireworms.

Figure 7

The chart displays the percentage of different nematode IJs in the outer circles after 24 h, depending on the temperature used in the experiment. The error bars correspond to the standard error. Capital letters denote statistically significant differences (p < 0.05) among the various nematode species at the same temperature, while small letters indicate statistically significant differences (p < 0.05) among the different temperatures within the same nematode species. The nematode species used in the experiment are as follows: Hb = H. bacteriophora; Sc = S. carpocapsae; Sf = S. feltiae, Om = O. myriophilus; Pp = P. papillosa.