Brachycorynella asparagi (Mordv.) Induced-Oxidative Stress and Antioxidative Defenses of Asparagus officinalis L

Abstract

The aim of this study was to investigate whether and to what extent oxidative stress is induced in leaves of one- and two-month-old plants of Asparagus officinalis L. cv. Argenteuil infested by Brachycorynella asparagi (Mordvilko) at a varied population size. The pest B. asparagi has been described as the most damaging species feeding on asparagus. Analyses using electron paramagnetic resonance (EPR) demonstrated generally higher concentrations of semiquinone radicals with g-values of 2.0045 ± 0.0005 and 2.0026 ± 0.0005 in Asparagus officinalis (A. officinalis) leaves after Brachycorynella asparagi (B. asparagi) infestation than in the control. Observations of leaves under a confocal microscope showed a post-infestation enhanced generation of the superoxide anion radical (O₂^•-) and hydrogen peroxide (H₂O₂) in comparison to the control. Strong fluctuations in Mn²⁺ ion levels detected by EPR spectroscopy versus time were detected in leaves infested by aphids, which may indicate the involvement of these ions in the control of O₂^•- production. An enhanced superoxide dismutase activity is an important element in leaf defense against oxidative stress. Visible symptoms were found in aphid-infested A. officinalis. Damage to leaves of one- and two-month-old A. officinalis plants by the aphid B. asparagi was dependent on the intensity, duration of infestation and plant age.

Keywords: Asparagus officinalis; Brachycorynella asparagi; antioxidant enzymes; manganese ions; oxidative stress; reactive oxygen species; semiquinone radicals.

Figure 1

Concentrations of semiquinone radicals with two g-values: 2.0045 ± 0.0005 and 2.0026 ± 0.0005 (a,b); and manganese ions with g-values of 2.00 (0.01) (c,d) in leaves of one- and two-month-old plants of Asparagus officinalis L. cv. Argenteuil infested by Brachycorynella asparagi (Mordvilko) at a varied population size (10, 20, and 30 aphids per plant). Values represent means and SE from three independent experiments. The data was statistically analyzed using analysis of ANOVA variance (p-values at α = 0.05). In individual figures significant differences are shown using asterisks.

Figure 2

Relative generation and cytochemical localisation of superoxide anion radical in leaves of one- (a) and two-month-old plants (b) of Asparagus officinalis L. cv. Argenteuil infested by Brachycorynella asparagi (Mordv.) at a varied population size (10, 20, and 30 aphids per plant); and 96-h leaves of Asparagus officinalis one-month-old plants infested by 30 aphids B. asparagi per plant (c). Yellow fluorescence originating from DHE (dihydroethidium) was observed under a Zeiss LSM 510 confocal microscope (objective magnification of 20× for a,b and 5×, 20× and 63×, respectively, for c). Scale bar 100 µm (a,b) and 50 µm (c).

Figure 2

Relative generation and cytochemical localisation of superoxide anion radical in leaves of one- (a) and two-month-old plants (b) of Asparagus officinalis L. cv. Argenteuil infested by Brachycorynella asparagi (Mordv.) at a varied population size (10, 20, and 30 aphids per plant); and 96-h leaves of Asparagus officinalis one-month-old plants infested by 30 aphids B. asparagi per plant (c). Yellow fluorescence originating from DHE (dihydroethidium) was observed under a Zeiss LSM 510 confocal microscope (objective magnification of 20× for a,b and 5×, 20× and 63×, respectively, for c). Scale bar 100 µm (a,b) and 50 µm (c).

Figure 3

Relative generation and cytochemical localization of hydrogen peroxide in leaves of one- (a) and two-month-old plants (b) of Asparagus officinalis L. cv. Argenteuil infested by Brachycorynella asparagi (Mordv.) at a varied population size (10, 20, and 30 aphids per plant); 48-h leaves of Asparagus officinalis one-month-old plants infested by 30 aphids B. asparagi per plant (c); and 96-h leaves of Asparagus officinalis two-month-old plants infested by 30 aphids (d). Green fluorescence originating from DCFH-DA (dichlorodihydro-fluorescein diacetate) was observed under a Zeiss LSM 510 confocal microscope (objective magnification of 20× for a,b and 5× and 20×, respectively, for c). Scale bar 100 µm (a–d).

Figure 3

Relative generation and cytochemical localization of hydrogen peroxide in leaves of one- (a) and two-month-old plants (b) of Asparagus officinalis L. cv. Argenteuil infested by Brachycorynella asparagi (Mordv.) at a varied population size (10, 20, and 30 aphids per plant); 48-h leaves of Asparagus officinalis one-month-old plants infested by 30 aphids B. asparagi per plant (c); and 96-h leaves of Asparagus officinalis two-month-old plants infested by 30 aphids (d). Green fluorescence originating from DCFH-DA (dichlorodihydro-fluorescein diacetate) was observed under a Zeiss LSM 510 confocal microscope (objective magnification of 20× for a,b and 5× and 20×, respectively, for c). Scale bar 100 µm (a–d).

Figure 3

Relative generation and cytochemical localization of hydrogen peroxide in leaves of one- (a) and two-month-old plants (b) of Asparagus officinalis L. cv. Argenteuil infested by Brachycorynella asparagi (Mordv.) at a varied population size (10, 20, and 30 aphids per plant); 48-h leaves of Asparagus officinalis one-month-old plants infested by 30 aphids B. asparagi per plant (c); and 96-h leaves of Asparagus officinalis two-month-old plants infested by 30 aphids (d). Green fluorescence originating from DCFH-DA (dichlorodihydro-fluorescein diacetate) was observed under a Zeiss LSM 510 confocal microscope (objective magnification of 20× for a,b and 5× and 20×, respectively, for c). Scale bar 100 µm (a–d).

Figure 4

Superoxide dismutase activity in leaves of one- (a) and two-month-old plants (b) of Asparagus officinalis L. cv. Argenteuil infested by Brachycorynella asparagi (Mordv.) at a varied population size (10, 20, 30 aphids per plant). Values represent means and SE from three independent experiments. The data was statistically analyzed using ANOVA (p-values at α = 0.05). In individual figures significant differences are shown using asterisks.