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Phytochemical responses to herbicide exposure and effects on herbivorous insects

6. The effect of herbivory on phytochemical profile

The present experiments were conducted to test if herbivores and herbicide together cause a (consequent) change in the content of selected phenolic compounds. It was observed in Chapter 4 that UV-B-radiation in general increases the amount of phenolic compounds in F. convolvulus. Rousseaux et al. (1998) observed that herbivorous insects eat more on plants that were not exposed to UV-B radiation. If this is the case for G. polygoni it may imply that an interaction between effects of UV-B and herbivory exists. It is therefore of interest to study if such interactions exist for compounds shown to increase due to UV-B and chlorsulfuron treatment.

6.1 Materials and methods

The effect of herbivore load was assessed by means of artificial defoliation in order to reduce the variability of the data due to for example mortality of the larvae or abnormal feeding behaviour. However, first a trial was conducted to verify that comparable responses are found in plants exposed to artificial and natural defoliation, respectively. The combined effect of herbivory and UV-B radiation was assessed in a greenhouse experiment.

6.1.1 Comparison of phytochemical responses to natural and simulated herbivory

Thirty F. convolvulus plants possessing approximately five leaves each were placed in a controlled-environment-chamber administered at 20°C, 16 h photoperiod, and a relative humidity of 70%. The following day, 10 larvae were placed on each of 10 plants. Ten other plants were exposed to simulated herbivory equal to the feeding activity by 10 G. polygoni-larvae (Table 1 presents the leaf area removed during the experiment based on a pilot experiment) and another 10 plants were left without leaf damage. Both natural and simulated herbivory were initiated on the third leaf counted from the bottom of the plant. After 6 days, the damaged leaves were harvested. Those exposed to natural herbivory were gently cleaned for faecal deposits with water before they were freeze-dried singly. The other leaves were freeze-dried without further handling. Early in development, larvae are more or less aggregated. As they grow, they become more dispersed on the leaves. Therefore, the artificial defoliation was conducted as follows (Table 6.1). On day 1, only one hole was made, and thereafter the number of holes created increased to 10 on day 3 (equal to the number of larvae). Hereafter, the size of the holes increased. If specimens died or disappeared (probably dead) they were replaced with specimens of the same size and age.

Table 6.1
Schematic presentation of the procedure for simulated leaf damage.

6.1.2 Effects of herbivore density on the content of selected phenolic compounds

As described above, there is a marked effect of herbivore density on the quality of the host plant to the herbivore when sprayed with chlorsulfuron. Therefore, an experiment was set up to measure the content of selected phenolic compounds in plants when manipulating the herbivore density and chlorsulfuron treatment.

Ninety-six F. convulvulus plants with five true leaves each, were placed in a controlled-environment-chamber. The conditions in the chamber were set at 20 °C, 16 h photoperiod and 70% RH. Half of the plants were sprayed with chlorsulfuron in a dosage of 0.5 times the recommended field rate in cereals (10 g ha^-1 formulated chlorsulfuron, i.e. 2 g a.i. ha^-1). The other half was sprayed with water. The day after treatment the plants were exposed to artificial defoliation. The defoliation (herbivory) was simulated by cutting out pieces of leaf each day equal to the feeding activity of 0, 10, 20, and 40 G. polygoni-larvae placed on the same leaf. Removal of leaf material was done on the third leaf from the bottom on plants with five leaves and was initiated on the first day after spraying. On day 7 after spraying, the treated ("eaten") leaf was harvested. All harvested leaves were freeze-dried singly and analysed for the selected phenolic compounds. All treatments were made as three independently prepared spray solutions with two samples per replicate.

6.1.3 Effects of herbivory and UV-B-light in combination

One hundred and twenty F. convulvulus seedlings were transplanted into pots and placed on watering tables in a greenhouse. Sixty potted plants were placed under UV-B exposure and another 60 pots placed without supplementary radiation. When the plants possessed eight leaves, 36 plants of similar size were selected from each treatment. The plants were then sprayed with chlorsulfuron in the following dosages: 0, 0.125 and 0.25 times the recommended field rate. Every treatment was replicated six times.

Four days after spraying, 10 newly hatched larvae were placed on the lower side of the third leaf counted from the bottom of the plant. The larvae were confined on the plant by a fine 1-mm mesh net formed as a bag. Seven days after spraying the leaf with larvae was harvested and freeze-dried for subsequent chemical analysis.

For plants receiving UV-B light, this was supplied by Phillips TL 12/ 40 W lamps placed 0.5 m apart and 1 m above the plants. The lamps were turned on for a period of 5 h every day, imitating the daily UV-B influx on sunny days in the beginning of July. A cellulose acetate filter was used to absorb light below 290 nm.

During the experiment, plants were watered from the bottom, the temperature in the greenhouse was maintained at a target temperature of 22°C with variations due to external weather, and the photoperiod was 16h daylight. The plants were supplied with additional light if light intensity dropped below 5 klux; light was switch off when intensity raised above 25 klux. The artificial light source gave an approximate photo flux density of 300 mE m^-2 sec^-1 at the soil surface. The plants were moved around during the experiment to avoid position effects.

6.1.4 Spraying procedure

Plants were sprayed with a pot sprayer designed for automatic and controlled spraying of larger plants (Kristensen pot sprayer, Ringsted, Denmark; l ´ w ´ h = 120 ´ 100 ´ 170 cm). Trials with chlorsulfuron were conducted with the surfactant Citowett (BASF) added to the spray solution (0.5% v/v). Control plants were sprayed with water. The sprayer was equipped with two Hardi flat fan nozzles type 411014 separated by 53 cm and used at a working pressure of 2 bar. The sprayer was calibrated to deliver a spray volume of 200 l ha^-1.

6.1.5 Chemical analyses

Six phenolic compounds have been identified from F. convolvulus, as described in Chapter 2. We analysed the plant material for these six compounds. The compounds identified were: 3-E-caffeoylquinic acid (compound 1), 1-E-caffeoyl-b -D-glucose (compound 2), 1-E-p-coumaroyl-b _D-glucose (compound 3), 2-E-caffeoyl-tartronic acid (compound 4), 2-E-caffeoyl-meso-tartaic acid (compound 5), and quercetin-3-O-b -D-glucuronide (compound 6). For determination of the phytochemical profile of the leaves, extraction with ethanol and kampfeol triosid was performed (Chapter 2). The phenolic compounds were separated by HPLC, and characterised by negative ion FAB-MS as well as 1- and 2-dimensional NMR techniques.

Natural and artificial defoliation was compared by testing the concentration of each compound in a t-test. A two-way ANOVA was performed to test for effects of herbivory and herbicide treatment on the content of each compound. The first test was done with interactions, but as the interaction was non-significant, tests with only main effects were used. Regression analysis was performed to describe the effect of herbivory on compound 2 and 3. The effect of UV-B radiation and herbivory in combination were tested by means of two-way ANOVA. Single comparisons were made with Tukey t-tests.

6.2 Results

6.2.1 Comparison of phytochemical responses to natural and simulated herbivory

No significant effects on the content of selected phenolic compounds were found of damaging the leaves by either artificial or natural herbivory. None of the treated leaves analysed for phenolic compounds were different from the control plants. (Table 2).

Table 2
Statistical analyses of the impact of type of leaf damage on the concentration of phenolic compounds (One-way ANOVA).

6.2.2 Effects of herbivore density on the content of selected phenolic compounds

The content of all analysed compounds changed significantly in response to herbicide treatment. Compounds 1, 4 and 6 decreased significantly (Tukey-t-test), and compounds 2 and 3 increased (Table 3 and 4). Only compound 2 showed significant changes in relation to herbivory. The concentration of compound 2 decreased with increased herbivory for both sprayed and unsprayed specimens (Fig 6.1A). Compound 3 was only found in detectable concentration in sprayed plants. For the sprayed plants, the concentration decreased with increasing herbivory (Fig 6.1B).

Table 3
Statistical analyses of the impact of herbicide treatment and artificial defoliation on the concentration of phenolic compounds (Two-way ANOVA without interactions).

Figure 6.1
Relationship between the degree of artificial defoliation and the concentrations of compound 2 (Fig. A) and compound 3 (Fig. B). The herbivore load is artificial defoliation equally the feeding of a specified number of G. polygoni larvae. Circles represent data from unsprayed plants and triangles represent plants treated with chlorsulfuron at a dosage of 0.5 times the recommended field rate. The concentration of the compounds is given in m moles g dry weight^-1. Error bars represent Standard Error of Mean

Table 4
The effect of chlorsulfuron treatment and simulated G. polygoni herbivory on the content of selected phenolic compounds in black bindweed (F. convolvulus). The analyses were made on the artificially damaged third leaf counted from the bottom of the plant.

6.2.3 Effects of herbivory and UV-B-light in combination on the phenolic compounds

Two-way ANOVAs were conducted for each compound and none showed significant interactions. Therefore, test were made with main effects only (i.e. herbicide treatment and UV-B radiation).

UV-B radiation and spraying with chlorsulfuron had a significant impact on the concentration of both compound 1 (p = 0.0063 and p < 0.0001 respectively in a two-way ANOVA) (Fig. 6.2) and compound 2 (p = 0.0091 and p = 0.0005; two-way ANOVA). Spraying caused a lower concentration of compound 1, but the two herbicide dosages were not different (p > 0.05; Tukey). However, the concentration was higher in plants exposed to UV-B radiation than in control plants. For compound 2, only the highest spray dosage caused a significant increase compared to the control (p < 0.05; Tukey t-test). The UV-B-radiation caused higher concentrations in exposed plants.

Compounds 3 and 4 were not affected by UV-B radiation (p = 0.0824 and p = 0.0890 respectively; two-way ANOVA). Herbicide treatment affected the concentration of compound 3 (p = 0.0074; two-way ANOVA). There was a tendency to reduced concentration of compound 4 with increasing herbicide treatment.

Exposure to UV-B radiation and herbicide spraying with chlorsulfuron had no effect on the concentration of compound 5 (p = 0.3901; p = 0.1016; two-way ANOVA).

UV-B-radiation had a significant effect on the concentration of compound 6 (p = 0.0012; two-way ANOVA). The concentration was higher in UV-B treated plants. Spraying with chlorsulfuron did not change the concentration of compound 6 (p = 0.1167; two-way ANOVA).

6.3 Discussion

The phytochemical response of the plants exposed to herbicide and to UV-B radiation showed the same trends as described for in Chapter 4, except that the herbicide caused a decrease in compound 4. The difference may be due to differences in timing of harvesting.

The absolute concentration of compound 2 was high in the experiment with UV-B radiation compared to the previously presented data from controlled environmental chambers. The first experiment was conducted in the greenhouse, which both have higher temperature fluctuations and probably also experience higher light intensities. The content of the phenolic compounds in this study with UV-exposure and herbivory were comparable to the UV study without herbivory (Table 4.6). It was also found that the levels for the phenolic compounds in the experiment with artificial defoliation were comparable to the control plants with herbivores in the "UV-B experiment", except again for compound 4 (Table 6.3 and Figure 6.1).

Figure 6.2
Relationship between herbicide dosage, UV-B radiation and the content of selected phenolic compounds. The open symbols represent plants were unexposed to UV-B radiation and closed symbols represent UV-B exposed plants. Bars represent standard error of mean.

It was the aim of this study to find if any of the selected compounds increased with herbicide dosage and herbivore load. No such combination was found. However, if we should proceed along the working hypothesis that phenolic compounds elicit the observed effects only two compounds are likely candidates i.e. compounds 2 and 3, which increase with herbicide treatment.

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