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Phytochemical responses to herbicide exposure and effects on herbivorous insects

4. Phytochemical responses to chlorsulfuron treatment under laboratory and field conditions

The apparently reduced food quality of F. convolvulus to G. polygoni larvae when the plant is sprayed with sulfonylurea herbicides or grown under field-like conditions compared to control plants grown in the laboratory may be a result of changes in the secondary metabolites of the plant.

The aim of this chapter was to study whether the phytochemical profile of F. convolvulus changes as a result of growth conditions and herbicide treatment. This was done by exposing the plant to herbicide spraying under laboratory as well as field-like conditions and analysing the resulting phytochemical contents.

4.1 Materials and methods

F. convolvulus plants were exposed to different dosages of chlorsulfuron under laboratory and field-like conditions to see if differences in growth conditions and herbicide treatment caused differences in selected phytochemicals.

F. convolvulus plants were grown in two different set-ups, i.e. in greenhouse and under semi-field conditions outside the greenhouse. The plants were sprayed with different dosages of chlorsulfuron. At the end of the experimental period, the content of phytochemicals in leaves was estimated.

For both exposure situations, seed dormancy was broken by at least three months storage in humid Sphagnum medium at 5° C. The seeds were then placed under light conditions at approximately 20° C to produce seedlings, and the seedlings were transplanted to 11-cm pots containing a standard growth medium (SM vækstmuld, Stenrøgel). After transplantation, the pots were kept in a greenhouse at approximately 22° C, and a daily light period of 16 h. Sunlight was supplemented with artificial light (300 m E m^-2 s^-1) when light intensity dropped below 5 klux. If light conditions exceeded 25 klux, the artificial light was shut off. Plants for the field experiment were grown on tables outside, and climatic information was used to calculate their physiological age (Physiological age = (measured temperature - 8° C) ´ time, calculated on basis of hourly climatic data).

Plants were sprayed with Glean 20 DF (Dupont) (20 % chlorsulfuron) plus the detergent Citowett (BASF AG) when they had 5-7 leaves. Dosages of 0, 0.5 and 1.0 times the recommended field rate (4 g a.i./ha) were applied. The spraying equipment was a pot sprayer (Christensen) with Hardi 411014 flat-fan nozzles, calibrated at 200 l/ha. There were three replicates, each consisting of 3 plants. Plants for the laboratory experiment were placed in a controlled-environment chamber at a photo flux density of 350 m E m^-2 s^-1, 20° C, 70% RH and photoperiod of 16 h. Plants for the field experiment were returned to the outdoor tables after spraying.

After 1, 2 and 4 days laboratory plants were harvested, and field plants were cut off after 4 and 7 days (physiological time). Leaves from top, middle and bottom of plants were freeze-dried for phytochemical analyses.

For determination of the phytochemical profile of the leaves, extraction, separation and characterisation were performed as described in Chapter 2. Middle leaves were analysed for all collection times, whereas top and bottom leaves were only analysed when collected after 4 days and 7 days (field plants only).

A further laboratory experiment was set up to study if the differences in phytochemical profile between laboratory and field conditions observed in the above experiments (see results below) could be explained by differences in light conditions.

F. convolvulus plants were produced as described for the above experiment and covered with 1 mm mesh. 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 (not UV light).

UV-B light (+/-) was combined with chlorsulfuron spraying in a 2´ 2 factorial design, with herbicide dosages of 0 and 0.125 times the recommended field rate, and treatments were replicated six times. Spraying took place when the plants had approximately 8 leaves and was performed with the pot sprayer described above. Spray solutions were not replicated. Four days after spraying, the third (middle) leaf of every plant was collected for analyses of phytochemicals, which were performed as described in Chapter 2.

Effects of the various variables on phytochemical content were analysed by analysis of variance (ANOVA or GLM procedures of the SAS Stat programme). First, tests of interactions were performed, and in case of no significance, analyses for main effects alone were carried out. Means were compared by Tukey’s HSD t-test. All analyses were evaluated at the 5 % significance level.

4.2 Results

Greenhouse experiments

Five phytochemicals were identified: three hydroxycinnamoyl derivatives (compounds 1, 2 and 4), one coumaroyl derivative (compound 3) and one flavonyl glucoside (compound 6) (cf. Chapter 2). The content of the single substances in the middle leaves at the three collection times is presented in Table 4.1.

Table 4.1
Phenolic content (m moles g^-1 dry weight, means ± s.e.) in F. convolvulus leaves from the middle of the plants at different harvest times (days) after herbicide treatment under laboratory conditions. p values indicate whether there is a significant effect of herbicide dosage.

Collection time

The concentration of compound 1 was unaffected by herbicide treatment after 1 and 2 days. When harvested 4 days after herbicide treatment, the content was significantly lower in treated leaves than in controls. The content of compound 2 in treated plants rose already at day two after herbicide treatment, and this effects was even stronger after 4 days. Compound 3 was never found in unsprayed plants. Induction of this compound started at day 2 after spraying, and was significant after 4 days. Compounds 4 and 6 were unaffected by herbicide treatment at all harvest times.

The analysis of variance showed significant effects of herbicide dosage on compounds 1, 2, and 3 (Table 4.2). Time of leaf harvest significantly affected the content of compounds 2, 3 and 6, and there was an interactive effect of herbicide dosage and harvest time on compounds 2, 3 and 6 (Table 4.2). The effects of herbicide treatment increased with time for compounds 2 and 3, whereas the interactive effect for compound 6 is "artificial", since this compound was not affected by herbicide dosage (Tables 4.1 and 4.2).

Table 4.2
Effects of herbicide dosage and harvest time on the phenolic content of middle leaves of F. convolvulus grown under laboratory conditions, as tested by analysis of variance.

Leaf position

The content of phenols at day four in leaves at different position of the plant is presented in Table 4.3 and Figure 4.1. Compounds 1, 2, 4 and 6 occurred in highest concentrations in the top leaves. Herbicide treatment caused the content of compound 1 to decrease in all types (ages) of leaves, whereas the content of compounds 2 and 3 rose in the lower and middle leaves, but not in the top (young) leaves. The level of compound 6 decreased in the top leaves decreased as a consequence of herbicide treatment. These trends are confirmed by the analysis of variance (Table 4): All compounds were significantly affected by leaf position, and all except compound 2 were affected by herbicide dosage. Furthermore, the interactive effect of dosage and leaf position on compounds 1, 3 and 6 confirms the different effects of herbicide treatment on phytochemicals in leaves in different positions.

Field experiment

The plants were generally much more compact/stunted than plants grown under laboratory conditions.

The same five phenolic compounds as described for laboratory studies were identified, i.e. compounds 1-4 and 6 (cf. Chapter 2). Generally, the phytochemical content of field-grown plants was higher than that of laboratory plants, but not for compound 4 (Table 4.5, Figure 4.1). In contrast to the laboratory plants, small concentrations of compound 3 were found in unsprayed leaves.

Figures for 4 and 7 days (physiological time) are not directly comparable, since they were analysed on two different occasions.

With a few exceptions, the vertical distributions of the phytochemicals followed the same pattern 4 and 7 days after herbicide treatment (Figure 4.1). Herbicide dosage had only few significant (p < 0.05) effects on phytochemical content when tested for the different substances, harvest times and leaf positions (Table 4.5).

Compound 1 was found in higher concentrations in the top than in the bottom of control plants. Chlorsulfuron treatment depressed this compound in top parts of the plant, both after 4 and 7 days, and the same herbicide effect was also found in the middle parts after 7 days.

Table 4.3
Phenolic concentrations (m moles mg^-1 dry weight, means ± s.e.) in bottom, middle and top leaves of F. convolvulus 4 days after chlorsulfuron treatment. The plants were grown under laboratory conditions. p-values indicate result of analysis of variance on effects of herbicide dosage.

Table 4.4
Results of analyses of variance of the effect of leaf position (bottom, middle, top) on F. convolvulus plants harvested 4 days after herbicide treatment. Plants were grown under laboratory conditions.

Compound 2 concentrations were higher in top and middle than in bottom leaves of control plants after 4 days, but tended to accumulate in the middle parts after 7 days. Four days after chlorsulfuron treatment, there was a strong tendency of an increased content in the bottom parts of the plants. After 7 days, however, there was a strong tendency of a decrease in the middle leaves.

Compound 3 was found in small concentrations in control plants. After chlorsulfuron treatment, the content was increased, and the main proportion was found in the top leaves at both harvest times.

Compound 4 was rather evenly distributed in control plants. Following chlorsulfuron treatment, no significant change was seen, but there was a tendency of decline in top leaves.

The content of compound 6 increased from bottom in control plants. Herbicide treatment with 0.5 and 0.25 times the field rate was followed by a decrease in leaves at all positions (for 4 days only significant for bottom leaves). At full field rate, this effect was less evident, especially in bottom leaves.

Table 4.5
Phytochemical concentrations (m moles g¹ dry weight, means ± s.e.) of leaves at different positions, 4 and 7 days (physiological time) after chlorsulfuron treatment of F. convolvulus plants grown under field conditions. p value indicates outcome of analysis of variance on effect of herbicide dosage on phytochemical content. N = 3 for 4 days and N = 6 (8) for 7 days.

Figure 4.1
Content (m moles g^-1 dw) of 5 phenolic compounds in F. convolvulus grown under field conditions (middle and right) and in laboratory (left), measured 4 and 7 days (field, physiological time) and 4 days (laboratory) after chlorsulfuron treatment as function of herbicide dosage (times field rate) in bottom, middle and top leaves. N.B. different scales on y-axes.

4.2.2 Effects of UV-B light on phytochemical response

The effects of combinations of UV-B light and chlorsulfuron treatment on the content of phytochemicals in leaves sampled from the middle of the plants are presented in Table 4.6. Six phenolic compounds were identified and quantified (compounds 1-6, cf. Chapter 2).

Unsprayed plants

In unsprayed plants, UV-B increased the content of compounds 1, 2 and 6; the content of compound 4 was unaffected (but very high compared to the above results), and compound 5 was slightly suppressed. Compound 3 was not found in unsprayed plants.

Table 4.6
Concentrations of phytochemicals (m moles g^-1 dry weight, means ± s.e.) in F. convolvulus 4 days after chlorsulfuron treatment. Plants were grown under laboratory conditions, with or without supply of UV-B light. p values indicate effects of UV-B supply as evaluated by analysis of variance.

Herbicide treatment

There was an interactive effect (p £ 0.015) of herbicide treatment and supply of UV-B light for all compounds, except compound 4 (p = 0.27). For compound 1, the effects of herbicide treatment and UV-B were contrary, the herbicide decreasing the content and UV-B increasing it. For compound 2, the increase in content caused by herbicide treatment was slightly diminished when UV-B was supplied. For compound 3, the induction by herbicide treatment was enhanced by UV-B. For compound 5, UV-B reversed the effect of herbicide treatment from positive to negative. For compound 6, the interactive effect seems artificial, since there was hardly any difference between sprayed and unsprayed plants, but a large positive impact of UV-B.

Herbicide treated plants only showed significant UV-B effects for compounds 5 and 6 (Table 4.6). In plants not receiving UV-B light, there was a significant effect of herbicide treatment on compound 3. All other compounds were unaffected by herbicide treatment. In UV-B treated plants, herbicide effects occurred for compounds 1 and 3 (p = 0.0032 and 0.022).

4.3 Discussion and conclusions

The observed effect of the rather short time-span of 1 to 4 days on the content of compounds 2, 3 (increase) and 6 (increase followed by decrease) (Table 4.1) shows that the occurrence (and distribution) of these compounds within F. convolvulus is very dynamic. Hence, the chosen time-window may be crucial for results and conclusions drawn from the various studies presented in the present report.

Phytochemicals were generally quite similarly distributed within unsprayed plants grown under laboratory and field conditions (Figure 4.1). However, levels were higher in field plants, except for compound 4 (Figure 4.1), indicating an effect of growth conditions on the selected phytochemicals.

Supply of UV-B light in the laboratory increased the similarity with field plants for compounds 1, 2 and 6 in middle leaves – most successfully for compound 2 (Table 4.6). Thus, light conditions seem an important factor for those compounds. UV-B did not affect compounds 3 and 4 in unsprayed plants.

When the plants were sprayed with chlorsulfuron, the phytochemical response differed somewhat between laboratory and field plants (Figure 4.1): For compound 2, the content was generally higher in field plants. This may partly be due to differences in light conditions, since compound 2 is increased by UV-B light, and this part of the light spectrum was absent under the standard laboratory conditions. The vertical distribution of compound 2 differed between sprayed field plants and sprayed laboratory plants, with an increase in middle leaves in laboratory plants, whereas the distribution in field plants was hardly affected. The response in compound 3 to spraying also differed between growth conditions: In laboratory plants, an increase/induction was found in middle and bottom leaves, whereas field plants responded by an increase in top leaves. Part of the explanation may be differences in light conditions between the two exposure situations. From Table 4.6, it is obvious that UV-B light increases the response to spraying in compound 3 in middle leaves, but effects on the vertical distribution is unknown. Levels of compound 6 were generally higher in field plants than in laboratory plants (Figure 4.1), and the vertical distribution was more homogeneous in field plants. Differences in light conditions may be part of the explanation for the differences in level and distribution, since compound 6 is increased by UV-B (Table 4.6).

All in all, it was shown that growth conditions affected not only concentrations and distribution of phytochemicals in unsprayed plants, but also the effect of herbicides on the same phytochemical compounds. Light conditions (UV-B) could explain part, but not all of the phytochemical difference between plants grown under laboratory and field conditions.

The main conclusions that may be drawn from the described experiments are:

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