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Phytochemical responses to herbicide exposure and effects on herbivorous insects

3. Impact of herbicides with different modes of action on phenolic compounds

The changes in composition of phenolic compounds in F. convolvulus leaves, observed after application of chlorsulfuron (Chapter 2), may be caused by general stress reactions of the plant and/or specific actions induced by this specific herbicide. The same responses may be induced by other herbicides with the same or different mode of action. An assay including herbicides representing six different modes of action was carried out in a greenhouse and in controlled-environment-chamber to further investigate how specific the response is. Two herbicides of the same family as chlorsulfuron were included in the experiment to test if the responses seen for chlorsulfuron are general for all three sulfonylurea herbicides.

3.1 Methods

Eight different herbicides were selected in order to represent six different modes of action and three sulfonylurea compounds (Table 3.1).

Table 3.1
Herbicides used in the study. Active ingredient, trade name and manufacturer, label rate, dosage used and mode of action.

The dosage applied was determined in a pilot study and chosen to be sufficient to produce a clear effect without killing the plant during the experimental period.

For each herbicide, three replicates of three F. convolvulus-plants were sprayed and two plants were sprayed with water. For the sulfonylurea herbicides, the detergent Citowett were added to all replicates including control.

Plants were sprayed at the five leaves stage in a pot sprayer (manufacturer Christensen, Slagelse, DK). The pot-sprayer was calibrated to deliver 200 l ha^-1 at 2 bar and 4.7 km h^-1, nozzle Hardy No 16. The plants were kept in a greenhouse at 12-20 ° C until spraying. After spraying, plants treated with herbicide were left for 24 h in a separate compartment of the greenhouse and subsequently all plants were moved to a growth chamber where the environment was set to 20 ° C, 16 h light, 70% RH. Seven days after spraying three leaves were cut of each plant, one leaf from the top, one from the middle, and one from the bottom of the plant. The leaves were freeze-dried for 24 h and stored in a freezer until chemical analyses were performed. Due to loss of leaves, a few replicates of bottom leaves and one middle leaf could not be sampled from plants sprayed with the chlorsulfuron.

The leaves were analysed for six phenolic compounds i.e. 3-O-E-caffeoylquinic acid (neochlorogenic acid) (compound 1), 1-O-E-caffeoyl-beta-D-glucose (compound 2), 3-O-E-p-coumaroyl-beta-D-glucose (compound 3), caffeoyl tartronic acid (compound 4), caffeoyl meso-tartronic acid (compound 5), quercetin-3-O-beta-D-glucuronide (compound 6). Chemical analysis and numbering of the compounds follows Chapter 2. Compound number 5 was only found in trace amounts.

Differences in mean concentration of the phenolic compounds in leaves were tested in a Tukey ‘s Studentized Range (HSD) Test by use of the SAS statistical Software Program assuming p=0.05 as level of significance.

3.2 Results

The content of compound 1 was highest in top leaves and decreased downward (Table 3.2). For all the herbicides tested application significantly reduced the concentration of compound 1 in the top leaves by 70-100% (Fig. 3.1). In the middle leaves the concentration was reduced significantly by c. 30-60% for pendimethalin, metolachlor, tribenuron methyl and chlorsulfuron. For dicamba, glyphosate, bromoxynil, and metsulfuron no changes were observed in middle leaves. In the bottom leaves no clear trends in changes of concentration could be registered for any of the tested herbicides.

Table 3.2
Phenolic compounds (m mol g^-1 dw) in F. convolvulus-plants sprayed with different herbicides. Leaves sampled from top, middle and bottom of plants seven days after spraying. Concentrations are given as means ± one standard error of mean.

The concentration of compound 2 was highest in middle and top leaves (Table 3.2). Tribenuron methyl and chlorsulfuron treatment increased concentration of this compound 2 to 3 times in bottom and middle leaves respectively (Fig. 3.2). In top leaves the same herbicides apparently reduced concentration of compound 2, although not significantly. Metsulfuron-treated plants followed the same pattern for top and bottom leaves but the reaction was weaker.

Compound 3 was only found in significant amounts in bottom and middle leaves sprayed with sulfonylurea herbicides (Fig. 3.3). The effect of metsulfuron was weaker than for the other two sulfonylureas.

Compound 4 occurred generally in high concentrations, highest in the top leaves (Table 3.2). All tested herbicides reduced the concentration of compound 4 in the top leaves (Fig. 3.4). In bottom and middle leaves the changes due to herbicides were insignificant except for tribenuron methyl, which reduced the amount of compound 4 in bottom leaves as well. The two other sulfonylurea compounds induced the same changes in concentration pattern in the plants, but the changes were not statistically significant.

The concentration of compound 6 compared to compound 4 is approximately the same in the top leaves but much lower in middle and bottom leaves (Table 3.2). All tested herbicides reduced concentration of compound 6 in the top leaves (Fig. 3.5). Differences in concentrations in the lower leaves are uncertain because of the small amounts present there (Table 3.1).

Figure 3.1
Relative content of compound 1 seven days after spraying in leaves from plants sprayed with different herbicides. 100%= control, i.e. water treatment. * indicates significant difference between treatment and control.

Figure 3.2
Relative content of compound 2 seven days after spraying in leaves from plants sprayed with different herbicides. 100%=water. * indicates significant difference between treatment and control.

Figure 3.3
Compound 3 (m mol mg-1 dw) seven days after spraying in leaves from plants sprayed with different herbicides. * indicates significant difference between treatment and control.

Figure 3.4
Relative content of compound 4 seven days after spraying in leaves from plants sprayed with different herbicides. 100%=water. * indicates significant difference between treatment and control.

Figure 3.5
Relative content of compound 6 seven days after spraying in leaves from plants sprayed with different herbicides. 100%=water. * indicates significant difference between treatment and control.

3.3 Discussion

All the tested herbicides induced a significant reduction of concentrations of compounds 1, 4 and 6 in top leaves. This reaction may be a more general herbicide stress response. The occurrence of the response solely in the top leaves may be explained by the age of the leaves, young leaves having a higher metabolism as they grow faster etc. It may also be influenced by the position of the leaves in relation to light intensity. The exposure of the top leaves compared to the lower leaves is probably higher when plants are sprayed from a nozzle above. Consequently, there may also be a dose - response relationship involved in the observed effect of leaf position. This should be combined with the different sensitivity of the plant to different herbicides.

The content of compound 2 increased 2 to 3 times in bottom and middle leaves of plants treated with tribenuron and chlorsulfuron. The effect of these two herbicides on top leaves was not significant but seemed to be opposite to the impact found in the lower leaves. Of the five phenolic compounds identified compound 2 was the most evenly distributed in control plants. This difference in distribution in the plant suggests that the compound have a different route of synthesis.

Compound 3 was only found in significant amounts in bottom and middle leaves from plants treated with the sulfonylurea herbicides. The importance of the sulfonylurea compounds mode of action is not clear to us and the effect may be linked to other characteristics of this herbicide group.

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