| Front page | | Contents | | Previous | | Next |

Phytochemical responses to herbicide exposure and effects on herbivorous insects

5. Dose dependence and persistence of chlorsulfuron-induced phytochemical changes

Changes in phytochemical composition of a plant may have impact on the fitness of the plant, for example in terms of plant growth and reproduction or changes in palatability of the plant to herbivores. The dose-response relationship and the durability of the response are of paramount importance to the potential impacts of the phyto-chemical changes. Therefore, a dose-assay and a time-assay were set up to investigate the changes in concentration of some phenolic constituents after spraying with chlorsulfuron, as a function of dosage and time, respectively.

5.1 Methods

F. convolvulus plants were grown in 11 cm plastic pots filled with standard pot soil. At the 5-7 leaves stage, the plants were sprayed in a pot-sprayer (Christensen, Slagelse, DK) with the herbicide Glean 20 DF (20% chlorsulfuron) and the detergent Citowett (0.05 v%). The pot-sprayer was mounted with Hardi nozzles no. 16 and adjusted to deliver 200 l ha^-1 at 2 bar. Six dosages were applied, viz. 0.03125, 0.0625, 0.125, 0.25 0.5, and 1 times the recommended field rate, which is 4 g chlorsulfuron ha^-1. For each dosage, five replicates (plants) were made. Two control groups were sprayed with water and Citowett. In total, 40 plants were used. After five days, one middle leaf (third leaf from the bottom) was abscised, freeze-dried and analysed for phenolic constituents.

In the time assay, two dosages corresponding to 0.25 and 0.5 times the recommended field rate were used. As controls we used plants sprayed with water and Citowett. Three replicates, each with three plants, were run per treatment per harvest time, and in total 108 plants were included in the assay. Harvest was carried out at four points in time, i.e. 4 days, 8 days, 16 days and 30 days after spraying.

At harvest, one leaf from the middle of each harvested plant was removed, freeze-dried and analysed for phenolic compounds. The surface area of all leaves was measured by use of an area meter (Li-Cor 3100, Lincoln, Nebraska) and dry weight of the plant not used for chemical analysis was obtained after freeze drying for 24 h.

The experiments were carried out from March to May 1998. Plants were grown in a green house. Twenty-four hours after spraying, the plants were moved to a controlled-environment chamber and kept at 20 C, 16:8 h light:dark, and 70% RH.

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.

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) and quercetin-3-O-beta-D-glucuronide (compound 6). Chemical analysis and numbering of the compounds follow Chapter 2. Compound 5 was not identified in any samples from the present study.

5.2 Results

Only the results dealing with compounds 2 and 3 are presented here.

At 0.03125 times the recommended field rate, were there is no significant changes in concentration of compounds 2 and 3 in the leaves, although concentration of compound 2 was elevated compared to the control. At 0.0625 times the recommended field rate, a clear increase was observed for both compounds. Compound 2 reached its maximum leaf concentration in plants sprayed with 0.125 – 0.5 times the recommended field rate and no further increase was seen (Fig. 5.1). Compound 3 followed the same pattern and reached maximum concentration at 0.25 times recommended field rate with a tendency to further increase at higher dosages.

Figure 5.1
Concentration of compounds 2 and 3 in middle leaves of F. convolvulus as a function of chlorsulfuron dosage (dosage in multipla of recommended field rate).

Results for all detected compounds are presented. Concentration of compounds 1, 4, and 6 in middle leaves decreased significantly with age in control plants. During the first 4 days of our observations, the concentration of compound 1 drops from 4.2 to 1.3 mg g^-1 dw. Compound 4 drops from 19.0 to 6.3 mg g^-1 dw and concentrations of compound 6 decreases from 4.5 to 1.2 mg g^-1 dw (Figure 5.2). The content of compound 1, 4 and 6 was reduced in leaves from sprayed plants but there was no difference between 0.25 and 0.5 tims the recommended field rate (Fig.5.2). The effect was just significant 4 days after spraying. After 8 days differences between treatments could not be detected anymore.

Figure 5.2
Concentrations (mg g^-1 dw) of compounds 1, 4 and 6 in middle leaves of F. convolvulus as a function of time (days) after spraying for two chlorsulfuron dosages, 0.25 and 0.5 times the recommended field rate.

The concentration of compound 2 was considerably higher in leaves from plants sprayed with 0.5 times the recommended field rate than in the control leaves (Fig. 5.3). This effect could not be detected 8 days after spraying. 0.25 of recommended field rate did not have any effect on the concentration of compound 2.

Figure 5.3
Concentrations (m g g^-1 dw) of compounds 2 and 3 in middle leaves of F. convolvulus as a function of time (days) after spraying with two chlorsulfuron dosages, 0.25 and 0.5 times the recommended field rate.

Compound 3 was only found in sprayed plants and the concentration in the leaves was dosage dependent (Fig. 5.3). The mean middle leaf concentration 4 days after spraying was 3.5 mg g^-1 dw (s.e = 0.91) in leaves sprayed with 0.5 times the lable rate dosage and 0.9 mg g^-1 dw (s.e =0.18) in leaves sprayed with 0.25 times the recommended field rate. After 8 days, the concentration of compound 3 in leaves treated with 0.5 times the recommended field rate decreased to the same level as in leaves treated with 0.25 times the recommended field rate. This level was approximately the same after 16 days. At day 30, compound 3 could still be detected in some but not all of the plants, and only in trace amounts.

The mean area of middle leaves from control plants increased steeply until day 16, whereafter growth ceased or some of the leaves started to loose area (Fig. 5.4). The growth of leaves exposed to chlorsulfuron was low and insignificant

Figure 5.4
Mean leaf area (cm²) of middle leaves from F. convolvulus as a function of time (days) after spraying with chlorsulfuron at three dosages: 0, 0.25, and 0.5 times the recommended field rate.

5.3 Discussion

The dose-response reaction of the concentration of compounds 2 and 3 in the leaves seems to be dominated by a sigmoid response mechanism within the investigated concentration range of chlorsulfuron (Fig. 5.1).

The reduction of the content of compounds 1, 4, and 6 in F. convolvulus leaves was consistent with what has been observed in the experiment with several different herbicides (Chapter 3). The herbicide-induced depression of these compounds made a significant difference between herbicide-exposed plants and unexposed plants. The difference disappeared with time and was not detectable after 8 days (Fig. 5.2) because the level of these compounds dropped in the control plants.

In leaves subjected to chlorsulfuron treatment at the highest dosage the concentration of compound 2 was considerably higher than in the control plants 4 days after spraying. Four days later this effect disappeared. Compound 3 was only found in chlorsulfuron-sprayed plants as seen before (Chapters 2 and 3). The concentration was dependent of chlorsulfuron dosage. At the highest dosage, concentration of compound 3 dropped steeply from day 4 to day 8 (Fig. 5.3). The decline was exactly parallel to the decline of compound 2 at the highest chlorsulfuron dosage. The concentration curves describing compounds 2 and 3 at the low chlorsulfuron dosage follow similar tracks.

Sixteen days after spraying, the chlorsulfuron treated plants still contained significant amounts of compound 3, but 30 days after spraying not all plants contained detectable amounts. Compound 3 seems very applicable as indicator for exposure to chlorsulfuron at 0.065 times the ideal recommended field rate (0.25 g per ha) until 16 days after actual exposure. Considering the ideal growing conditions during the experiment, which made the plants grow very fast and finish there life cycle fast, it may be difficult to find any indicator substance with a longer lifetime. Under field conditions with lower average temperatures the indicator may even live longer. It is surprising that 0.25 times the recommended field rate of chlorsulfuron appeared to induce a different concentration development of compound 3 in time in the middle leaves compared to 0.5 times the recommended field rate. Again, the pattern was consistent with compound 2. At 0.5 times the recommended field rate the concentrations of compounds 2 and 3 were relatively high after 4 days but then the major part of this peak concentration was metabolised during the next 4 days. However, at the low dosage (0.25 times the recommended field rate) there were no signs of a net degradation from day 4 to day 16 of compound 3! It should be mentioned that at the applied herbicide dosages, the growth of the plants is severely inhibited (Fig. 5.3). Thus, there is hardly any dilution of herbicide or constituents taking place in the sprayed plants.

| Front page | | Contents | | Previous | | Next | | Top |