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Phytochemical responses to herbicide exposure and effects on herbivorous insects

7. Phenolic compounds and mortality of herbivorous larvae

The observed increased in mortality of G. polygoni larvae with herbivore density and dosage of the herbicide chlorsulfuron has led us to the suggestion that F. convolvulus possesses a herbivore-induced chemical defence, which is enhanced by the chlorsulfuron treatment. It is our hypothesis that phenolic compounds could be active in this relationship. We take a starting point in those compounds identified in Chapter 2 which in Chapter 4 have been shown to increase with herbicide treatment, i.e. compound 2 and compound 3. For these compounds we will establish correlations between the concentration and the number of surviving G. polygoni larvae. Therefore, this chapter presents an experiment designed to document correlation between these phenolic compounds and larval survival.

7.1 Materials and methods

The content of selected phenols in F. convolvulus plants treated in four different ways was measured for the third leaf on day 1, 4, 7 and 10 after treatment. The treatments encompassed: 1) herbicide treatment 2) herbivory, 3) both herbicide treatment and herbivory 4) no treatment plants. Herbicide treatment implies that plants were sprayed with 0.5 times the recommended field rate of chlorsulfuron (i.e. 2 g ha^-1). Herbivory was introduced to adding 20 newly hatched G. polygoni-larvae to the plant one day after spraying. The larvae were placed on the lower side of the third leaf counted from the bottom of the plant. Untreated plants were sprayed with water.

The experiment was conducted as a controlled-environment-chamber experiment. Seedlings of black bindweed (F. convolvulus) were transplanted singly to pots and placed in the greenhouse until they possessed five true leaves. Each treatment was replicated three times. All plants were confined in polyurethane cylinders until the adult beetles emerged from the soil.

Simultaneously with the chemical analysis, the survival of the larvae were registered. Upon harvest, the larvae residing on the leaves were moved to another leaf. The harvested leaves were cleaned for faecal deposits and freeze-dried before the chemical analysis. A schematic presentation of the experiment is given in Table 1.

Table 7.1
Schematic presentation of the experimental design

7.1.1 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.

7.1.2 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.

An ANOVA was performed to test for effects of herbivory and herbicide treatment and time on the content of each compound. The first test was done with interactions, but as interactions were non-significant, a test with only main effects was used. Regression analysis was performed to describe the realtionship between compound 2 or compound 3 and larval survival. Single comparisons were made with Tukey t-tests.

7.2 Results

7.2.1 Phytochemical responses of F. convolvulus to chlorsulfuron, herbivory and time

All compounds were significantly affected by the herbicide treatment. However, only the concentration of compound 2 and compound 3 increased with herbicide treatment (Tukey t-test), whereas all the other compounds (i.e. compounds 1, 4 and 6) decreased when treated with chlorsulfuron (Tukey t-test) (Table 7.2). There was a significant effect of herbivory on the content of compound 4, which increased with herbivory.

7.2.2 Relationship between content of phenolic compounds and insect survival

The data presented in Table 7.2 and Figure 7.1 are combined of leaves harvested in intervals of three days after spraying. In order to ensure that the data were not biased due to harvesting date, the whole data set was tested in a one-way anova with the principal factor being time and the concentration of compounds 2 and 3 as covariates. The time variable was not significant and the regression analysis also showed that only compound 3 had significant impact on the survival of G. polygoni larvae, i.e. the rate of decrease with dosage was significant different from 0 (t-test, p=0.0367). Despite this result, we chose to look at both compounds 2 and 3. These two compounds have repeatedly been shown to increase with herbicide dosage/treatment, and their structure is very similar. Thus, they may have the same mode of action if they are responsible for the observed reduction in larval survival. The regression analysis of survival as a function of concentration of compound 3 revealed the following equation: {Compound 3 = 13.32 - 0.725 ´ Dosage, r = 0.635, p = 0.005}. Compound 2 did correlate with the survival of the larvae, but not in the same degree as compound 3 {Compound 2 = 14.11 - 0.441 ´ Dosage, r = 0.505, p = 0.033}. A regression analysis of larval survival and the total amount of both compound 2 and 3 also showed a significant correlation between these two parameters, i.e. {Compound 2+3 = 14.53 - 0.366 ´ Dosage, r = 0.644, p = 0.004}. The total amount has a slightly better correlation than that for compound 3 and a much better one than that for compound 2.

Table 7.2
Concentration of phenolic compounds in black bindweed (F. convolvulus) in relation to time (days) since herbicide treatment, herbicide treatment with chlorsulfuron and feeding by 20 G. polygoni larvae.

Figure 7.1
The relationship between the number of surviving G. polygoni larvae and the concentration of the compounds 2, 3 and both added. The initial number of larvae was 20.

7.3 Discussion

There is some variation in the dose-response relationship, because the larvae probably did not stay on the third leaf for their entire development and there are differences in phytochemical concentrations between different strata of the plant (see Chapter 4 for example). Furthermore, the relation between the compounds and the survival of the larvae expresses effects on the larvae population over a longer period, whereas the chemical data represent spot checks of the content over the development. In order to adjust for this, a model for the intake of the compounds is needed. The large variation may also express that these compounds co-vary with some unidentified compounds that are the main causal reason for the mortality of the larvae.

The slightly better performance of the linear model when both compounds 2 and 3 are used is presumable due to the fact that compound 3 was not found in untreated plants, but G. polygoni did die to some degree in the controls which all contained compound 2. If bioassays cannot verify that these two compound are active in the increased mortality one should look for compounds that are found in detectable concentration in control plants and changes with chlorsulfuron treatment or for compounds that alter the host recognition behaviour.

The mortality found in this experiment,s was lower than that reported earlier (Kjær and Elmegaard, 1996). A possible explanation of this discrepancy is differences in design. In the former experiment, the beetles were added as eggs. This means that the larvae hatch and start feeding later, when the amounts of compounds 2 and 3 are higher due to herbicide treatment. This hypothesis is supported by the fact that compound 3 is not found in measurable quantities in sprayed leaves until day 4 after spraying (see Chapter 4 and Table 7.1).

Finally, other mortality factors not necessarily related to the herbicide treatment and not accounted for in the present set-up may influence the variation. This includes the presence of tolerant ecotypes of plants. Kjær et al. (1998b) observed that plants exposed to high concentrations of copper resulted in a differentiation of the growth rate and the mortality of F. convolvulus. This indicates that tolerant ecotypes might exist in F. convolvulus.

The data presented in this chapter therefore shows that the mortality of G. polygoni larvae are positively correlated to the concentration of compounds 2 and 3 in the leaves of F. convolvulus.

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