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Phytochemical responses to herbicide exposure and effects on herbivorous insects

Summary and conclusions

Background

Secondary plant metabolites are known to affect herbivorous insects. Effects of increased/induced metabolites on insects may be positive, through stimulation of consumption or negative, due to toxicity of the compound(s) or deterrent effects owing to changes in odour or taste.

Phenolic compounds are often involved in plant defence against herbivores, and the level of these compounds in plants may change after herbivore damage or herbicide treatment. Some phenolic compounds have also been shown to stimulate feeding or oviposition of insects.

The content and composition of phytochemicals may change as a consequence of e.g. chemical treatment, climatic stress, or herbivory. Consequently, not only herbicide treatment itself, but also the activity of herbivores as well the conditions under which testing takes place may affect the internal response in plants.

It has been observed that the food quality of Black bindweed (Fallopia convolvulus) to larvae of the leaf beetle Gastrophysa polygoni may be affected by spraying with sulfonylurea herbicides. It was shown that G. polygoni- larvae had an increased mortality on sprayed plants and that mortality was dependent on chlorsulfuron dose and herbivore density. It is our hypothesis that a herbivore-induced chemical defence, which was enhanced by the chlorsulfuron treatment, caused the observed increase in mortality. Furthermore, we suggest that phenolic compounds are active in this relationship.

Aim

The work presented in this report was initiated in order to investigate the possible role of phenolic compounds in the interactions between F. convolvulus, G. polygoni and the herbicide chlorsulfuron. The aim was to identify and quantify phenolic compound occurring in significant concentrations in leaves of F. convolvulus and/or compounds for which the concentrations responded to herbivore or herbicide stress. The impact of growth stage, herbivore load, herbicide dose and growth conditions were included in the different experiments. Furthermore, the project aimed at describing the effect of sulfonylurea herbicides on other plant-insect relationships and if other types of herbicides could have a similar effect.

Experimental data

Six phenolic compounds were isolated and identified from F. convolvulus leaves 3-O-E-caffeoylquinic acid (neochlorogenic acid)(1), 1-O-E-caffeoyl-beta-D-glucose (2), 3-O-E-p-coumaroyl-beta-D-glucose (3), caffeoyl tartronic acid (4), caffeoyl meso-tartronic acid (5), quercetin-3-O-beta-D-glucuronide (6) (Fig. 2.1). Compound 3 was isolated from leaves treated with the herbicide, chlorsulfuron, other compounds 1, 2, 4, 5, and 6 were isolated from untreated leaves. Compounds 1, 2, 4 and 5 were esters of caffeic acid while 3 was an ester of p-coumaric acid, and 6 was a flavonoid.

Eight herbicides with different modes of action were tested for their impact on the concentrations of the selected phenolic compounds. It was found that all tested herbicides induced a significant reduction of concentrations of compounds 1, 4 and 6 in top leaves (Table 3.2). This reaction may be a more general herbicide stress response.

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. Compound 3 was found to increase only in the bottom and middle leaves of plants treated with the sulfonylurea herbicides. It was only found in trace amounts in plants treated with other types of herbicides.

A closer examination was made of the impact of chlorsulfuron in combination with a range of other influential factors. These factors encompass time, UV-B radiation, herbivory and chlorsulfuron dose.

One to 4 days after herbicide application the content of compounds 2, 3 and 6 increased (Table 4.1). Hereafter a gradual decrease followed until day 30 after spraying when levels were comparable with the content in unsprayed plants.

Supply of UV-B light in the laboratory increased the similarity with field plants for compounds 1, 2 and 6 in middle leaves. Thus, light conditions are an important parameter for these compounds. UV-B light did not affect the concentrations of compounds 3 and 4 in unsprayed plants.

Experiments with varying numbers of herbivores per plant showed that the content of compounds 1, 2, 3 and 4 in general was reduced with increasing density of herbivores. The decrease was, however, mainly observed at the highest density (Table 5.4).

The dose-response reaction of the concentration of compounds 2 and 3 in the leaves described a sigmoid response 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 for several other herbicides. The herbicide-induced depression of these compounds caused a significant difference between herbicide-exposed plants and unexposed plants.

The many experiments conducted generally showed a large degree of comparability in the concentration of phenolic compounds for plants grown under similar conditions, such as dosage, time since spraying, UV-radiation etc.

An experiment carried out in controlled environment showed a weak correlation between the concentrations of the compounds 2 and 3 and the survival of G. polygoni larvae. In order to improve this correlation and describe larval intake for these compounds a mathematical model was developed. The model aims at describing the leaf concentration of compounds 2 and 3 over time and under different environmental conditions. The model was used to estimate the cumulated larval intake of the selected phenolic compounds during development from egg to pupae. Intake was then related to the observed mortality of the larvae on both chlorsulfuron-sprayed and unsprayed plants. This calculation improved the correlation between larval survival and the concentrations of compounds 2 and 3 considerably.

Differences between greenhouse- and field-grown plants

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

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 response in concentrations of 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 situations.

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 concentrations of the phytochemical compounds. Light conditions (UV-B) could explain some, but not all of the phytochemical difference between plants grown under laboratory and field conditions.

Indicator of herbicide exposure

Compound 3 seems applicable as indicator for exposure of F. convolvulus to sulfonylurea herbicides. The compound is only present in trace amounts in control plants, and the concentration increases in plants sprayed with 0.067 times the recommended field rate of chlorsulfuron (0.25 g a.i. ha^-1) or more. The compound is present at least until 16 days after actual exposure. Considering the growing conditions during the experiment, which made the plants grow very fast, it may be difficult to find any indicator substance with a longer lifetime. Under field conditions with lower average temperatures the indicator is expected to persist for a longer time. Finally, concentration of the compound was unaffected in plants treated with other types of herbicides.

Herbicide treatment and the performance of herbivorous insects

In addition to the Fallopia-Gastrophysa test system two other plant-herbivore systems were tested, namely the large white butterfly on oilseed rape and the cereal aphid on winter wheat plants. The following sulfonylurea herbicides were tested on the three plant-herbivore-systems: chlorsulfuron, metsulfuron and tribenuron. It was the intention to study whether other sulfonylurea herbicides had similar effects as those observed for chlorsulfuron.

A tendency of reduced survival was observed for G. polygoni feeding on hosts treated with sulfonylurea herbicides other than chlorsulfuron. This indicates that the effects observed for chlorsulfuron may be expected also for these herbicides, if higher dosages are used.

Larvae of the large white butterfly larvae (Pieris brassicae) were not directly affected by any of the herbicides in the dosages tested, but the host plant lost the leaves at very low dosages. An effect is therefore expected for specimens in adjacent non-crop habitats due to reduction of food resources. The experiments furthermore suggest that that the butterfly will not increase its pest status following spray drift from adjacent fields and that the use of reduced dosages of herbicide are unlikely to benefit insects associated with Brassica napus in the fields.

Furthermore, there were no indications of that metsulfuron treatment changed the quality of the winter wheat plants, as the cereal aphid (Sitobion avenae) developed and reproduced equally well on treated and untreated plants. Winter wheat is tolerant to sulfonylurea herbicides.