Phytochemical responses to herbicide exposure and effects on herbivorous insects
1. General introduction
| 1.1 | Organisms studied |
| 1.2 | Types of experiments performed |
Herbicides are widely used in conventional agriculture. On average, a Danish agricultural field is sprayed with herbicides 1.37 times a year . Spraying a field with herbicides may increase pest populations. This is found especially for synthetic auxins (review by Campbell (1988)). Most of these studies concerns aphid species ((Hintz and Schultz, 1969; Oka and Pimentel, 1974; Oka and Pimentel, 1976; Oka, 1979)) or species living in the meristematic tissues of the plant (Ingram and Charpentier, 1947; Ighii, 1963). For other feeding guilds, like foliar feeders, the response is more variable, but with a tendency to reduced performance of the herbivore (Agnello et al., 1986a; Agnello et al., 1986b; Agnello et al., 1986c; Holopainen et al., 1991; Meisner et al., 1987). The positive effects on aphids has been attributed both to a reduced predation in field studies, probably due to toxic effects of the herbicide on predators (Adams and Drew, 1969; Adams and Drew, 1965)), and to an improved food quality of treated host plants (Maxwell and Harwood, 1960; Oka and Pimentel, 1974).
Plants present in the spray drift zone (adjacent crops as well as wild plants) may be affected by the herbicide. If the food quality of the plants for herbivorous insects is changed, unintended and unwanted effects may occur. In adjacent crops, an improved food quality may result in an increased pest population with the possible need of additional insecticide spraying. For plants present in natural habitats near to the sprayed field, negative effects on herbivorous insects may disturb ecosystems or may have negative effects on species of public concerns (e.g. butterflies).
The proportion of the herbicide-sprayed area treated with sulfonylurea herbicides is increasing. One study (Kjær and Elmegaard, 1996) has indicated that this group of herbicides may have detrimental effects on leaf chewing herbivores. The food quality of Fallopia convolvulus to Gastrophysa polygoni larvae may be affected by spraying with (low dosages of) sulfonylurea herbicides (Kjær and Elmegaard, 1996), which, under field conditions, may result from herbicide drift from the sprayed fields into adjacent areas. Furthermore, G. polygoni has been found to thrive better on F. convolvulus plants grown in laboratory than on plants grown under field conditions (unpublished data). The phenoxy acid dichlorporp has been shown to reduce the survival of G. polygoni at low dosages, i.e. 0.167 times the recommended field rate (Madsen, 1995).
The sulfonylurea herbicides are a class of herbicides that act by inhibiting the first enzyme specific to the branched chain amino acid biosynthetic pathway (Chaleff and Mauvais, 1984). Furthermore, it has been shown that this is accompanied by a reduced translocation of photosynthetate (Bestman et al., 1990; Devine et al., 1990; Vanden Borne et al., 1988). As a consequence of this inhibition (Ralphs et al., 1998; Suttle and Schreiner, 1982; Suttle et al., 1983) have found an increased concentration of secondary plant compounds in tall larkspur, soybean and sunflower. The compounds affected encompassed both phenolics and alkaloids. These herbicides are selective against broad-leaved species, i.e. dicotyledons. The differences in susceptibility of plants have been found to rely primarily on differences in the capacity for metabolism of the herbicides (Hageman and Beherens, 1984; Hall et al., 1992).
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. These types of impact have been described for a range of compounds including both primary and secondary plant metabolites (Harborne, 1993; Rosenthal and Berenbaum, 1992). The secondary plant compounds with these properties involve, among others, the alkaloids, terpenes, phenols and glucosinolates. We have chosen to focus on the phenolic compounds in this report because they are often found to be involved in plant defence against herbivores (Horwath and Stamp, 1993; Shaver and Lukefahr, 1969). Furthermore, the level of these compounds in plants may change after herbivore damage (Dixon and Paiva, 1995; Watermann and Mole, 1994) or herbicide treatment (Devine et al., 1993; Duke and Hoagland, 1978; Lydon and Duke, 1989; Lydon and Duke, 1993). Some phenolic compounds have also been shown to stimulate feeding or oviposition of insects (Feeny et al., 1988; Harbourne and Grayer, 1993; Shaver et al., 1998) including a Japanese Gastrophysa-species that is stimulated to feeds on another Polygonaceous by certain phenolic compounds (Matsuda, 1976; Matsuda, 1978; Matsuda, 1981).
The content and composition of phytochemicals may change as a consequence of e.g. chemical treatment, climatic stress, or herbivory as describe above. Consequently, not only herbicide treatment itself, but also the activity of herbivores as well the conditions under which testing takes place (laboratory versus field) may affect the internal response in plants (Watermann and Mole, 1981).
It is our hypothesis that the observed increase in mortality of G. polygoni larvae with herbivore density and dosage of the herbicide chlorsulfuron is caused by a herbivore induced chemical defence, which is enhanced by the chlorsulfuron treatment. This hypothesis is based on the reduced translocation in plants treated with sulfonylurea herbicides and the density dependent response of the larvae. Furthermore, we suggest that phenolic compounds are active in this relationship. This suggestion is based on the phytotoxic potential of phenolic compounds (Shaver and Lukefahr, 1969) and their role in host recognition processes of herbivores.
The present 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 large concentrations in leaves of F. convolvulus and/or responding to herbivore and herbicide stress of the plant. The impact of growth stage, herbivore load, herbicide dosage and growth conditions were included in the different experiments.
The interactions between herbicide treatment, phytochemical changes in plants and herbivore response were addressed and the main focal points were:
 | The effect of sulfonyl-urea herbicides on selected crops and weeds |
| Indirect effects of herbicide treatment on selected insects utilising the above plants as hosts |
| The possible relationship between any effects on plants and insects following herbicide treatment and the phenolic profile of the plants |
| The effect of herbivory on selected phytochemicals |
| Comparison of laboratory and field observations |
| Risk assessment of herbicide drift to non-target organisms in areas adjacent to treated fields |
| The potential of phytochemicals as indicators of plant exposure to sulfonyl-urea herbicides |
1.1 Organisms studied
Three plant – herbivore systems representing different feeding guilds were included in the project in order to study how common side-effects of herbicides are on herbivores. The chosen organisms are all likely to be exposed to sulfonylurea herbicides due to spray drift into adjacent crops or semi-natural areas. Butterflies were represented by the Brassica napus – Pieris bassicae system, aphids by the Triticum aestivum – Sitobion avenae system, and leaf beetles by the F. convolvulus – G. polygoni system. The former two are described in more detail in Chapter 10, whereas the F. convolvulus – G. polygoni system is used in several chapters and therefore is presented below.
F. convolvulus (L.) A. Löve is one of the main host plants for the leaf beetle G. polygoni (Coleoptera: Chrysomelidae) (Sotherton, 1982). F. convolvulus is a strict annual weed species. It often climbs adjacent crop plants. Under greenhouse conditions the plant reproduces mainly by self-fertilisation. It has no leaf loss during development, but continues to grow until senescence, when all leaves dies almost synchronously and the seeds ripen. Details of the life cycle of F. convolvulus are presented by (Hume et al., 1983).
G. polygoni L. is a chrysomelid beetle utilising mainly two food plants, viz. F. convolvulus and P. aviculare L. It eats the foliage of the plants in all three larval instars and as adults. The larvae pupate in the soil.
F. convolvulus is an important weed in agricultural fields, and herbicides are used in order to control it. On the other hand, seeds of this plant as well as all stages of the beetle may serve as important protein sources for young chicks of the partridge (Southwood, 1969) and other farmland birds (Potts, 1986). Numbers of the farmland birds have declined dramatically during the last 20-40 years, and attempts have therefore been made to use reduced amounts of herbicides in field margins in order to increase the food sources available to farmland birds. The success of these attempts depends on the food quality of plants treated with sublethal dosages of herbicides. F. convolvulus plants sprayed with low dosages of the herbicide chlorsulfuron and at the same time heavily loaded with herbivores have been shown to provide food of lowered quality to young G. polygoni larvae (Kjær and Elmegaard, 1996). These results suggest that combined effects of herbicide and herbivore stress may induce a defensive reaction in the plant, while there are only minor effects when the two factors act alone.
1.2 Types of experiments performed
In the course of the project period a range of experiments has been performed. The main factors included in the experiments presented are outlined schematically below in Figure 1.1.
Figure 1.1
Schematic outline of the "study system" involved in the report.
In order to assess the phytochemical reaction of the plants, separation, identification and quantification of the quantitative most important phenolic compounds in Fallopia convolvulus were performed, as presented in Chapter 2.
Plants were sprayed with herbicide(s), and the resulting phytochemicals were measured to describe the correlation between herbicide treatment and changes in the phytochemical profile. First, different herbicides with different modes of action were applied to identify phytochemicals that may be unique to plants treated with sulfonylurea herbicides (Chapter 3). Secondly, a dose-response relationship between chlorsulfuron dosage and the content of selected phytochemicals was aimed at (Chapter 5). Thirdly, the time-course of the observed phytochemical changes following chlorsulfuron treatment was studied by harvesting leaves at different time intervals after spraying, to get an idea of induction and persistence (Chapters 2, 4, 5 and 7). Fourthly, studies of phytochemical concentrations in leaves at different positions (i.e. of different age) were conducted separately (Chapters 3 and 4) in order to examine whether old or young plant parts were more affected by herbicide treatment, and thus to find indications of possible mechanisms the observed effects on phytochemicals.
The possible differences in phytochemical response to herbicide treatment between plants grown under laboratory conditions and plants grown under outdoor conditions were studied in Chapter 4 by comparing plants grown indoor and outdoor after chlorsulfuron spraying. Since one of the major factor differing between the mentioned growth conditions and known to affects phytochemicals is UV light, this factor was included in the laboratory studies.
The influence of herbivory on the phytochemical profile of F. convolvulus was studied in Chapter 6. The experiments comprised both natural (Gastrophysa polygoni) and simulated herbivory.
Subsequently, effects on beetle survival of the changes in phytochemicals induced by chlorsulfuron treatment of the plant were studied by introducing the larvae on chlorsulfuron treated leaves (Chapter 7). Furthermore, the (possibly indirect) effects of sulfonylurea herbicide treatment of three different host plants on the associated herbivorous insects were studied by applying three herbicides in different dosages before introduction of the herbivore (Chapter 10). Both short-term and chronic effects on the herbivores were studied.
Since herbicide drift into adjacent field or natural areas is a likely exposure route for both plants and herbivorous insects, this aspect was studied by exposing F. convolvulus to chlorsulfuron in the field at different distances to the spray track (Chapter 9). Subsequently, the plants were transferred to the greenhouse and toxic effects as well as effects on the phytochemical profile were quantified. The influence of differences in spray droplet size on growth effects was studied in a greenhouse experiment (Chapter 9).
In many experiments a range of variables were varied simultaneously, because this allows for comparisons between experiments. Identification and quantitative analysis of selected phenolic compounds
The aim of this chapter is to identify the most important phenolic compounds in F. convolvulus leaves and to develop a reliable quantitative method for detection of these compounds in plant tissues.