On integrating molecular and ecological studies of plant resistance: variety of mechanisms and breadth of antagonists

We are grateful that an attempt has been made (Heil 1999) to place the issue of systemic acquired resistance (SAR) within an ecological context. However, although this review raises some interesting points for further action and provides food for thought, we believe that the author may have underestimated both the changes in conceptual frameworks that are needed in order to incorporate SAR within such a context, and the efforts already being made in this area. The investigation of induced resistance mechanisms in plants is a fast-moving area of research and several excellent reviews on aspects considered in Heil (1999) have subsequently appeared (e.g. Agrawal et al. 1999; Bostock 1999; Maleck & Dietrich 1999; Pieterse & van Loon 1999). Indeed, a further review could usefully explore the evidence available for the points that Heil raises at the beginning of his article. Our article does not purport to be such a review; rather we wish to raise, in reply, two important points that have been generally overlooked in this debate. First, any understanding of SAR in its ecological context involves integrating this resistance within the framework of the other mechanisms by which the plant modifies its interactions with consumers. Second, this wider context for SAR must consider the potential for very broad interphylum effects. We demonstrate these points from our studies on the interactions between the large perennial herb Rumex obtusifolius and its consumers, notably the leaf-feeding chrysomelid beetle Gastrophysa viridula and the leaf-infecting rust fungus Uromyces rumicis. This is one of the few tripartite systems that has been intensively studied and that can also provide information on multiple, interacting resistances in plants. Current molecular studies of SAR exist in an ecological vacuum. They often use young parts, even cotyledons, generally of fast-growing annuals (e.g. Arabidopsis) or crop plants that are grown under strictly defined, controlled-environment conditions that usually differ substantially from conditions in the field. We understand that there are good biochemical reasons for concentrating on plant parts with such properties, but it will make the transition to ecological studies all the more difficult. Furthermore, most laboratory studies of SAR considered this phenomenon in isolation, i.e. without reference to the broader defence mechanisms of the plant. The plant is likely to have a number of strategies to deal with predators, whose potential diversity is evident, for example, in Rumex where we have found three resistance mechanisms. Age-specific constitutive resistance to infection by U. rumicis is observed in undamaged and uninfected young developing leaves of R. obtusifolius (Hatcher et al. 1995a); however, these leaves become susceptible to the fungus as they age. Localized induced resistance against rust has been demonstrated following artificial damage, insect chewing damage and rust infection (Hatcher et al. 1994b; 1995a). Resistance is induced when leaves are ‘challenged’ in these ways but is localized to undamaged areas of the affected leaf. Systemic induced resistance against rust (i.e. resistance in unchallenged leaves on challenged plants) follows insect chewing damage and rust infection, but cannot be stimulated by artificial damage (Hatcher et al. 1994b; 1995a). In addition to these resistance mechanisms, a fourth, resource depletion, may lead to resistance, although it may not have been selected for as such. For example, infection by the fungus U. rumicis causes a depletion in leaf nitrogen and changes in carbohydrate composition (Hatcher et al. 1995b; Hatcher & Ayres 1998) that are correlated with the reduced growth rates and fecundity of G. viridula fed on these leaves (Hatcher et al. 1994a). Interactions between R. obtusifolius and its consumers are also influenced by tolerance, especially in older plants that are able to regrow foliage rapidly after complete defoliation, for example by mowing (Niggli et al. 1993). By analogy with other systems, regrowth foliage may be better, worse or no different for insect consumers than the primary foliage (van der Meijden et al. 1988; Krause & Raffa 1992; Brown & Weis 1995). Such changes are currently still being defined in the R. obtusifolius system, but are one example of many possible interactions between the mechanisms that modify the interactions of plants with their consumers. Thus, we suggest that the more interesting ecological questions include the coexistence and interaction of the plant's various possible resistance mechanisms and how they affect interactions between the plant and its consumers. A number of important issues highlighted in Heil (1999), including the costs of SAR and its place in the framework of established theories of plant defence, must surely be assessed within this broader context, and not by focusing on SAR in isolation. By concentrating on pathogen-induced SAR, Heil (1999) did not do justice to studies of insect-induced defence mechanisms. Far from being ‘…mainly focused on ecological problems…’ there is a long history here of molecular and biochemical investigations paralleling the ecological work, which is excellently described and discussed by Karban & Baldwin (1997). Furthermore, in considering possible links between pathogen-induced and herbivore-induced resistances, Heil (1999) concentrated on a rather narrow range of the possible interactions, and thus underestimated the scope for ecologically significant interactions. Since Karban et al.'s (1987) ground-breaking study of interphylum resistance in cotton, several studies have reported resistance induced by an insect that has also affected subsequent infection by a plant pathogenic fungus (reviewed in de Nooij et al. 1992; Hatcher 1995; Hatcher & Ayres 1997). We agree with Heil (1999) that an ecological viewpoint can provide a framework for understanding physiological and biochemical findings, but disagree with his emphasis. For example, Heil (1999) takes a rather narrow view of cross-phyla induced resistance which he considers to be a mechanism by which plants are defended against secondary infection of wounds created by feeding. In Rumex, SAR induced by G. viridula grazing is not only effective against the rust U. rumicis but also against several fungal pathogens in both laboratory (Hatcher et al. 1994b; 1995a) and field tests (Hatcher & Paul 2000). Here it is worth noting two points: first, that the species involved, U. rumicis, Venturia rumicis and Ramularia rubella, are pathogens that do not need wounds in order to enter leaves; and second, that even for pathogens that do require wounds, localized resistance would be a more appropriate defensive mechanism than SAR. In addition, given Heil's comments that ‘…defence induced systemically by a single pathogen…gives resistance against many others…’, it is important to appreciate that there was no evidence that field infection of R. obtusifolius by any of the fungi induced SAR or localized resistance against the other fungal species (Hatcher & Paul 2000). Clearly, predicting the specificity of SAR in nature may be more difficult than expected from laboratory-based studies. Heil (1999) also suggests that ‘…herbivores seldom use heavily infected plant parts as a food source…’ and uses this as a reason as to why it is unlikely that antiherbivore defence will be induced by signals derived from pathogen attack, as this would lead to the synthesis of superfluous defence molecules. Although herbivores avoid infected tissues in a number of insect–fungus systems (Hatcher 1995), including the beetle G. viridula and the rust U. rumicis (Hatcher et al. 1994a; Hatcher 1995), some other herbivores prefer feeding on fungus-infected tissue (Leach 1940; Wheeler & Blackwell 1984; Ramsell & Paul 1990) and the absence of fungal-induced plant resistance against these herbivores could be due to the lack of selection pressure. Reduced selection pressure might occur because herbivores are feeding on diseased tissue that is already ‘lost’ in terms of its contribution to the plant's physiological processes, making grazing neutral in terms of host fitness. Indeed, if herbivores selectively consume the pathogen, as occurs with some rust–herbivore interactions (e.g. Ramsell & Paul 1990), grazing may even increase host fitness. Taking too narrow a view of SAR not only overlooks a whole body of ecological research but it also risks missing the point of some very interesting molecular work that is starting to demonstrate links between the pathogen-induced salicylic acid (SA) SAR pathway and the herbivore-induced octadecanoid or jasmonate (JA) pathway. The biochemistry and molecular biology of such interactions remain controversial. Some studies have suggested that stimulation of one pathway also facilitates the other. For example, the wounding of one leaf of young rice plants caused both a strong accumulation of jasmonic acid and SAR to infection by the rice blast fungus Magnaporthe grisea (Schweizer et al. 1998). However, there are now many examples of the SA and JA pathways inhibiting each other. Early in vitro studies suggesting that the two SAR pathways may be mutually inhibitory (e.g. Doares et al. 1995) have received some support in planta by the recent report (Felton et al. 1999) which showed that tobacco in which pathogen-induced SAR was suppressed expressed greater grazing-induced resistance to larvae of Heliothis virescens. Likewise, Thaler et al. (1999) demonstrated in field-grown tomato that the salicylate mimic benzothiadiazole reduces the jasmonate-induced expression of the enzyme polyphenol-oxidase, which is linked to antiherbivore defence and also reduces host-plant resistance to larvae of Spodoptera exigua. Conversely, treating plants with jasmonic acid reduces resistance to pathogen infection. In addition, treatment with salicylic acid prevented wounded plants from accumulating proteinase inhibitors and polyphenol oxidase, both of which may be involved in defence mechanisms triggered by jasmonic acid (Stout et al. 1998, 1999). This increasing molecular evidence of the ‘cross-talk’ between these different pathways of induced resistances (reviewed by Bostock 1999; Pieterse & van Loon 1999; Stout & Bostock 1999) and the range of interactions that have been shown, provide an intriguing context for ecological studies that is only just beginning to be explored. There is increasing evidence that insects and fungi may stimulate the SA and JA pathways, respectively (Inbar et al. 1998; Stout et al. 1998, 1999; Thomma et al. 1998; Stout & Bostock 1999); that is, instigate the ‘wrong’ response. This led Karban & Kuc (1999) to suggest that if responses mediated by JA and SA are generally antagonistic to each other, then perhaps insects that stimulate SA and pathogens that stimulate JA are indirectly depressing the plant's responses that would be most effective against them, thereby increasing their own fitness. This has been developed by Felton & Eichenseer (1999) who found that glucose oxidase from the saliva of Heliothis zea stimulated the SA pathway in soybean and induced resistance against Pseudomonas syringae and Cercospora sojinae. They suggest that this action of insect saliva may represent an adaptation to suppress the early events associated with the host-plant system of recognition and defence signal transduction (Felton & Eichenseer 1999), a suggestion supported by the work of Korth & Dixon (1997). If such manipulation of host-induced defences by herbivores proves to be widespread, it highlights the quandary of why SA and JA pathways are mutually inhibitory. Are there physiological constraints that limit simultaneous induction of both pathways, or are there other selective pressures of which we remain unaware? We suggest that this is a fruitful area of investigation for both ecologists and molecular biologists. The above investigations of interactions between pathways have been driven mainly by interest in biochemical ‘cross-talk’. However, other interactions may be mediated entirely at the ecological level, between resistance mechanisms that need not have any biochemical similarity. For example, in R. obtusifolius, resource depletion by the rust U. rumicis leads to a greater amount of damage to the plant because it induces G. viridula to move from the infected leaves to those healthy younger leaves that are normally protected against the fungus by age-specific resistance (unpublished data). This negative effect (i.e. the increased feeding of G. viridula on younger leaves) must affect fitness to a smaller extent than the main effect of age-specific resistance towards the fungus, which is to allow the plant to outgrow a fungal infection. We are currently attempting to elucidate the molecular mechanisms behind the induced resistances that we have already demonstrated ecologically in Rumex. However, our field experiments provide an initial ecological answer to the final question raised by Heil (1999), ‘…do plants exist in nature that are older than seedlings that have not already been induced?’. This echoes the suggestion of previous authors that a state of permanent insect-induced (Karban & Myers 1989; Karban & Baldwin 1997) or fungus-induced (Heath 1995) resistance may be the norm. We have demonstrated that induced resistance in the field is a quantitative response to variation in herbivory within the natural range experienced by the plant, and hence we suggest that plants in the field may always be induced, although they are not always fully induced (Hatcher & Paul 2000). Although many of the biochemical studies referred to above originate from the last couple of years, ecological studies on these interactions predate them by several years. These ecological studies were themselves preceded by a considerable literature on the relationships that are mainly, but not entirely, directly between insects and plant pathogens (see Leach 1935, 1940 for earlier literature reviews). Indeed, it should have been possible for these earlier ecological studies that suggested interphylum induced resistance to inform molecular biologists of the possible range of interactions between resistance pathways, rather than vice versa. It still is. Overall, be it pathogen-induced SAR or herbivore-induced resistance, we argue that the continuation of ‘intradisciplinary’ approaches will continue to hinder real understanding of the ecology of SAR. In conclusion, we reiterate Karban & Baldwin's (1997; p. 245) suggestion that ‘It seems important to us that ecologists begin to appreciate the advances that biochemists have made and that molecular biologists begin to think a bit more like ecologists and evolutionary biologists’. We thank Jane Taylor for helpful comments on an earlier draft of this article. We thank the Natural Environment Research Council for funding our work on Rumex tripartite interactions over the years. Received 27 May 1999 revision accepted 6 February 2000