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- Materials and Methods
Hyperaccumulation in plants is the phenomenon that individuals of certain species accumulate metals or metalloids to concentrations several orders of magnitude higher than those found in other species on the same site (Baker & Brooks, 1989). Elements that have been reported to be hyperaccumulated include As, Cd, Co, Cu, Mn, Ni, Pb, Se, and Zn (Reeves & Baker, 2000; Guerinot & Salt, 2001). Selenium hyperaccumulator species have been reported in the genera Astragalus (Fabaceae), Stanleya (Brassicaceae), Oonopsis (Asteraceae) and Xylorhiza (Asteraceae) (Beath et al., 1939a,b; Brown & Shrift, 1982). These Se hyperaccumulators occur mainly on naturally seleniferous soils such as in the western USA and typically contain 0.1% of d. wt (1000 mg kg−1) Se in their tissue (Feist & Parker, 2001). Ingestion of these ‘locoweeds’ causes disease and death in animals (Rosenfeld & Beath, 1964). The toxicity of Se is thought to be due to its chemical similarity to sulfur, leading to nonspecific replacement of S by Se in proteins and other sulfur compounds (Stadtman, 1990; Anderson, 1993). On the other hand, Se is also an essential element for many organisms, as a component of seleno-enzymes and seleno-tRNAs (Stadtman, 1990). Whether Se is essential for higher plants is still unknown (Fu et al., 2002; Novoselov et al., 2002).
It is not clear why plants hyperaccumulate these metals or metalloids. Several hypotheses have been proposed: metal tolerance, inadvertent uptake, allelopathy, drought resistance, and protection from herbivory and/or infection (Reeves et al., 1981; Boyd & Martens, 1992). No evidence was found for a role of Ni hyperaccumulation in drought resistance (Whiting et al., 2003). There is also no consistent correlation between metal tolerance and accumulation; the two traits often segregate independently (Macnair et al., 1999).
There is substantial evidence for a protective role of Ni and Zn against invertebrate herbivory. Zinc was shown to protect the Zn hyperaccumulator Thlaspi caerulescens from herbivory by caterpillars, slugs and locusts (Pollard & Baker, 1997; Jhee et al., 1999). Nickel protected the Ni hyperaccumulator Streptanthus polygaloides from caterpillar herbivory (Boyd et al., 1994; Martens & Boyd, 1994, 2002; Boyd & Moar, 1999). Nickel also protected Senecio coronatus from herbivory by snails (Boyd et al., 2002). No Ni protection was found against aphids, perhaps because the phloem Ni concentration was subtoxic (Boyd & Martens, 1999). There is also evidence that Ni can protect plants from microbial infection by fungi (Pythium, Erisyphe polygoni, Alternaria brassicicola) and bacteria (Xanthomonas) (Boyd et al., 1994; Ghaderian et al., 2000). By contrast, Ni enhanced the susceptibility to turnip mosaic virus (Davis et al., 2001).
There is also evidence that Se can protect plants from biotic stresses. The moth caterpillar Spodoptera exigua showed a preference to feed on artificial diet without Se, and Se was toxic at levels of 50 mg kg−1 d. wt feed (Trumble et al., 1998; Vickerman & Trumble, 1999). In the field, varieties of Atriplex that accumulated more Se showed reduced insect growth and survival of Spodoptera exigua (Vickerman et al., 2002). Feeding deterrence by Se to caterpillars was also found for the cabbage looper (Trichoplusia ni), who preferred to feed on Brassica juncea plants without Se rather than on plants containing Se at 465 mg kg−1 d. wt (Bañuelos et al., 2002). Selenium was shown to protect plants from herbivory by Pieris rapae caterpillars both due to deterrence and toxicity, but to promote feeding by the snail Mesodon ferrissi (Hanson et al., 2003). In addition to protecting plants from invertebrate herbivory, Se may also protect them from fungal infection (Hanson et al., 2003) and from feeding by mammals (Franke & Potter, 1936).
In the present study, the hypothesis was tested that Se can protect plants from feeding by invertebrate phloem-feeders. The green peach aphid (Myzus persicae) was used as a generalist phloem feeder. M. persicae is a European native that is now worldwide in distribution. Its host range comprises over 40 plant families including Brassicaceae (Heathcote, 1962). Brassica juncea (Indian mustard) was used as a model plant species since it is quite susceptible to aphid herbivory, tolerant to Se, and a good Se accumulator. B. juncea is thought to originate in central Asia but now grows as a crop or weed worldwide including Asia, the Americas, Africa and Australia.
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The objective of this study was to investigate whether Se offers plants any protection from phloem feeding herbivores. B. juncea and the generalist aphid M. persicae were chosen as a model system for this study. Both species have a worldwide distribution, and Brassica species are known hosts to this aphid. B. juncea and M. persicae thus may interact in natural and agricultural settings. The results presented here show that Se accumulation in B. juncea is effective against colonization by M. persicae. The protective effect of Se was both due to deterrence and toxicity: the aphids avoided Se leaves when given the choice, and when forced to feed on Se leaves the aphids died, likely due to Se ingestion. As little as 2 mg kg−1 d. wt of Se in leaves was enough to be toxic, and 10 mg kg−1 d. wt was enough to deter aphids (no lower levels were tested).
These results fit into a pattern of earlier reports that Se in plants can protect them from invertebrate herbivory (Trumble et al., 1998; Vickerman & Trumble, 1999; Bañuelos et al., 2002; Vickerman et al., 2002; Hanson et al., 2003). Selenium was also shown to protect plants against fungal infection (Hanson et al., 2003). These results shed light on the possible evolution of Se hyperaccumulation. The leaf Se concentration effective against aphid colonization in these experiments (10 mg kg−1 d. wt) is two orders of magnitude below those typically observed in the field in Se hyperaccumulators (Beath et al., 1939a,b; Feist & Parker, 2001). It is therefore feasible that the Se levels occurring in plants in the field are protecting the plants from herbivory by aphids. Indeed, similar results were obtained for the hyperaccumulator Stanleya pinnata. In a small-scale study, glasshouse-grown S. pinnata plants that were colonized by aphids were treated systemically with Se (20 µm selenate), or without Se as a control. In the absence of Se there was a 211% increase in aphid population size over a 7 d-period, while the application of Se resulted in a 28% decrease. Thus, aphids may be a natural herbivore of this hyperaccumulator and Se may protect the plant from aphid colonization in the field as well. Together, these results support the hypothesis that Se hyperaccumulation functions as a defence mechanism against invertebrate herbivory, and that protection from invertebrate herbivory may have driven the evolution of this trait.
The observation that the aphids contained Se after feeding on phloem fluid of selenate-treated plants suggests that Se was transported through the phloem. This is in agreement with the idea that selenate is assimilated into organic forms in the leaf and transported in the phloem to roots and other organs as organic Se (Pilon-Smits et al., 1999). The finding that plant Se accumulation is toxic to this phloem feeder is different from the report by Boyd and Martens (1999) that Ni accumulation in Streptanthus polygaloides offered no protection against the pea aphid (Acyrthosiphon pisum). This difference may be because Ni is less toxic to aphids, but also because the phloem Ni concentration may be lower than that of Se.
As already mentioned by Beath (1959), Se may have application as an insecticide. Our studies showed that systemically supplied Se as 0.1 µm selenate effectively inhibits aphid population growth. Topical application did not have as pronounced an effect: spraying with 20 µm selenate gave similar reduction in aphid population growth to 0.1 µm systemically applied selenate. The reason why spraying the plants with Se was less toxic to the aphids than systemic application of Se may be that when sprayed the Se is not necessarily ingested by the animals, in contrast to systemically applied Se, and Se needs to be ingested to be toxic. Also, in the systemic experiment Se was bioconcentrated by the plants to values c. 20-fold higher than in the external medium. Finally, the form of Se that the aphids consumed may have been different in the systemic experiment compared to the spraying fluid. As mentioned, Se is thought to be assimilated into organic forms in plant leaves and then transported in the phloem; organic Se is more toxic to animals than selenate (Wilber, 1980). Based on these findings, Se may indeed be useful as an insecticide, and systemic application appears to be more efficient than topical application. However, systemically applied Se leads to Se accumulation in plant tissue (e.g. 2 mg kg−1 d. wt when supplied with 0.1 µm selenate), which may be undesirable depending on the use of the plant material. On the other hand, Se is an essential nutrient for humans and animals, and Se deficiency occurs in many areas of the world, especially in livestock. Thus, Se application to crops could function in the dual roles of insecticide and food fortifier, provided the plant Se levels are monitored carefully and if needed the Se-containing material is mixed with low-Se material to prevent toxicity. The interaction of B. juncea with herbivores is also of applied significance in view of the fact that monocultures of this species are currently being grown for phytoremediation of Se (Bañuelos et al., 2002).