Polymorphism in a flea beetle for the ability to use an atypical host plant

The flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae: Alticinae) is polymorphic for its ability to use Barbarea vulgaris R. Br. (Brassicaceae) as a host plant. The genetic factors influencing this ability show both sex-linked and autosomal inheritance. Evidence was found for the presence of major genes such as those found in earlier studies, but also of genes with a smaller effect which have not previously been found. Although the ability to survive on B. vulgaris exists in most populations in eastern Denmark, it is usually at a low frequency. Beetles collected on B. vulgaris, however, usually produced larvae that survived on this plant. The inheritance and the abundance of the ability to use B. vulgaris are discussed in the context of the evolution of the interaction between P. nemorum and its atypical host plant.     

Temporal and host-related variation in frequencies of genes that enable Phyllotreta nemorum to utilize a novel host plant, Barbarea vulgaris

The flea beetle, Phyllotreta nemorum L. (Coleoptera: Chrysomelidae), is an intermediate specialist feeding on a small number of plants within the family Brassicaceae. The most commonly used host plant is Sinapis arvensis L., whereas the species is found more rarely on Cardaria draba (L.) Desv., Barbarea vulgaris R.Br., and cultivated radish (Raphanus sativus L.). The interaction between flea beetles and Barbarea vulgaris ssp. arcuata (Opiz.) Simkovics seems to offer a good opportunity for experimental studies of coevolution. The plant is polymorphic, as it contains one type (the P‐type) that is susceptible to all flea beetle genotypes, and another type (the G‐type) that is resistant to some genotypes. At the same time, the flea beetle is also polymorphic, as some genotypes can utilize the G‐type whereas others cannot. The ability to utilize the G‐type of B. vulgaris ssp. arcuata is controlled by major dominant genes (R‐genes). The present investigation measured the frequencies of flea beetles with R‐genes in populations living on different host plants in 2 years (1999 and 2003). Frequencies of beetles with R‐genes were high in populations living on the G‐type of B. vulgaris ssp. arcuata in both years. Frequencies of beetles with R‐genes were lower in populations living on other host plants, and declining frequencies were observed in five out of six populations living on S. arvensis. Selection in favour of R‐genes in populations living on B. vulgaris is the most likely mechanism to account for the observed differences in the relative abundance of R‐genes in flea beetle populations utilizing different host plants. A geographic mosaic with differential levels of interactions between flea beetles and their host plants was demonstrated.     

Cucurbitacin E and I in Iberis amara: Feeding inhibitors for Phyllotreta nemorum

Cucurbitacin E and cucurbitacin I have been isolated from green parts of Iberis amara and identified by TLC, UV and MS. It is shown that cucurbitacins act as feeding inhibitors for the flea beetle Phyllotreta nemorum. The most potent feeding inhibitors in green parts of I. amara towards P. nemorum are cucurbitacin E and I, and the concentrations of these compounds in the plant are found to be high enough to prevent feeding of the flea beetle.     

Phylogeny of an Albugo sp. infecting Barbarea vulgaris in Denmark and its frequency of symptom development in natural populations of two evolutionary divergent plant types

The oomycete Albugo candida has long been considered a broad spectrum generalist pathogen, but recent studies suggest that it is diverged into several more specialized species in addition to the generalist Albugo candida sensu stricto. Whereas these species cause the disease white blister rust in many crucifer plants, asymptomatic endophytic infections may be important in the epidemiology of others. One of the plant species attacked by Albugo sp. is the wild crucifer Barbarea vulgaris ssp. arcuata, which is diverged into two phytochemically and genetically different types with different geographical distributions in Europe. These were previously shown to differ strongly in propensity to develop white rust upon controlled infections in the greenhouse. Here, we analyse the phylogenetic relatedness of this local Albugo sp. field isolate to other species and lines of Albugo spp., including others collected on B. vulgaris. We further ask whether the difference in incidence of white rust between the two types of B. vulgaris are also expressed in natural populations.     

Compensation of seed production after severe injury in the short-lived herb Barbarea vulgaris

A pot experiment with the common ruderal herb Barbarea vulgaris (Brassicaceae) was set up to elucidate to what extent short-lived species sprouting from roots regenerate and compensate for seed production after damage. We tested if sprouting from roots ensures survival after severe aboveground biomass damage, but the number of seeds produced declines with increasing severity of injury, decreasing nutrient availability and progress in the life cycle at the time of injury.Plants of B. vulgaris were cultivated in a 3-year garden experiment at two nutrient levels (high vs. low). During the experiment, two levels of injury severity were applied: high (removal of all aboveground biomass) and low (removal of aboveground biomass leaving basal axillary buds intact). Damage was applied at four life-cycle phases: young rosette, overwintered rosette, flowering plant and fruiting plant. All injured plants survived and resprouted irrespective of life-cycle phase, severity of injury and nutrient availability. Injury significantly affected seed production and also the plants’ life cycle. Plants injured in the second year of their life (overwintered rosette, flowering plant and fruiting plant) postponed reproduction to the third season (in the case of high injury severity) or their seed production was lower than in intact plants (in the case of low injury severity). In plants injured in the first life year, seed production and life cycle were not influenced. Nutrient level only marginally affected resprouting after injury and seed production.The experiment showed that the ability to sprout from roots enables plants to survive a 100% loss of aboveground biomass, and to keep some seed production or even compensate it. The short-lived ruderal species B. vulgaris successfully copes with severe disturbance by resprouting and does not rely only on its seed bank.     

Host plant use of Phyllotreta nemorum: do coadapted gene complexes play a role?

The view of (insect) populations as assemblages of local subpopulations connected by gene flow is gaining ground. In such structured populations, local adaptation may occur. In phytophagous insects, one way in which local adaptation has been demonstrated is by performing reciprocal transplant experiments where performance of insects on native and novel host plants are compared. Trade-offs are assumed to be responsible for a negative correlation in performance on alternative host plants. Due to mixed results of these experiments, the importance of trade-offs in host plant use of phytophagous insects has been under discussion. Here we propose that another genetic mechanism, the evolution of coadapted gene complexes, might also be associated with local adaptation. In this case, however, transplant experiments might not reveal any local adaptation until hybridization takes place. We review the results we have obtained in our work on the host plant use of the flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae: Alticinae), and propose a hypothesis involving coadapted genes to explain the distribution of genes that render P. nemorum resistant to defences of one of its host plants, Barbarea vulgaris R. Br. (Cruciferae).     

Pleiotropic effects associated with an allele enabling the flea beetle <Emphasis Type="Italic">Phyllotreta nemorum </Emphasis>to use <Emphasis Type="Italic">Barbarea vulgaris</Emphasis> as a host plant

In the Danish region of Kværkeby, a mutation in an, as yet, unknown single autosomal gene has resulted in a dominant resistance (R-) allele in the flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae: Alticinae). It enables the beetle to overcome the defences of Barbarea vulgaris ssp. arcuata (Opiz.) Simkovics G-type (Brassicaceae) and use it as a host plant. In this study, we investigated the pleiotropic effects associated with the presence of this particular R-allele in female P. nemorum. These females had the R-allele backcrossed into the genetic background of non-resistant beetles. The effects were investigated under both favourable and stressful conditions (cold shock). The presence of the R-allele in a non-resistant genetic background caused a very high mortality in resistant individuals during the early stages of development under both conditions, but it did not affect the adult life-history traits longevity, body size and fecundity, under both conditions. Regardless of temperature treatment, resistant females in general were found to lay significantly more eggs. Developmental stability, as measured by tibia length fluctuating asymmetry, was not correlated with overall developmental stress in this study.     

Non‐random segregation of an autosomal gene in males of the flea beetle,Phyllotreta nemorum: implications for colonization of a novel host plant

The flea beetle, Phyllotreta nemorum (L.) (Coleoptera: Chrysomelidae: Alticinae), is currently expanding its host plant range in Europe. The ability to utilize a novel host plant, Barbarea vulgaris R. Br. (Brassicaceae), is controlled by major dominant genes named R‐genes. The present study used extensive crossing experiments to illustrate a peculiar mode of inheritance of the R‐gene in a population from Delemont (Switzerland). When resistant males from Delemont are mated with recessive females from a laboratory line, the female F₁ offspring contains the R‐allele and is able to utilize B. vulgaris, whereas the male offspring contains the r‐allele and is unable to utilize the plant. This outcome suggests X‐linkage of the R‐gene, but further crossing experiments demonstrated that this was not the case. When the R‐gene is present in offspring from males from a laboratory line that originates from Taastrup (Denmark), it is transmitted to female and male offspring in equal proportions as a normal autosomal gene. The results demonstrate a polymorphism in segregation patterns of an autosomal R‐gene in P. nemorum males. Males from Delemont contain a factor which causes non‐random segregation of the R‐gene (NRS‐factor). This factor is inherited patrilineally (from fathers to sons). Males with the NRS‐factor transmit the R‐gene to their female offspring, whereas males without the NRS‐factor transmit the R‐gene to female and male offspring in equal proportions. Various models for the non‐random segregation of autosomes in P. nemorum males are discussed – e.g., fusions between autosomes and sex chromosomes, and genomic imprinting. The implications of various modes of inheritance of R‐genes for the ability of P. nemorum populations to colonize novel patches of B. vulgaris are discussed.     

Detection of refuge from enemies through phenological mismatching in multitrophic interactions requires season-wide estimation of host abundance

The concept of “enemy-free space” (EFS) refers to ways of living that reduce or eliminate the vulnerability of a species to natural enemies. It has been invoked to explain host shifts of phytophagous insects. A demonstrated cause of EFS is escape from enemies in time, through phenological mismatching of herbivore development and enemy occurrence, leading to low percentages of predation/parasitism of herbivores occurring at a certain time. The mere measurement of percentage parasitism, however, is not sufficient to demonstrate EFS in certain cases. Here we present such a case, where parasitism was studied of a phytophagous insect (Phyllotreta nemorum), using two different host plant species in the field: an atypical, relatively rarely used, plant (Barbarea vulgaris), and a more widely used one (Sinapis arvensis). At one location we found a paradoxical result: on each separate sampling day throughout the season the percentage of parasitism of P. nemorum using a patch of B. vulgaris was not significantly different from, or even significantly higher than on a nearby patch of S. arvensis. The overall season-wide proportion parasitism of the flea beetle cohort using the B. vulgaris patch, however, was lower. We conclude that, in the year and at the location we studied, the patch of B. vulgaris provided enemy-free space to the herbivore in the form of a temporal refuge, and that the importance of enemy-free space in the use of an atypical host plant should be evaluated on the basis of season-wide sampling, including estimation of host population size.     

Genetic diversity and similarity in the Barbarea vulgaris complex (Brassicaceae)

Two subspecies of Barbarea vulgaris are taxonomically recognized as ssp. vulgaris and ssp. arcuata. In addition, two types of Barbarea vulgaris ssp. arcuata occurs in Denmark. The G‐type is resistant to an herbivorous flea beetle (Phyllotreta nemorum) whereas the P‐type is susceptible. A previous study suggested that the P‐type evolved by a loss of resistance from a resistant progenitor. We analyzed the genetic relatedness among eight Barbarea taxa: B. vulgaris spp. vulgaris, B. vulgaris ssp. arcuata G‐ and P‐types, hybrids between the types, B. verna, B. intermedia, B. stricta, B. orthoceras and B. australis, using AFLP and SSR markers. A clear distinction between the G‐ and P‐types was revealed. Both were distinct from B. vulgaris ssp. vulgaris, the G‐type less so than the P‐type. Barbarea verna and B. intermedia formed unambiguous clusters, whereas the remaining taxa produced less discrete groupings. Possible evolutionary scenarios for flea‐beetle resistance and susceptibility are discussed, including lineage sorting from a polymorphic ancestral population, and de novo loss of resistance in the P‐type of B. vulgaris ssp. arcuata.     

Identification of Defense Compounds in Barbarea vulgaris against the Herbivore Phyllotreta nemorum by an Ecometabolomic Approach

Winter cress (Barbarea vulgaris) is resistant to a range of insect species. Some B. vulgaris genotypes are resistant, whereas others are susceptible, to herbivory by flea beetle larvae (Phyllotreta nemorum). Metabolites involved in resistance to herbivory by flea beetles were identified using an ecometabolomic approach. An F2 population representing the whole range from full susceptibility to full resistance to flea beetle larvae was generated by a cross between a susceptible and a resistant B. vulgaris plant. This F2 offspring was evaluated with a bioassay measuring the ability of susceptible flea beetle larvae to survive on each plant. Metabolites that correlated negatively with larvae survival were identified through correlation, cluster, and principal component analyses. Two main clusters of metabolites that correlate negatively with larvae survival were identified. Principal component analysis grouped resistant and susceptible plants as well as correlated metabolites. Known saponins, such as hederagenin cellobioside and oleanolic acid cellobioside, as well as two other saponins correlated significantly with plant resistance. This study shows the potential of metabolomics to identify bioactive compounds involved in plant defense.Plants are sessile organisms that have developed various strategies to adapt to or counteract abiotic and biotic stress. The ability to accumulate low-molecular-weight bioactive compounds, often referred to as allelochemicals, secondary metabolites, or bioactive natural products, provides a chemical defense against herbivorous insects used by plants. As a result of natural selection, insects often develop mechanisms to adapt to such compounds and eventually manage to break the resistance.The interaction between Barbarea vulgaris (Brassicaceae) and the flea beetle Phyllotreta nemorum (Coleoptera: Chrysomelidae) is a unique model system to study chemical defenses in plants and counteradaptations in insects (Nielsen, 1997a; de Jong et al., 2000; Agerbirk et al., 2001, 2003b; Nielsen and de Jong, 2005). B. vulgaris, a biennial or short-lived perennial wild crucifer (MacDonald and Cavers, 1991), is polymorphic with respect to insect resistance: the pubescent P-type is susceptible to all known flea beetle genotypes, whereas the glabrous G-type is resistant to most common genotypes of the insect (Nielsen, 1997a, 1997b; Agerbirk et al., 2003a). In contrast, P. nemorum is polymorphic with respect to plant defenses (Breuker et al., 2005; Nielsen and de Jong, 2005).B. vulgaris has a potential as an oil crop for use at northern latitudes (Börjesdotter, 1999) and is considered to be an important genetic resource for food and agriculture (International Treaty on Plant Genetic Resources for Food and Agriculture; ftp://ftp.fao.org/ag/cgrfa/it/ITPGRe.pdf). It has been used for salads and garnishes as well as a medicinal plant (Senatore et al., 2000). B. vulgaris has a wide native distribution area (Eurasia) and is furthermore naturalized in North America, Africa, Australia, New Zealand, and Japan as a weed (Hegi, 1958; MacDonald and Cavers, 1991; Tachibana et al., 2002). The subspecies arcuata is by far the most common Barbarea taxon in Denmark and comprises two morphologically, biochemically, and cytologically deviating genotypes, P and G, which differ by glucosinolate profiles, flea beetle resistance, and leaf pubescence (Agerbirk et al., 2003b; Fig. 1). B. vulgaris is a diploid; the G-type has 2n = 16 chromosomes, while the P-type has 2n = 16 or 2n = 18 chromosomes (Ørgaard and Linde-Laursen, 2008). B. vulgaris is phylogenetically positioned between Arabidopsis (Arabidopsis thaliana) and allopolyploid oil seed rape (Brassica napus; Bailey et al., 2006). Accordingly, research on plant-insect interaction in B. vulgaris may be applied to B. napus.Open in a separate windowFigure 1.Rosette leaves of P- and G-types of B. vulgaris subspecies arcuata. The P-type has hairs, while the G-type does not.Glucosinolates constitute a group of defense compounds present in crucifers and play a key role in host selection by crucifer specialists (Renwick, 2002). These compounds are feeding and oviposition stimulants for a number of specialist insects, which have become adapted to such compounds as an outcome of long-standing coevolutionary interactions with host plants containing them (Renwick, 2002; Thompson, 2005). Therefore, glucosinolates no longer offer efficient protection against many specialist insects, and the relationship between glucosinolate profiles of plants and their suitability as food for insects is not simple (Nielsen et al., 2001; Poelman et al., 2008; van Leur et al., 2008). The P-type B. vulgaris contains the R-isomer of 2-hydroxy-2-phenylethylglucosinolate, whereas the G-type contains the S-isomer. However, the differences in glucosinolate profiles between the P- and G-types are not related to resistance to flea beetles (Agerbirk et al., 2003b).As a putative response to renewed selection pressure from herbivorous insects, a number of crucifers have evolved a second generation of defense secondary compounds (e.g. cucurbitacins in Iberis species, cardenolides in Cheirantus and Erysimum species, and saponins in B. vulgaris). These compounds are feeding deterrents for a number of insect species (Nielsen, 1978; Renwick, 2002; Shinoda et al., 2002; Agerbirk et al., 2003a). Until now, Barbarea is the only crucifer known to contain saponins. Two saponins, oleanolic acid cellobioside (3-O-β-cellobiosyloleanolic acid) and hederagenin cellobioside (3-O-β-cellobiosylhederagenin), have been identified in B. vulgaris (Shinoda et al., 2002; Agerbirk et al., 2003a). The restricted distribution of such saponins in crucifers suggests that they originated later than the glucosinolates, which have a much wider distribution in the family.Saponins are triterpenoid glycosides widely distributed in higher plants (Hostettmann and Marston, 1995; Sparg et al., 2004; Vincken et al., 2007). They are constituents of many plant drugs and folk medicines and possess a wide range of biological activities, including antifungal, antibacterial, molluscicidal, and insecticidal activities (Hostettmann and Marston, 1995; Sparg et al., 2004; Chwalek et al., 2006; Güçlü-Ustündağ and Mazza, 2007; Gauthier et al., 2009). The toxicity of saponins to fungi and insects is thought to be a result of their ability to form complexes with sterols in the plasma membrane, thus destroying the cellular semipermeability and leading to cell death. Although saponins are toxic to cold-blooded animals, their oral toxicity to mammals is low (for review, see Hostettmann and Marston, 1995; Sparg et al., 2004; Güçlü-Ustündağ and Mazza, 2007).Hederagenin cellobioside has been identified as an active defense compound of B. vulgaris against the world-wide pest diamondback moth (Shinoda et al., 2002), which has become resistant to most insecticides. Oleanolic acid cellobioside concentration has been shown to correlate with resistance of B. vulgaris to the diamondback moth (Agerbirk et al., 2003a). This compound is present in the resistant G-type plant, and its concentration declines in autumn at the same time as the decline in resistance toward diamondback moth (Agerbirk et al., 2001, 2003b). The impact of the two saponins on defense against flea beetles, a major pest in oil seed rape, has not been reported previously.The objective of this study was to develop an unbiased strategy to identify metabolites that correlate with resistance to flea beetle larvae in B. vulgaris and to provide knowledge that may facilitate more efficient and sustainable breeding for resistance toward insect pests. The results presented in this study are significant for understanding chemical plant defense against insects and may be utilized in future crop protection breeding by screening for the presence of similar bioactive compounds, biosynthetic enzymes, and genetic markers or transfer of resistance components to crop plants.     

Effects of leaf and root herbivory by potential insect biological control agents on the performance of invasive Vincetoxicum spp.

The European leaf-feeding moth Abrostola asclepiadis and root-feeding beetle Eumolpus asclepiadeus are promising biological control agents for two European swallow-worts (Vincetoxicum rossicum and Vincetoxicum nigrum) in North America, however, their impact on plant performance is uncertain. Densities of each herbivore were manipulated in a common garden to determine whether leaf and root herbivory affect the performance of these plants. During the second year of the experiment, V. rossicum and V. nigrum unexpectedly became infected with the fungal pathogens Ascochyta sp. and Cercospora sp. (Ascomycota), respectively. Although pathogen infection mainly reduced shoot height and delayed reproduction, herbivore effects on plant growth were still evident. Leaf herbivory by A. asclepiadis had no effect on plant growth 1 year after defoliation. Root herbivory by E. asclepiadeus reduced shoot height and plant biomass and decreased the ability of plants to compensate for pathogen attack. Pathogen infection prevented detection of herbivore effect on reproduction. Due to its substantial impact on plant biomass, E. asclepiadeus should be further evaluated as a biological control agent against Vincetoxicum spp. populations invading open habitats in North America. Further research is needed to evaluate the impact of A. asclepiadis in combination with E. asclepiadeus and plant competition under high and low light conditions.     

Impact of two specialist insect herbivores on reproduction of horse nettle,Solanum carolinense

The frequency of coevolution as a process of strong mutual interaction between a single plant and herbivore species has been questioned in light of more commonly observed, complex relationships between a plant and a suite of herbivore species. Despite recognition of the possibility of diffuse coevolution, relatively few studies have examined ecological responses of plants to herbivores in complex associations. We studied the impact of two specialist herbivores, the horse nettle beetle, Leptinotarsa juncta, and the eggplant flea beetle, Epitrix fuscula, on reproduction of their host, Solanum carolinense. Our study involved field and controlled-environment experimental tests of the impact on sexual and potential asexual reproduction of attack by individuals of the two herbivore species, individually and in combination. Field tests demonstrated that under normal levels of phytophagous insect attack, horse nettle plants experienced a reduction in fruit production of more than 75% compared with plants from which insects were excluded. In controlled-environment experiments using enclosure-exclosure cages, the horse nettle's two principal herbivores, the flea beetle and the horse nettle beetle, caused decreases in sexual reproduction similar to those observed in the field, and a reduction in potential asexual reproduction, represented by root biomass. Attack by each herbivore reduced the numbers of fruits produced, and root growth, when feeding in isolation. When both species were feeding together, fruit production, but not root growth, was lower than when either beetle species fed alone. Ecological interactions between horse nettle and its two primary herbivores necessary for diffuse coevolution to occur were evident from an overall analysis of the statistical interactions between the two herbivores for combined assessment of fruit and vegetative traits. For either of these traits alone, the interactions necessary to promote diffuse coevolution apparently were lacking.     

Genetic and sexual separation between insect resistant and susceptible Barbarea vulgaris plants in Denmark

Co‐evolution between herbivores and plants is believed to be one of the processes creating Earth’s biodiversity. However, it is difficult to disentangle to what extent diversification is really driven by herbivores or by other historical‐geographical processes like allopatric isolation. In the cruciferous plant Barbarea vulgaris, some Danish individuals are resistant to herbivory by flea beetles (Phyllotreta nemorum), whereas others are not. The flea beetles are, in parallel, either resistant or susceptible to the plants defenses. To understand the historical‐evolutionary framework of these interactions, we tested how genetically divergent resistant and susceptible plants are, using microsatellite markers. To test whether they are reproductively fully compatible, resistant and susceptible plants were grown intermixed in an outdoor experiment, and the paternity of open‐pollinated offspring was determined by analysis of molecular markers. Resistant and susceptible Danish plants were genetically strongly differentiated and produced significantly fewer hybrids than expected from random mating or nearest neighbour mating. Our results suggest that the two types belong to different evolutionary lineages that have been (partly) isolated at some time, during which genetic and reproductive divergence evolved. A parsimonious scenario could be that the two plant types were isolated in different refugia during the previous ice age, from which they migrated into and met in Denmark and possibly neighbouring regions. If so, resistance and susceptibility has for unknown reasons become associated with the different evolutionary lineages.     

Isolation of polymorphic microsatellite loci from the flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae)

Ten microsatellite markers for the flea beetle Phyllotreta nemorum were developed using di‐ and trinucleotide repeat‐enriched libraries. Each of these primer pairs were characterized on 96 individuals. Expected heterozygosities ranged between 0.11 and 0.84 and the number of alleles ranged between two and 14 per locus. These microsatellite markers are the first published for any Phyllotreta species.     

Host specificity ofDisonycha argentinensis [Col.: Chrysomelidae], an agent for the biological control ofalternanthera philoxeroides (Alligator weed) in Australia

The biology and host specificity of a South American flea beetle,Disonycha argentinensis Jacoby was investigated in quarantine facilities in Australia. Damage from feeding by larvae and adults ofD. argentinensis onAlternanthera philoxeroides (Mart.) Griseb (alligator weed) indicated that it might be a useful biological control agent for the plant when growing in terrestrial situations. Complete development of the immature stages took place only onAlternanthera philoxeroides. Adults produced a few feeding scars onBeta vulgaris L. (beetroot) andAmaranthus spinosus L. (spiny amaranth) but failed to oviposit. Host specificity studies indicated that establishment of this beetle in Australia would be without significant risk to nontarget plant species.D. argentinensis was first released in Australia in 1980.     

Are Phytochelatins Involved in Differential Metal Tolerance or Do They Merely Reflect Metal-Imposed Strain?

Plants from nontolerant and copper-tolerant populations of Silene vulgaris both produce phytochelatins upon exposure to copper. The threshold copper concentration for induction of phytochelatin and the copper concentration at which maximum phytochelatin contents occurs increase proportionally with the level of tolerance to copper. When exposed to their own highest no-effect concentration or 50%-effect concentration of copper for root growth, tolerant and nontolerant plants exhibit equal phytochelatin contents in the root apex, which is the primary copper target. This also holds for distinctly tolerant nonsegregating F₃ families, derived from a single cross of a nontolerant plant to a tolerant one. Therefore, the phytochelatin content of the root apex can be used as a quantitative tolerance-independent measure of the degree of toxicity experienced by the plant. Differential copper tolerance in S. vulgaris does not appear to rely on differential phytochelatin production.     

Exogenous application of plant defense hormones alters the effects of live soils on plant performance

The overall effect of a live soil inoculum collected from nature on plant biomass is often negative. One hypothesis to explain this phenomenon is that the overall net pathogenic effect of soil microbial communities reduces plant performance. Induced plant defenses triggered by the application of the plant hormones jasmonic acid (JA) and salicylic acid (SA) may help to mitigate this pathogenic effect of live soil. However, little is known about how such hormonal application to the plant affects the soil and how this, in turn, impacts plant growth. We grew four plant species in sterilized and inoculated live soil and exposed their leaves to two hormonal treatments (JA and SA). Two species (Jacobaea vulgaris and Cirsium vulgare) were negatively affected by soil inoculation. In these two species foliar application of SA increased biomass in live soil but not in sterilized soil. Two other species (Trifolium repens and Daucus carota) were not affected by soil inoculum and for these two species foliar application of SA reduced plant biomass in both the sterilized and live soil. Application of JA reduced plant biomass in both soils for all species. We subsequently carried out a multiple generation experiment for one of the plant species, J. vulgaris. In each generation, the live soil was a mixture of 10% soil from the previous generation and 90% sterilized soil and the same hormonal treatments were applied. The negative effects of live soil on plant biomass were similar in all four generations, and this negative effect was mitigated by the application of SA. Our research suggests that the application of SA can mitigate the negative effects of live soil on plant growth. Although the inoculum of soil containing a natural live soil microbial community had a strong negative effect on the growth of J. vulgaris, we found no evidence for an increase or decrease in negative plant-soil feedback in either the control or the SA treated plants. Also plant performance did not decrease consistently with succeeding generations.     

Local variation in conspecific plant density influences plant–soil feedback in a natural grassland

Several studies have argued that under field conditions plant–soil feedback may be related to the local density of a plant species, but plant–soil feedback is often studied by comparing conspecific and heterospecific soils or by using mixed soil samples collected from different locations and plant densities. We examined whether the growth of the early successional species Jacobaea vulgaris in soil collected from the field is related to the local variation in plant density of this species. In a grassland restoration site, we selected eight 8 m × 8 m plots, four with high and four with low densities of J. vulgaris plants. In 16 subplots in each plot we recorded the density and size of J. vulgaris, and characteristics of the vegetation and the soil chemistry. Soil collected from each subplot was used in a greenhouse pot-experiment to study the growth of J. vulgaris, both in pure field soil and in sterile soil inoculated with a small part of field soil.In the field, flowering J. vulgaris plants were taller, the percentage of rosette plants was higher and seed density was larger in High- than in Low-density plots. In the pot experiment, J. vulgaris had a negative plant–soil feedback, but biomass was also lower in soil collected from High- than from Low-density plots, although only when growing in inoculated soil. Regression analyses showed that J. vulgaris biomass of plants growing in pure soil was related to soil nutrients, but also to J. vulgaris density in the field.We conclude that in the field there is local variation in the negative plant–soil feedback of J. vulgaris and that this variation can be explained by the local density of J. vulgaris, but also by other factors such as nutrient availability.     

Effects of belowground vertical distribution of a herbivore on plant biomass and survival in Lolium perenne

Root herbivory affects plant performance, but the effects are not well understood. We tested the effects of the vertical distribution of a root-feeding beetle larva (Anomala cuprea) by restricting its access to the top, middle, or bottom zone in pots of perennial ryegrass (Lolium perenne) or by allowing unrestricted access. We predicted that plant mortality, biomass, and biomass allocation should change with the zone of root herbivory, because both the magnitude of root loss and the consequences of such loss are specific to the point of damage. Seven of nine plants died in each treatment in which the larvae had access to the top zone. In contrast, no plants died when larvae occupied the middle or bottom zones. Plants were killed when the larvae grazed the root base and severed the shoots from the roots. Moreover, total plant biomass and biomass allocation to roots were significantly lower when the larvae were confined to the top and middle feeding zones. The greatest number of roots were removed when the larvae occupied the top feeding zone. Thus, the vertical distribution of a belowground herbivore is crucially important to plant fate. In nature, most belowground herbivores are concentrated near the soil surface, and thus the effects of belowground herbivory are often more severe than the effects of aboveground herbivory.