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.     

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).     

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.     

Consequences of combined herbivore feeding and pathogen infection for fitness of Barbarea vulgaris plants

Plants are often attacked by pathogens and insects. Their combined impact on plant performance and fitness depends on complicated three-way interactions and the plant’s ability to compensate for resource losses. Here, we evaluate the response of Barbarea vulgaris, a wild crucifer, to combined attack by an oomycete Albugo sp., a plant pathogen causing white rust, and a flea beetle, Phyllotreta nemorum. Plants from two B. vulgaris types that differ in resistance to P. nemorum were exposed to Albugo and P. nemorum alone and in combination and then monitored for pathogen infection, herbivore damage, defence compounds, nutritional quality, biomass and seed production. Albugo developed infections in the insect-resistant plants, whereas insect-susceptible plants were scarcely infected. Concentrations of Albugo DNA were higher in plants also exposed to herbivory; similarly, flea beetle larvae caused more damage on Albugo-infected plants. Concentrations of saponins and glucosinolates strongly increased when the plants were exposed to P. nemorum and when the insect-susceptible plants were exposed to Albugo, and some of these compounds increased even more in the combined treatment. The biomass of young insect-susceptible plants was lower following exposure to flea beetles, and the number of leaves of both plant types was negatively affected by combined exposure. After flowering, however, adult plants produced similar numbers of viable seeds, irrespective of treatment. Our findings support the concept that pathogens and herbivores can affect each other’s performance on a host plant and that the plant reacts by inducing specific and general defences. However, plants may be able to compensate for biomass loss from single and combined attacks over time.     

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.     

UDP-Glycosyltransferases from the UGT73C Subfamily in Barbarea vulgaris Catalyze Sapogenin 3-O-Glucosylation in Saponin-Mediated Insect Resistance

Triterpenoid saponins are bioactive metabolites that have evolved recurrently in plants, presumably for defense. Their biosynthesis is poorly understood, as is the relationship between bioactivity and structure. Barbarea vulgaris is the only crucifer known to produce saponins. Hederagenin and oleanolic acid cellobioside make some B. vulgaris plants resistant to important insect pests, while other, susceptible plants produce different saponins. Resistance could be caused by glucosylation of the sapogenins. We identified four family 1 glycosyltransferases (UGTs) that catalyze 3-O-glucosylation of the sapogenins oleanolic acid and hederagenin. Among these, UGT73C10 and UGT73C11 show highest activity, substrate specificity and regiospecificity, and are under positive selection, while UGT73C12 and UGT73C13 show lower substrate specificity and regiospecificity and are under purifying selection. The expression of UGT73C10 and UGT73C11 in different B. vulgaris organs correlates with saponin abundance. Monoglucosylated hederagenin and oleanolic acid were produced in vitro and tested for effects on P. nemorum. 3-O-β-d-Glc hederagenin strongly deterred feeding, while 3-O-β-d-Glc oleanolic acid only had a minor effect, showing that hydroxylation of C23 is important for resistance to this herbivore. The closest homolog in Arabidopsis thaliana, UGT73C5, only showed weak activity toward sapogenins. This indicates that UGT73C10 and UGT73C11 have neofunctionalized to specifically glucosylate sapogenins at the C3 position and demonstrates that C3 monoglucosylation activates resistance. As the UGTs from both the resistant and susceptible types of B. vulgaris glucosylate sapogenins and are not located in the known quantitative trait loci for resistance, the difference between the susceptible and resistant plant types is determined at an earlier stage in saponin biosynthesis.Triterpenoid saponins are a heterogeneous group of bioactive metabolites found in many species of the plant kingdom. The general conception is that saponins are involved in plant defense against antagonists such as fungi (Papadopoulou et al., 1999), mollusks (Nihei et al., 2005), and insects (Dowd et al., 2011). Saponins consist of a triterpenoid aglycone (sapogenin) linked to usually one or more sugar moieties. This combination of a hydrophobic sapogenin and hydrophilic sugars makes saponins amphiphilic and enables them to integrate into biological membrane systems. There, they form complexes with membrane sterols and reorganize the lipid bilayer, which may result in membrane damage (Augustin et al., 2011).However, our knowledge of the biosynthesis of saponins, and the genes and enzymes involved, is limited. The current conception is that the precursor 2,3-oxidosqualene is cyclized to a limited number of core structures, which are subsequently decorated with functional groups, and finally activated by adding glycosyl groups (Augustin et al., 2011). These key steps are considered to be catalyzed by three multigene families: (1) oxidosqualene cyclases (OSCs) forming the core structures, (2) cytochromes P450 adding the majority of functional groups, and (3) family 1 glycosyltransferases (UGTs) adding sugars. This allows for a vast structural complexity, some of which probably evolved by sequential gene duplication followed by functional diversification (Osbourn, 2010). A major challenge is thus to understand the processes of saponin biosynthesis, which structural variants of saponins play a role in defense against biotic antagonists, and how saponin biosynthesis evolved in different plant taxa. This knowledge is also of interest for biotechnological production and the use of saponins as protection agents against agricultural pests as well as for pharmacological and industrial uses as bactericides (De Leo et al., 2006), anticancerogens (Musende et al., 2009), and adjuvants (Sun et al., 2009).Barbarea vulgaris (winter cress) is a wild crucifer from the Cardamineae tribe of the Brassicaceae family. It is the only species in this economically important family known to produce saponins. B. vulgaris has further diverged into two separate evolutionary lineages (types; Hauser et al., 2012; Toneatto et al., 2012) that produce different saponins, glucosinolates, and flavonoids (Agerbirk et al., 2003b; Dalby-Brown et al., 2011; Kuzina et al., 2011). Saponins of the one plant type make plants resistant to the yellow-striped flea beetle (Phyllotreta nemorum), diamondback moth (Plutella xylostella), and other important crucifer specialist herbivores (Renwick, 2002); therefore, it has been suggested to utilize such plants as a trap crop to diminish insect damage (Badenes-Perez et al., 2005). The other plant type is not resistant to these herbivores. B. vulgaris, therefore, is ideal as a model species to study saponin biosynthesis, insect resistance, and its evolution, as we can contrast genes, enzymes, and their products between closely related but divergent plant types.Insect resistance of the one plant type, called G because it has glabrous leaves, correlates with the content of especially hederagenin cellobioside, oleanolic acid cellobioside, 4-epi-hederagenin cellobioside, and gypsogenin cellobioside (Shinoda et al., 2002; Agerbirk et al., 2003a; Kuzina et al., 2009; Fig. 1). These saponins are absent in the susceptible plant type, called P because it has pubescent leaves, which contains saponins of unknown structures and function (Kuzina et al., 2011). The sapogenins (aglycones) of the resistance-causing saponins hederagenin and oleanolic acid cellobioside do not deter feeding by P. nemorum, which highlights the importance of glycosylation of saponins for resistance (Nielsen et al., 2010). Therefore, the presence or absence of sapogenin glycosyltransferases could be a determining factor for the difference in resistance between the insect resistant G-type and the susceptible P-type of B. vulgaris.Open in a separate windowFigure 1.Chemical structures of the four known G-type B. vulgaris saponins that correlate with resistance to P. nemorum and other herbivores. The cellobioside and sapogenin parts of the saponin are underlined, and relevant carbon positions are numbered.Some P. nemorum genotypes are resistant to the saponin defense of B. vulgaris (Nielsen, 1997b, 1999). Resistance is coded by dominant R genes (Nielsen et al., 2010; Nielsen 2012): larvae and adults of resistant genotypes (RR or Rr) are able to feed on G-type foliage and utilize B. vulgaris as host plant (de Jong et al., 2009), whereas larvae of the susceptible genotype (rr) die and adult beetles stop feeding on G-type foliage. Larvae and adults of all known P. nemorum genotypes can feed on P-type B. vulgaris (Fig. 2).Open in a separate windowFigure 2.Feeding behavior of adult P. nemorum that are either susceptible (ST) or resistant (AK) toward the saponin-based defense of G-type B. vulgaris; the P-type produces different saponins and is not resistant against P. nemorum. Potential feeding is shown by green arrows, and termination of feeding briefly after initiation is indicated by a red dashed arrow. Larvae of the ST line die if fed on G-type plants.In this study, we asked which enzymes are involved in glucosylation of sapogenins in B. vulgaris, whether saponins with a single C3 glucosyl group are biologically active, and whether the difference between the insect resistant and susceptible types of B. vulgaris is caused by different glucosyltransferases.We report the identification of two UDP-glycosyltransferases, UGT73C10 and UGT73C11, which have high catalytic activity and substrate specificity and regiospecificity for catalyzing 3-O-glucosylation of the sapogenins oleanolic acid and hederagenin. The products, 3-O-β-d-glucopyranosyl hederagenin and 3-O-β-d-glucopyranosyl oleanolic acid, are predicted precursors of hederagenin and oleanolic acid cellobioside, respectively. The expression patterns of UGT73C10 and UGT73C11 in different organs of B. vulgaris correlate with saponin abundance, and monoglucosylated sapogenins, especially 3-O-β-d-glucopyranosyl hederagenin, deter feeding by P. nemorum. Our results thus show that glucosylation with even a single glucosyl group activates the resistance function of these sapogenins. However, since the UGTs are present and active in both the insect-resistant and -susceptible types of B. vulgaris, we cannot explain the difference in resistance by different glucosylation abilities. Instead, the difference between the susceptible and resistant types must be determined at an earlier stage in saponin biosynthesis.     

Can colonization by an endophytic fungus transform a plant into a challenging host for insect herbivores?

Endophytic growth of arthropod pathogenic fungi can parasitize insect herbivores without causing damage to the crop. However, studies addressing this tritrophic interaction are absent. Here, the endophytic arthropod pathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordyciptaceae), the polyphagous two-spotted spider mite Tetranychus urticae Koch (Trombidiformes: Tetranychidae), and its preferred plant host Phaseolus vulgaris L. (Fabales: Fabaceae) were selected to study the multi-kingdom interactions among plants, arthropods, and entomopathogenic fungi. Real-Time PCR analysis of nine defense-related genes revealed that a broad range of plant defense mechanisms is activated in response to the endophytic growth of B. bassiana. Moreover, we studied the molecular mechanism adapted by the two-spotted spider mite that underlies resistance. The analysis of 41 detoxification genes revealed that relatively moderate, high, and few numbers of genes were changed in the adults, nymphs, and eggs stages of T. urticae, respectively, after inoculation on colonized tissues of P. vulgaris. The endophytic growth of B. bassiana can have a negative effect on the growth and performance of the pest, in a developmental stage-dependent manner, by priming plant defense pathways. In parallel, the herbivore induces a broad range of detoxification genes that could potentially be involved in adaptation to endophytically colonized plant tissues.     

Nodulation and Nitrogen Fixation Efficacy of Rhizobium fredii with Phaseolus vulgaris Genotypes

Phaseolus plant introduction (PI) genotypes (consisting of 684 P. vulgaris, 26 P. acutifolius, 39 P. lunatus, and 5 P. coccineus accessions) were evaluated for their ability to form effective symbioses with strains of six slow-growing (Bradyrhizobium) and four fast-growing (Rhizobium fredii) soybean rhizobia. Of the 684 P. vulgaris genotypes examined, three PIs were found to form effective nitrogen-fixing symbioses with the R. fredii strains. While none of the Bradyrhizobium strains nodulated any of the genotypes tested, some produced large numbers of undifferentiated root proliferations (hypertrophies). A symbiotic plasmid-cured R. fredii strain failed to nodulate the P. vulgaris PIs and cultivars, suggesting that P. vulgaris host range genes are Sym plasmid borne in the fast-growing soybean rhizobia.     

Narrow- and Broad-Host-Range Symbiotic Plasmids of Rhizobium spp. Strains That Nodulate Phaseolus vulgaris

Agrobacterium transconjugants containing symbiotic plasmids from different Rhizobium spp. strains that nodulate Phaseolus vulgaris were obtained. All transconjugants conserved the parental nodulation host range. Symbiotic (Sym) plasmids of Rhizobium strains isolated originally from P. vulgaris nodules, which had a broad nodulation host range, and single-copy nitrogenase genes conferred a Fix⁺ phenotype to the Agrobacterium transconjugants. A Fix⁻ phenotype was obtained with Sym plasmids of strains isolated from P. vulgaris nodules that had a narrow host range and reiterated nif genes, as well as with Sym plasmids of strains isolated from other legumes that presented single nif genes and a broad nodulation host range. This indicates that different types of Sym plasmids can confer the ability to establish an effective symbiosis with P. vulgaris.     

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.     

HYBRIDIZATION STUDIES ON THE HOST RACES OF EUROSTA SOLIDAGINIS: IMPLICATIONS FOR SYMPATRIC SPECIATION

We studied the inheritance of survival ability in host-associated populations of the tephritid fly, Eurosta solidaginis, to test predictions of sympatric speciation models. Eurosta solidaginis induces galls on two species of goldenrod, Solidago altissima and S. gigantea. The host-associated populations have been hypothesized to be host races that originated in sympatry (Craig et al. 1993). We found evidence for disruptive selection for host use, which is a critical assumption of sympatric speciation models. Each host race had higher survival rates on their host plant than on the alternative host. F₁ and backcross hybrids also had lower survival rates than the pure host-race flies on their host plant. Since assortative mating occurs due to host-plant preference (Craig et al. 1993) this would select for divergence in host preference. Low hybrid survival could have been due to strong genetic incompatibilities of the populations or due to host adaptation by each population. Strong genetic incompatibilities would result in poor survival on all host plants, while host adaptation could result in low overall survival with high hybrid survival on some host plants with particularly “benign” environments. High survival of F₁, F₂, and backcross hybrids on some plant genotypes in some years supported the host adaptation hypothesis. F₁ flies mated and oviposited normally and produced viable F₂ and backcross hybrids indicating gene flow is possible between the host races. A few flies developed and emerged on the alternative host plant. This demonstrates that genes necessary to utilize the alternative host exist in both host races. This could have facilitated the origin of one of the populations via a host shift from the ancestral host. The inheritance of survival ability appears to be an autosomal trait. We did not find evidence that survival ability was maternally influenced or sex linked.     

Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors

Background and aims

Burkholderia tuberum STM678^T was isolated from a South African legume, but did not renodulate this plant. Until a reliable host is found, studies on this and other interesting beta-rhizobia cannot advance. We investigated B. tuberum STM678^T’s ability to induce Fix⁺ nodules on a small-seeded, easy-to-propagate legume (Macroptilium atropurpureum). Previous studies demonstrated that B. tuberum elicited either Fix^- or Fix⁺ nodules on siratro, but the reasons for this difference were unexplored.

Methods

Experiments to promote effective siratro nodule formation under different environmental conditions were performed. B. tuberum STM678^T’s ability to withstand high temperatures and desiccation was checked as well as its potential for promoting plant growth via mechanisms in addition to nitrogen fixation, e.g., phosphate solubilization and siderophore production. Potential genes for these activities were found in the sequenced genomes.

Results

Higher temperatures and reduced watering resulted in reliable, effective nodulation on siratro. Burkholderia spp. solubilize phosphate and produce siderophores. Genes encoding proteins potentially involved in these growth-promoting activities were detected and are described.

Conclusions

Siratro is an excellent model plant for B. tuberum STM678^T. We identified genes that might be involved in the ability of diazotrophic Burkholderia species to survive harsh conditions, solubilize phosphate, and produce siderophores.     

Oviposition preference of Anthocoris nemorum and A. nemoralis for apple and pear

Anthocoris nemorum L. and Anthocoris nemoralis Fabricius (Heteroptera: Anthocoridae) are important predators of insect pests in pome fruit. Females insert their eggs in leaf tissue. The females’ choice of oviposition site is important for the subsequent distribution of nymphs on host plants. Oviposition preference for apple and pear leaves was tested in the laboratory in four experiments (experiments 1–4). In three experiments it was tested whether simulated insect damage to leaves (experiments 5 and 6) or the presence of prey (experiment 7) influenced oviposition preference. The effect of the presence of prey was only tested for A. nemorum on apple leaves. There was a highly significant anthocorid species × plant interaction for the number of eggs laid on apple and pear leaves. Anthocoris nemorum laid more eggs on apple than on pear leaves, while A. nemoralis preferred pear. Anthocoris nemorum's preference for apple increased over the 6‐week period in which experiments 1–4 were performed, from 66% to 91% eggs laid on apple leaves. No change over time in preference was found for A. nemoralis. Across experiments 1–4, the majority of A. nemorum eggs were laid near leaf margins, whereas eggs of A. nemoralis were more commonly found in the leaf centre, 5 mm or more from the margin, with a highly significant leaf region × species interaction. There was no significant difference in preference for leaf side between A. nemorum and A. nemoralis, but there was a highly significant plant × leaf side × experiment interaction. Thus, more eggs were laid on the ventral than on the dorsal side of pear leaves in experiment 4, while significantly more eggs were laid on the dorsal side of apple leaves in experiments 3 and 4. Choice tests between damaged and healthy leaves showed that A. nemorum laid significantly more eggs on the damaged leaves, while A. nemoralis preferred healthy leaves. Anthocoris nemorum showed a near‐significant preference for ovipositing on leaves with eggs of Operophtera brumata (Lepidoptera: Geometridae). The oviposition preferences found correspond to the natural distribution of these predators in apple and pear orchards. The preference of A. nemorum for leaf margins, and of A. nemoralis for the leaf centre as an oviposition site, supports earlier observations. A preference for leaf side for oviposition site has not been reported earlier. Preference for damaged leaves could help A. nemorum to locate prey in a field situation.     

Dynamics of diamondback moth oviposition in the presence of a highly preferred non-suitable host

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), highly prefers to oviposit on yellow rocket, Barbarea vulgaris (R. Br.) (Cruciferae) var. arcuata, despite larvae not being able to survive on it, suggesting it may have potential as a trap crop. In a no‐choice greenhouse experiment, P. xylostella laid 28% more eggs on B. vulgaris than on cabbage. Within the B. vulgaris plant, P. xylostella laid 3.7 times more eggs on younger than older leaves. Furthermore, we demonstrated that in the presence of B. vulgaris volatiles, P. xylostella laid 23% more eggs on cabbage plants than when B. vulgaris volatiles were absent. Because increased oogenesis in the presence of B. vulgaris could complicate the use of this host as a trap crop for P. xylostella, we wanted to examine levels of oogenesis in varying mixtures of cabbage and B. vulgaris. In outdoor screenhouse experiments, P. xylostella laid a decreasing percentage of eggs on cabbage as the percentage of B. vulgaris increased. However, the total number of eggs laid on cabbage did not differ among treatments, suggesting that the presence of B. vulgaris may have stimulated P. xylostella oviposition. In the field, total oviposition in cabbage plots containing B. vulgaris was 6.3 times higher than in cabbage plots without B. vulgaris. However, in plots with B. vulgaris, P. xylostella laid 99% of the eggs on B. vulgaris and oviposition on cabbage plants was 6.2 times lower than in the plots without B. vulgaris. The results of this study are discussed according to P. xylostella egg‐laying behavior and life history as it relates to its interaction with B. vulgaris.     

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.     

Genomic basis of symbiovar mimosae in Rhizobium etli

Background

Symbiosis genes (nod and nif) involved in nodulation and nitrogen fixation in legumes are plasmid-borne in Rhizobium. Rhizobial symbiotic variants (symbiovars) with distinct host specificity would depend on the type of symbiosis plasmid. In Rhizobium etli or in Rhizobium phaseoli, symbiovar phaseoli strains have the capacity to form nodules in Phaseolus vulgaris while symbiovar mimosae confers a broad host range including different mimosa trees.

Results

We report on the genome of R. etli symbiovar mimosae strain Mim1 and its comparison to that from R. etli symbiovar phaseoli strain CFN42. Differences were found in plasmids especially in the symbiosis plasmid, not only in nod gene sequences but in nod gene content. Differences in Nod factors deduced from the presence of nod genes, in secretion systems or ACC-deaminase could help explain the distinct host specificity. Genes involved in P. vulgaris exudate uptake were not found in symbiovar mimosae but hup genes (involved in hydrogen uptake) were found. Plasmid pRetCFN42a was partially contained in Mim1 and a plasmid (pRetMim1c) was found only in Mim1. Chromids were well conserved.

Conclusions

The genomic differences between the two symbiovars, mimosae and phaseoli may explain different host specificity. With the genomic analysis presented, the term symbiovar is validated. Furthermore, our data support that the generalist symbiovar mimosae may be older than the specialist symbiovar phaseoli.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-575) contains supplementary material, which is available to authorized users.     

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.