Research has shown that soil-borne beneficial microorganisms can enhance plant growth, productivity, and resistance against pests and pathogens and could thus serve as a sustainable alternative to agrochemicals. To date, however, the effect of soil-beneficial microbes under commercial crop production has been little assessed. We here investigated the effect of root inoculation with nine well-characterized bacterial and fungal strains and two consortia on tomato performance under intensive tomato crop management practices. We measured the impact of these root inoculations on plant growth, fruit quality, yield, and pest and pathogen incidence. While most microbial strains showed weak effects, we found that the fungal strains Trichoderma afroharzianum T22 and Funneliformis mosseae significantly increased marketable tomato yield. Moreover, we found that inoculation with most of the fungal strains led to a significant reduction in the incidence of the devastating leaf-mining pest Tuta absoluta, while this effect was not observed for bacterial inoculants. In addition, we found that microbial inoculations did not impact the incidence of introduced natural pest enemies, supporting their compatibility with well-established integrated pest management strategies in horticulture. In summary, the observed general positive effects of soil microbes on tomato yield and resistance reinforce the move toward broader adoption of microbial inoculants in future crop production, ultimately improving agricultural sustainability.

Abstract Research is showing that soil-borne beneficial microorganisms can enhance plant growth, productivity, and resistance against pests and pathogens, and could thus serve as a sustainable alternative to agrochemicals. To date, however, the effect of soil beneficial microbes under commercial crop production has not been fully assessed. We here investigated the effect of root inoculation with 11 well-characterized bacterial and fungal strains on tomato performance under intensive tomato crop management practices. We measured the impact of these strains on plant growth, fruit quality, yield, and pest and pathogen incidence. While most microbial strains showed weak effects, we found that the fungal strains Trichoderma afroharzianum T22 and Funneliformis mosseae significantly increased marketable tomato yield. Moreover, we found that inoculation with most of the fungal strains led to a significant reduction in the incidence of the devastating leaf mining pest Tuta absoluta, while this effect was not observed for bacterial inoculants. In addition, we found that microbial inoculations did not impact the incidence of introduced natural enemies, supporting their compatibility with well-established integrated pest management strategies in horticulture. In sum, the observed general positive effects of soil microbes on tomato yield and resistance reinforce the move toward a broader adoption of microbial inoculants in future crop production, ultimately improving agricultural sustainability.

BACKGROUNDMultiple and simultaneous attacks by pathogens and insect pests frequently occur in nature. Plants respond to biotic stresses by activating distinct defense mechanisms, but little is known about how plants cope with multiple stresses. The focus of this study was the combined interaction of fungal infection caused by Leptosphaeria maculans (synonym Plenodomus lingam) and arthropod infestation by the diamondback moth (Plutella xylostella) in oilseed rape (Brassica napus). We hypothesized that infection by the fungal pathogen L. maculans could alter oilseed rape palatability to P. xylostella-chewing caterpillars. Feeding preference tests were complemented with analyses of defense gene transcription, and levels of glucosinolates (GLSs) and volatile organic compounds (VOCs) in L. maculans-inoculated and non-inoculated (control) leaves to determine possible causes of larval choice.RESULTSCaterpillars preferred true leaves to cotyledons, hence true leaves were used for further experiments. True leaves inoculated with L. maculans were more palatable to caterpillars over control leaves during the early stage of infection at 3 days post inoculation (dpi), but this preference disappeared in the later stages of infection at 7 dpi. In parallel, genes involved in the salicylic acid and ethylene pathways were up-regulated in L. maculans-inoculated leaves at 3 and 7 dpi; L. maculans increased the level of total aliphatic GLSs, specifically glucobrassicanapin, and decreased the level of glucoiberin at 3 dpi and altered the content of specific VOCs. A group of 55 VOCs with the highest variability between treatments was identified.CONCLUSIONWe suggest that the P. xylostella preference for L. maculans-inoculated leaves in the early stage of disease development could be caused by the underlying mechanisms leading to changes in metabolic composition. Further research should pinpoint the compounds responsible for driving larval preference and evaluate whether the behavior of the adult moths, i.e. the stage that makes the first choice regarding host plant selection in field conditions, correlates with our results on larval host acceptance. (c) 2024 Society of Chemical Industry. Fungal infection of Brassica napus by Leptosphaeria maculans increases plant palatability to Plutella xylostella caterpillars during early but not later stages of infection. To explain this finding, involvement of plant signaling pathways, analyses of glucosinolates and volatile organic compounds.dagger image

Plants live in association with a multitude of microorganisms, some of which may improve the plant’s defense toward herbivores. In a previous study, we showed that Metarhizium brunneum, a fungus mostly known as an insect pathogen, can associate with cabbage roots and prime an increased myrosinase activity upon attack by Plutella xylostella larvae. Here, we ask whether another Metarhizium species, M. robertsii, also primes plant defense and whether this involves leaf glucosinolate content in addition to myrosinase activity. In addition, we tested whether priming reverses after removal of larvae. M. robertsii established and could be reisolated from roots at the end of the experiment, 43 days after inoculation. Before larval herbivory, leaf glucosinolate content and myrosinase activity did not differ between fungal-inoculated and control plants. Immediately after herbivory, in contrast, both the glucosinolate content and myrosinase activity were higher in inoculated plants than in control plants and less leaf material was consumed of the inoculated plants. Twenty-four hours after herbivory, glucosinolates, and myrosinase activity had decreased again to levels like before herbivory. Total biomass and shoot–root ratio were not affected by M. robertsii colonization. In conclusion, Metarhizium fungi can associate with Brassica roots and prime above-ground glucosinolate content and myrosinase activity. The increased defense levels were down-regulated 24 h after herbivory. Root-associated Metarhizium species may thus affect above-ground herbivores indirectly by host plant priming, in addition to their well-known direct effects as insect pathogens.

Many fungi live intimately associated with plants and may benefit or harm the host plant. Improved knowledge of such interactions is needed for increasing plant health and crop productivity by implementation of fungal inoculants. Co-inoculations of different beneficial fungi offer the possibility to understand complex plant-microbe interactions that may be functionally complementary for improved plant production and protection. Here, we studied the individual and combined effects of the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae with three isolates of entomopathogenic fungi (EPF), representing Metarhizium brunneum, M. robertsii and Beauveria bassiana, on protection against the foliar phytopathogen Botrytis cinerea and on plant growth. Seedlings of tomato (Solanum lycopersicum L. var. Moneymaker) were inoculated in the substrate with AMF or EPF alone and in dual combinations under greenhouse conditions. Inoculation with the different EPF isolates reduced lesion sizes of B. cinerea on inoculated tomato leaves, but only in the experimental repetition that showed highest level of disease severity. The AMF F. mosseae had no additional effect on B. cinerea lesion size in combinations with EPF. In the experimental repetition with least disease severity, the AMF treatment led to limited increase of B. cinerea lesion sizes. In general, F. mosseae caused an increase in plant biomass, and the co-inoculations of AMF and EPF did in some combinations increase plant growth. Below-ground interactions between AMF and EPF were observed, as the presence of AMF in the roots was associated with a decrease of EPF root colonization densities. However, AMF colonization rates were unaffected by EPF presence. The study indicated a functional complementarity between EPF and AMF by suppressing phytopathogens and increasing plant growth, respectively. However, it further revealed the challenge of obtaining consistent results of plant-microbe-phytopathogen interactions, which must be overcome for future implementation of beneficial fungi as inoculants in plant production.

Barbarea, winter-cress, is a genus of 29 species in Brassicaceae, the mustard family, which has emerged as a model for evolution of plant defence and specialised metabolites. Notably, some Barbarea species have evolved the ability to produce triterpenoid saponins as the only ones in Brassicaceae, some of which make plants resistant to important herbivores. Resistance has, however, been lost in a distinct group of plants within B. vulgaris ssp. arcuata, which is genetically strongly diverged from other B. vulgaris plants. This divergence is not reflected present in taxonomy. Thus, a phylogeny is needed to understand evolution and defence in Barbarea. Here, we analysed the nuclear ITS and the plastid matK, ndhF, rps16, and psbA-trnH DNA regions from seven out of 29 Barbarea species, 57 accessions of B. vulgaris, 10 accessions of other Barbarea species, and eight outgroup species, in addition to sequences available from GenBank. All Barbarea species formed a highly supported monophyletic group, separated from sister genera. Several clades seem to have radiated within the genus with no simple branching pattern, and discordant nuclear and plastid DNA phylogenies indicate reticulate evolution and chloroplast capture. One of the complex patterns may have resulted from chloroplast capture of a non-Nordic Barbarea species not included in the study. Two pairs of species were almost identical, B. australis and B. grayi, and B. orthoceras and B. stricta. Despite hybridization, chloroplast capture, and incongruence among the plastid and nuclear DNA data, the high level of intraspecific diversity, coupled with lineage specificity, lead us to recognize three groups of Barbarea vulgaris: G-type (glabrous) and P-type (pubescent) individuals of the current B. vulgaris ssp. arcuata as two distinct groups and the current B. vulgaris ssp. vulgaris as the third. Despite the high molecular diversity below species level, the evolutionary history of the saponin-based resistance remains unsettled due to unresolved basal branching.

Aphids, the phloem sap feeders, probe into leaf tissues and activate a complex network of plant defense responses. Phytohormonal signaling plays a major role in this network; however, the dynamics of the signal spreading is yet to be clarified. Despite the growing knowledge about transcriptomic changes upon infestation, results often differ due to sampling, varying strongly between the tissues collected at the single feeding site, individual leaves, pooled infested leaves, or whole plant rosettes. This study focuses on activation of salicylic acid (SA) and jasmonic acid (JA) signals in Arabidopsis leaves during infestation by cabbage aphid (Brevicoryne brassicae) in high spatio-temporal resolution. We used genetically encoded fluorescent biosensors, histochemistry, and quantitative reverse transcription-PCR to precisely map activation of distinct branches of phytohormonal signaling. We found a rapid induction of SA and JA signaling markers in cells surrounding stylet puncture, colocalizing with callose deposition. For both PR1 and JAZ10, we detected activation at 24 h postinfestation (hpi), increasing and spreading along the veins until 72 hpi and, to a lesser extent, within the epidermal pavement cells. The SA signaling wave appeared in parallel with JA-associated signaling and continued to increase in time. Our results first show a local activation of SA- and JA-related responses after stylet penetration of Arabidopsis leaves and bring a detailed insight into the spatio-temporal complexity of plant defense activation during specialist aphid attack.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The escalating food demand and loss to herbivores has led to increasing interest in using resistance-inducing microbes for pest control. Here, we evaluated whether root-inoculation with fungi that are otherwise known as entomopathogens improves tomato (Solanum lycopersicum) leaflets' reaction to herbivory by Spodoptera exigua (beet armyworm) larvae using chlorophyll fluorescence imaging. Plants were inoculated with Metarhizium brunneum or Beauveria bassiana, and photosystem II reactions were evaluated before and after larval feeding. Before herbivory, the fraction of absorbed light energy used for photochemistry (ΦPSII) was lower in M. brunneum-inoculated than in control plants, but not in B. bassiana-inoculated plants. After herbivory, however, ΦPSII increased in the fungal-inoculated plants compared with that before herbivory, similar to the reaction of control plants. At the same time, the fraction of energy dissipated as heat (ΦNPQ) decreased in the inoculated plants, resulting in an increased fraction of nonregulated energy loss (ΦNO) in M. brunneum. This indicates an increased singlet oxygen (1O2) formation not detected in B. bassiana-inoculated plants, showing that the two entomopathogenic fungi differentially modulate the leaflets' response to herbivory. Overall, our results show that M. brunneum inoculation had a negative effect on the photosynthetic efficiency before herbivory, while B. bassiana inoculation had no significant effect. However, S. exigua leaf biting activated the same compensatory PSII response mechanism in tomato plants of both fungal-inoculated treatments as in control plants.

We review glucosinolate (GSL) diversity and analyze phylogeny in the crucifer tribe Cardamineae as well as selected species from Brassicaceae (tribe Brassiceae) and Resedaceae. Some GSLs occur widely, while there is a scattered distribution of many less common GSLs, tentatively sorted into three classes: ancient, intermediate and more recently evolved. The number of conclusively identified GSLs in the tribe (53 GSLs) constitute 60% of all GSLs known with certainty from any plant (89 GSLs) and apparently unique GSLs in the tribe constitute 10 of those GSLs conclusively identified (19%). Intraspecific, qualitative GSL polymorphism is known from at least four species in the tribe. The most ancient GSL biosynthesis in Brassicales probably involved biosynthesis from Phe, Val, Leu, Ile and possibly Trp, and hydroxylation at the β-position. From a broad comparison of families in Brassicales and tribes in Brassicaceae, we estimate that a common ancestor of the tribe Cardamineae and the family Brassicaceae exhibited GSL biosynthesis from Phe, Val, Ile, Leu, possibly Tyr, Trp and homoPhe (ancient GSLs), as well as homologs of Met and possibly homoIle (intermediate age GSLs). From the comparison of phylogeny and GSL diversity, we also suggest that hydroxylation and subsequent methylation of indole GSLs and usual modifications of Met-derived GSLs (formation of sulfinyls, sulfonyls and alkenyls) occur due to conserved biochemical mechanisms and was present in a common ancestor of the family. Apparent loss of homologs of Met as biosynthetic precursors was deduced in the entire genus Barbarea and was frequent in Cardamine (e.g. C. pratensis, C. diphylla, C. concatenata, possibly C. amara). The loss was often associated with appearance of significant levels of unique or rare GSLs as well as recapitulation of ancient types of GSLs. Biosynthetic traits interpreted as de novo evolution included hydroxylation at rare positions, acylation at the thioglucose and use of dihomoIle and possibly homoIle as biosynthetic precursors. Biochemical aspects of the deduced evolution are discussed and testable hypotheses proposed. Biosyntheses from Val, Leu, Ile, Phe, Trp, homoPhe and homologs of Met are increasingly well understood, while GSL biosynthesis from mono- and dihomoIle is poorly understood. Overall, interpretation of known diversity suggests that evolution of GSL biosynthesis often seems to recapitulate ancient biosynthesis. In contrast, unprecedented GSL biosynthetic innovation seems to be rare.

A library of ion trap MS2 spectra and HPLC retention times reported here allowed distinction in plants of at least 70 known glucosinolates (GSLs) and some additional proposed GSLs. We determined GSL profiles of selected members of the tribe Cardamineae (Brassicaceae) as well as Reseda (Resedaceae) used as outgroup in evolutionary studies. We included several accessions of each species and a range of organs, and paid attention to minor peaks and GSLs not detected. In this way, we obtained GSL profiles of Barbarea australis, Barbarea grayi, Planodes virginica selected for its apparent intermediacy between Barbarea and the remaining tribe and family, and Rorippa sylvestris and Nasturtium officinale, for which the presence of acyl derivatives of GSLs was previously untested. We also screened Armoracia rusticana, with a remarkably diverse GSL profile, the emerging model species Cardamine hirsuta, for which we discovered a GSL polymorphism, and Reseda luteola and Reseda odorata. The potential for aliphatic GSL biosynthesis in Barbarea vulgaris was of interest, and we subjected P-type and G-type B. vulgaris to several induction regimes in an attempt to induce aliphatic GSL. However, aliphatic GSLs were not detected in any of the B. vulgaris types. We characterized the investigated chemotypes phylogenetically, based on nuclear rDNA internal transcribed spacer (ITS) sequences, in order to understand their relation to the species B. vulgaris in general, and found them to be representative of the species as it occurs in Europe, as far as documented in available ITS-sequence repositories. In short, we provide GSL profiles of a wide variety of tribe Cardamineae plants and conclude aliphatic GSLs to be absent or below our limit of detection in two major evolutionary lines of B. vulgaris. Concerning analytical chemistry, we conclude that availability of authentic reference compounds or reference materials is critical for reliable GSL analysis and characterize two publicly available reference materials: seeds of P. virginica and N. officinale.

Plants have evolved a variety of defences to reduce losses to herbivores and pathogens. The benefits of these may, however, be modified by resistance evolution in antagonists, changes in antagonist fauna, context-dependent "costs of defence", and by interactions between antagonists. In Barbarea vulgaris (Brassicaceae), the so-called "G-type" produces triterpenoid saponins that deter important specialist insect herbivores, whereas the "P-type" produces other saponins and are not insect-resistant. In contrast, P-type plants are predominantly resistant to the biotroph pathogen Albugo sp., causing white blister rust, whilst most G-type plants are susceptible. In a field experiment with F-3 hybrids between G and P-plants, we tested whether the two resistances are functionally coupled, leads to less disease and herbivory and to better plant performance, and whether insect herbivores and the pathogen interact in their effects on plant performance. The Albugo and insect resistances varied continuously between the F-3 plants and mapped to different linkage groups, indicating independent mechanisms and evolution. Plants with high Albugo resistance produced more biomass and survived better than more susceptible plants. Albugo DNA was detected in surface-sterilized green siliques, indicating systemic and sometimes non-symptomatic infection. Plants with high insect resistance were slightly less damaged by herbivores, but did not grow or survive better than more susceptible plants. Interactions between Albugo and insect herbivores did not affect plant performance. In contrast to the Albugo resistance, which clearly benefited the plants, our results show that the saponin-based insect resistance did convey any benefit under the given conditions despite its deterrent effects in controlled experiments.

In addition to direct tissue consumption, herbivory may affect other important plant processes. Here, we evaluated the effects of short-time leaf feeding by Spodoptera exigua larvae on the photosynthetic efficiency of tomato plants, using chlorophyll a fluorescence imaging analysis. After 15 min of feeding, the light used for photochemistry at photosystem II (PSII) (ΦPSII), and the regulated heat loss at PSII (ΦNPQ) decreased locally at the feeding zones, accompanied by increased non-regulated energy losses (ΦNO) that indicated increased singlet oxygen (1O2) formation. In contrast, in zones neighboring the feeding zones and in the rest of the leaf, ΦPSII increased due to a decreased ΦNPQ. This suggests that leaf areas not directly affected by herbivory compensate for the photosynthetic losses by increasing the fraction of open PSII reaction centers (qp) and the efficiency of these centers (Fv’/Fm’), because of decreased non-photochemical quenching (NPQ). This compensatory reaction mechanism may be signaled by singlet oxygen formed at the feeding zone. PSII functionality at the feeding zones began to balance with the rest of the leaf 3 h after feeding, in parallel with decreased compensatory responses. Thus, 3 h after feeding, PSII efficiency at the whole-leaf level was the same as before feeding, indicating that the plant managed to overcome the feeding effects with no or minor photosynthetic costs.

Plants evolved in close contact with a myriad of microorganisms, some of which formed associations with their roots, benefitting from carbohydrates and other plant resources. In exchange, they evolved to influence important plant functions, e.g. defense against insect herbivores and other antagonists. Here, we test whether a fungus, Metarhizium brunneum, which is mostly known as an insect pathogen, can also associate with plant roots and contribute to above-ground plant defense. Cauliflower (Brassica oleracea var. botrytis) seeds were sown together with M. brunneum-inoculated rice grains, and the resulting plants subjected to leaf herbivory by the specialist Plutella xylostella. Activity of myrosinases, the enzymes activating glucosinolates, was measured before and after herbivory; larval consumption and plant weight at the end of experiments. Metarhizium brunneum clearly established in the plant roots, and after herbivory myrosinase activity was substantially higher in M. brunneum-treated plants than in controls; before herbivory, M. brunneum-treated and control plants did not differ. Leaf consumption was slightly lower in the M. brunneum-treated plants whereas total biomass and allocation to above- or below-ground parts was not affected by the Metarhizium treatment. Thus, M. brunneum associates with roots and primes the plant for a stronger or faster increase in myrosinase activity upon herbivory. Consistent with this, myrosinase function has been suggested to be rate-limiting for induction of the glucosinolate-myrosinase defense system. Our results show that M. brunneum, in addition to being an insect pathogen, can associate with plant roots and prime plant defense.

According to coevolution theory, plant chemical defences are continually evolving in response to selection by herbivores. Unique to the Brassicales, a few species in the Barbarea genus produce triterpenoid saponins that are highly deterrent to some specialist insect herbivores. One species, B. vulgaris, has diverged into two chemotypes, the G- and P-type, of which the P-type seems to have lost the saponin-based insect resistance by producing different saponin structures; it also produces different glucosinolates and other potential defence traits. Here, we examined the preference and performance of a larger set of specialist and generalist herbivores on the two plant types, including three generalist mollusc (Arion vulgaris, Deroceras sp., Cepaea sp.) as well as three specialist (Phaedon cochleariae, Athalia rosae, Pieris napi oleraceae) and two generalist (Mamestra brassicae, Myzus persicae) insect herbivores. Five out of six herbivore species preferred leaves of the P-type for feeding, and most of them also survived and/or grew better on the P-type, or preferred it for oviposition. In contrast, larvae of M. brassicae showed no preference and performed equally well on the two plant types; the leaf beetle P. cochleariae preferred the G-type for oviposition, which was, however, not reflecting larval performance. Overall, the defences of the P-type against herbivores seem not to be as effective as those of the G-type, which is surprising given its large geographical distribution, overlapping with that of the G-type in Scandinavia and Finland. This suggests that additional ecological interactions determine the success of the two chemotypes.

Plants continuously evolve new defense compounds. One class of such compounds is triterpenoid saponins. A few species in the Barbarea genus produce saponins as the only ones in the large crucifer family. However, the molecular mechanism behind saponin biosynthesis and their role in plant defense remains unclear. We used pathway reconstitution in planta, enzymatic production of saponins in vitro, insect feeding assays, and bioinformatics to identify a missing gene involved in saponin biosynthesis and saponin-based herbivore defense. A tandem repeat of eight CYP72A cytochromes P450 colocalise with a quantitative trait locus (QTL) for saponin accumulation and flea beetle resistance in Barbarea vulgaris. We found that CYP72A552 oxidises oleanolic acid at position C-23 to hederagenin. In vitro-produced hederagenin monoglucosides reduced larval feeding by up to 90% and caused 75% larval mortality of the major crucifer pest diamondback moth and the tobacco hornworm. Sequence analysis indicated that CYP72A552 evolved through gene duplication and has been under strong selection pressure. In conclusion, CYP72A552 has evolved to catalyse the formation of hederagenin-based saponins that mediate plant defense against herbivores. Our study highlights the evolution of chemical novelties by gene duplication and selection for enzyme innovations, and the importance of chemical modification in plant defense evolution.

The genus Barbarea has emerged as a model for evolution and ecology of plant defense compounds, due to its unusual glucosinolate profile and production of saponins, unique to the Brassicaceae. One species, B. vulgaris, includes two ‘types’, G-type and P-type that differ in trichome density, and their glucosinolate and saponin profiles. A key difference is the stereochemistry of hydroxylation of their common phenethylglucosinolate backbone, leading to epimeric glucobarbarins. Here we report a draft genome sequence of the G-type, and re-sequencing of the P-type for comparison. This enables us to identify candidate genes underlying glucosinolate diversity, trichome density, and study the genetics of biochemical variation for glucosinolate and saponins. B. vulgaris is resistant to the diamondback moth, and may be exploited for “dead-end” trap cropping where glucosinolates stimulate oviposition and saponins deter larvae to the extent that they die. The B. vulgaris genome will promote the study of mechanisms in ecological biochemistry to benefit crop resistance breeding.

Hybridisation barriers are likely to evolve during allopatric separation of populations, in parallel with divergent adaptation to different conditions in the two ranges. If the populations secondarily come into contact, limited interbreeding between them may affect the subsequent spread of the two lineages and their adaptations in the region of sympatry. Barbarea vulgaris include two genetically divergent lineages that differ in secondary metabolites and resistances to insects and a pathogen. The two plant types grow in different Eurasian ranges but co-occur in Denmark and neighbouring countries, posing the question why they have not merged and the resistances spread from one type into the range of the other. Here, we tested whether a hybridisation barrier contribute to this. Different proportions of plants of the two types were placed in net tents and pollinated by flies, and paternity of the resulting seeds determined with genetic markers. Lower proportions of fruits and seeds developed successfully in mixtures with higher proportions of heterotypic plants (i.e. of the other plant type). When combined with results on offspring paternity in a statistical analysis, we found that heterotypic pollen was much less successful in fertilizing embryos and that heterotypic seeds survived less frequently than the contypic. Mature F-1 hybrids in addition produced lower proportions of mature pollen. The two B. vulgaris plant types are thus separated by substantial prezygotic and postzygotic barriers, as strong as reported for crosses between closely related but taxonomically recognised plant species. This may explain why the two B. vulgaris types have not merged in sympatry and why genes for insect-resistance have not introgressed to any extent from one to the other.

Evolution of ecotypic differentiation and maintenance of divergence in sympatry is more likely if populations occupy different habitats. Two genetically divergent chemotypes of Barbarea vulgaris (Brassicaceae) with contrasting insect and disease resistances (G-and P-type) occupy different geographical ranges in Eurasia but co-occur in Denmark. Here, they grow mostly in separate populations, posing the question why they have not merged. In a 2-year reciprocal transplant experiment, we tested whether the two plant types prefer different habitats and are adapted to these, possibly influenced by selection by herbivores. Vegetation and soil analyses indicated that the G-type occurs in slightly drier, less productive and more alkaline sites than the P-type. Plants planted at sites of their own chemotype had a higher biomass in the first year than plants of the nonresident chemotype, but were more damaged by beetle herbivory. This suggests that herbivores have adapted genetically or plastically to the resident chemotype. G-plants were less damaged by molluscs than P-plants, which may be caused by their content of saponins or glucosinolates. Levels of glucosinolates in siliques were higher in G-plants, but varied strongly among sites. Survival, reproduction and biomass in the second year did not differ between plants growing at sites of their own chemotype and plants of the other chemotype. Thus, the two chemotypes of B. vulgaris are adapted to different habitats to some extent, which could contribute to maintain their differentiation in sympatry. Herbivory by some insects, however, may counteract this.

The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs).