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Metabolomic studies on root uptake and transformation of bioactive compounds, like cereal benzoxazinoids (BXs) in non-BX producing plants, are very limited. Therefore, a targeted mass-spectrometry-based metabolomics study was performed to elucidate the root uptake of BXs in white clover (

Translocation of metabolites between different plant species provides important hints in understanding the fate of bioactive root exudates. In the present study, targeted and untargeted mass spectrometry-based metabolomics was applied to elucidate the transfer of bioactive compounds between rye and several crops and weed species. Our results demonstrated that benzoxazinoids (BXs) synthesized by rye were taken up by roots of neighbouring plant species and translocated into their shoots. Furthermore, we showed that roots of rye plants took up compounds originating from neighbouring plants. Among the compounds taken up by rye roots, wogonin was detected in the rye shoot, which indicated a root-to-shoot translocation of this compound. Elucidating the transfer of bioactive compounds between plants is essential for understanding plant–plant interactions, developing natural pesticides and understanding their modes of action.

This study aimed to elucidate the chemical response of hairy vetch (Vicia villosa Roth) to interspecific competition by rye (Secale cereale L.). Hairy vetch plants were grown as target plants in a mixture with different densities of rye plants (0, 1, 2, and 4). Twelve flavonoids in the shoot, root, and root exudate of hairy vetch plants were quantified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Kaempferol 3-O-{β-D-rhamnosyl-(1->6)-[β-D-xylosyl)-(1->2)]-β-D-galactoside} (Kaempferol-Rha-Xyl-Gal) was the predominant flavonoid found in shoots, roots, and root exudate of hairy vetch plant. Co-cultivation of hairy vetch with one rye plant increased the concentration of kaempferol-Rha-Xyl-Gal in hairy vetch root exudates. However, the concentration was reduced when co-cultivated with 2 or 4 rye plants. This study revealed that low levels of competition increased root exudation of kaempferol-Rha-Xyl-Gal, while higher levels of competition negatively affected production and root exudation of kaempferol-Rha-Xyl-Gal by hairy vetch.

Plants compete with their neighbors about the limited resources available to them. Plants under induced stress resulting from competition may alter their metabolome to increase their resilience or enhance their defense mechanisms. In the present study, rye (Secale cereale) plants were cocultivated with different densities (3, 12, and 18 plants per pot) of Austrian pea (Pisum sativum subsp. arvense), hairy vetch (Vicia villosa), and Alexandrian clover (Trifolium alexandrinum L.) to elucidate the changes in the rye metabolome in response to the different levels of competition. Global metabolic profiling by liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS), liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) was performed on rye plants, and the acquired data were analyzed using uni- and multivariate statistics. Targeted analysis showed that a high level of competition reduced the concentration of aglycone benzoxazinoids (BXs) and increased glycoside BXs in rye roots. Untargeted metabolomics analysis indicated an increase in the rye root content of the allelopathic compounds 4-hydroxybenzoic acid and uracil in response to competition. Untargeted analysis of rye shoots revealed that the plant competition increased the d-pyroglutamic acid, which is an elicitor of reactive oxygen species (ROS). Our results have enhanced the knowledge of the biochemical response of plant species to cocultivation.

Entomopathogenic fungi (EPF) can display a plant-associated lifestyle as endophytes. Seed application of EPF can affect insect herbivory above ground, but the mechanisms behind this are not documented. Here we applied three EPF isolates, Beauveria bassiana, Metarhizium brunneum and M. robertsii, as seed inoculation of wheat and bean, and evaluated the effects on population growth of aphids, Rhopalosiphum padi and Aphis fabae, respectively. In wheat and bean leaves, we quantified benzoxazinoids and flavonoids, respectively, in response to EPF inoculation and aphid infestation to elucidate the role of specific plant secondary metabolites (PSMs) in plant–fungus–herbivore interactions. Inoculations of wheat and bean with M. robertsii and B. bassiana reduced aphid populations compared with control treatments, whereas M. brunneum unexpectedly increased the populations of both aphids. Concentrations of the majority of PSMs were differentially altered in EPF-treated plants infested with aphids. Changes in aphid numbers were associated with PSMs regulation rather than EPF endophytic colonisation capacity. This study links the effects of EPF seed inoculations against aphids with unique PSM accumulation patterns in planta. The understanding of PSM regulation in tri-trophic interactions is important for the future development of EPF for pest management.

Plants have evolved advanced chemical defense mechanisms, including root exudation, which enable them to respond to changes occurring in their surroundings rapidly. Yet, it remains unresolved how root exudation affects belowground plant-plant interactions. The objective of this study was to elucidate the fate of benzoxazinoids (BXs) exuded from the roots of rye (Secale cereale L.) plants grown with hairy vetch (Vicia villosa). A rapid method that allows nondestructive and reproducible chemical profiling of the root exudates was developed. Targeted chemical analysis with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was performed to investigate the changes in the composition and concentration of BXs in the rye plant, and its root exudate in response to cocultivation with hairy vetch. Furthermore, hairy vetch plants were screened for the possible uptake of BXs from the rhizosphere and their translocation to the shoot. Rye significantly increased the production and root exudation of BXs, in particular 2-β-d-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-glc) and 2-β-d-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), in response to cocultivation with hairy vetch. DIBOA-glc and DIMBOA-glc were absorbed by the roots of the cocultivated hairy vetch plants and translocated to the shoots. These findings will strongly improve our understanding of the exudation of BXs from the rye plant and their role in interaction with other plant species.

Introduction: Benzoxazinoids (BXs) are plant phytochemicals that have both defensive properties in plants and therapeutic effects in humans. The presence of BXs has been largely studied in the Poaceae family (monocots). To study the presence or absence of BXs in dicotyledons and monocotyledons outside the Poaceae family, parts of 24 plant species at several growth stages were selected for analysis, some of which were already known to contain BXs.Objectives: To devise a step-wise mass spectrometry-based approach for confirming the presence of BXs in plant samples, and to use the method to explore the status of BXs in selected plant species.Experimental: Plant samples were extracted using accelerated solvent extraction and analyzed using triple-quadrupole liquid chromatography-mass spectrometry.Results: The use of different columns, double mass transitions, and ion ratios proved to be a robust tool for confirming the presence of BXs in different plant species. By this method, the presence of BXs was confirmed in three of the 24 species. Double-hexose forms of BXs, which have not been reported before in dicotyledons, were confirmed to be present in the dicotyledon plants Acanthus mollis and Lamium galeobdolon, and the presence of BXs in the seeds of Consolida orientalis is reported for the first time here. High concentrations of BXs were found in the aerial parts of Acanthus mollis and Lamium galeobdolon, at 20 and 32 μmol/g plant dry weight, respectively.Conclusions: The stepwise approach described in this work confirmed the presence of BXs in new samples.

Benzoxazinoids (BXs) are phytochemicals that exert plant-protecting, allelopathic, and human-health-promoting effects. Here, we present a data-dependent acquisition-mass spectrometry based method to locate and annotate BX-conjugates in different fractions of a plant extract. This allows for isolation of compounds present in a much smaller percentage than common for natural products yet still sufficient for subsequent identification through nuclear magnetic resonance (NMR) spectroscopy. The presence of BXs has been extensively studied in monocotyledons, whereas only a limited number of studies have focused on dicotyledons. Here, the presence of hitherto unknown BXs in the roots of the dicotyledonous plant Acanthus mollis L. has been determined. Two acetylated glycosylated BXs, compound 1 2-(6-acetyl-β-D-glucopyranoside)-2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one) and compound 2 (-2-(6-acetyl-β-D-glucopyranoside)-2-hydroxy-2H-1,4-benzoxazin-3(4H)-one), and a BX-derived glucoside carbamate olide, compound 3 (2-hydroxy-6-(2-deoxy-β-D-glucopyranosyloxy)-phenyl) carbamic acid 2′-olide), were isolated and identified by NMR. These previously unknown BXs may contribute to the reported biological activities of A. mollis L.

BackgroundAlthough it is well established that plant metabolomes mediate microbiome assembly, the question of how metabolome-microbiome interactions may prevent pathogen invasion remains to be answered. To address this question, we studied microbiome and metabolome profiles of two Arabidopsis thaliana accessions, Columbia-0 (Col-0) and Landsberg erecta (Ler-0) with differential resistance profiles to the fungal pathogen Fusarium oxysporum f.sp. mathioli (FOM). We used amplicon sequencing to characterize bacterial (16S) and fungal (ITS2) communities, and we used targeted metabolite analysis across 5 stages of FOM host progression. ResultsWe found that microbiome and metabolome profiles were markedly altered in FOM-inoculated and non-inoculated samples of resistant Col-0 and susceptible Ler-0. Co-occurrence network analysis revealed robust microbial networks in the resistant Col-0 compared to the susceptible Ler-0, during FOM infection. Specific metabolites and microbial OTUs (including indicator and hub OTUs) correlated in both non-inoculated and inoculated Col-0 and Ler-0. The glucosinolates 4-hydroxyglucobrassicin, neoglucobrassicin and indole-3 carbinol, but also phenolic compounds were active in structuring the A. thaliana-microbiome. ConclusionsOur results highlight the interactive effects of host resistance and its associated microbiota on Fusarium infection and progression. These findings shed significant insights into plant inter-omics dynamics during pathogen invasion and could possibly facilitate the exploitation of microbiomes for plant disease control.

Background and objectives: Integrated weed management in commercial wheat production is urgently needed due to increasing herbicide resistance and production costs. Benzoxazinoids (BXs), which include benzoxazinones and benzoxazolinones, are unique bioactive metabolites produced by certain members of the Poaceae including maize, wheat, rye and some dicots. BXs play important roles in plant defence and are causal agents of allelopathic interference. We investigated the role of genetics, environment and crop growth stage on BX abundance in the roots and rhizoplane of selected commercial wheat cultivars, and quantified their microbial transformation products (aminophenoxazinones) in roots and rhizosphere soils. Methods: Cultivar trials of competitive wheat (Triticum aestivum L.) genotypes were conducted in two moderate to low rainfall (449–572 mm) locations in southeastern Australia in 2015 and 2016. Replicated shoot, root, rhizoplane, and rhizosphere soil samples were collected for metabolic profiling at selected crop phenological stages, extracted and further analysed for known benzoxazinoid metabolites by liquid chromatography coupled with high resolution mass spectrometry. Results: Fifteen BXs and related microbially derived aminophenoxazinones were detected in wheat shoots, roots, rhizoplanes and rhizosphere soils in both years and locations. MBOA, HMBOA and HMBOA-Glc were the three most abundant BX metabolites in wheat tissues, with the heritage cultivar Federation producing the highest levels of MBOA. The phytotoxic aminophenoxazinones APO and AMPO were the most abundant BX microbial transformation products and were detected in wheat roots, rhizoplanes and rhizospheres. Abundance varied with cultivar, growth stage, location and year. Conclusions: Microbially-produced aminophenoxazinones generated from both heritage and modern wheat root exudates were detected and quantified in rhizosphere soils, with abundance dependent on cultivar, growth stage, and season. Concentrations of microbial metabolites APO, AMPO, and AAPO were higher in the rhizosphere of young wheat seedlings in contrast to that of mature plants suggesting that phenoxazinone production was upregulated early in the season. Our findings demonstrate that BX metabolites at all life stages of wheat potentially undergo rapid biotransformation to aminophenoxazinones under field conditions, resulting in ecologically relevant concentrations sufficient for weed suppression by certain wheat cultivars.

Phytochemicals or phenolic compounds are important natural bioactive molecules that plants accumulate in response to environmental conditions and may exert benef icial effects on health by protecting humans against many diseases. The aim of this work was to analyze the influence of biotic and abiotic stress on the accumulation of flavonoids and phenolic acids on leaves of two cultivars of Amaranthus hypochondriacus, which are differentiated by the colour of their leaves (red or green). Phenolic compounds were extracted using the accelerated solvent extraction (ASE) method and their identif ication was carried out by LC-MS analysis. Rutin was the main flavonoid in amaranth leaves; the highest concentrations were found in green leaves when plants were subjected to stress (9715 μg g-1). Phenolic acids were minor compounds; ferulic acid was only present in red leaves (0.5 μg g-1) and p-coumaric acid only in green leaves (0.7 μg g-1). Our results indicate that leaves from A. hypochondriacus, the main species that produce edible seeds, are a good source of phytochemical compounds and their accumulation could be driven by the growing conditions.

Knowledge about agricultural crops and their mechanisms is crucial in a time focused on climate. Flavonoids are plant secondary metabolites present in many plant species, including narrow-leafed lupin (Lupinus angustifolius L.). To gain a better understanding of the biological effect of flavonoids in the soil, the link between roots and soil must be elucidated. Plants were grown in a long-term crop rotation field experiment, where roots and rhizosphere soil were sampled under L. angustifolius and reference spring barley (Hordeum vulgare L.). Three high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods were developed and validated for the identification and quantification of 27 flavonoids in roots and rhizosphere soil, with flavonoids present in the soil representing exudation. Soil extraction using acetonitrile (ACN) and methanol (MeOH) was compared to 70% MeOH. In addition, to elucidate the bioavailable flavonoids, water-based extractions were investigated. The optimal soil extraction method was using 70% MeOH, achieving acceptable recoveries for several compounds. L. angustifolius roots contained 14 of 27 flavonoids at concentrations of 0.05–736 μg/g DW; genistein, genistin, and luteone being the most abundant. Four flavonoids were detected in L. angustifolius soil, but not in H. vulgare soil. None of the water-based extractions resulted in flavonoid quantities similar to those obtained with 70% MeOH extraction. L. angustifolius roots contained larger quantities of flavonoids than H. vulgare roots. The presence of four flavonoids in L. angustifolius soil, and not in H. vulgare soil, suggests that the flavonoids were exuded from the L. angustifolius roots. Sonication using 70% MeOH was an efficient soil extraction method, and enabled quantification of the flavonoid pool present in soil at the sampling time point.

The honey bee pollen/nectar diet is rich in bioactive phytochemicals and recent studies have demonstrated the potential of phytochemicals to influence honey bee disease resistance. To unravel the role of dietary phytochemicals in honey bee health it is essential to understand phytochemical uptake, bioavailability, and metabolism but presently limited knowledge exists. With this study we aim to build a knowledge foundation. For 5 days, we continuously fed honey bees on eight individual phytochemicals and measured the concentrations in whole and dissected bees by HPLC-MS/MS. Ample phytochemical metabolization was observed, and only 6–30% of the consumed quantities were recovered. Clear differences in metabolization rates were evident, with atropine, aucubin, and triptolide displaying significantly slower metabolism. Phytochemical gut uptake was also demonstrated, and oral bioavailability was 4–31%, with the highest percentages observed for amygdalin, triptolide, and aucubin. We conclude that differences in the chemical properties and structure impact phytochemical uptake and metabolism.

Although the effects of plant secondary metabolites on plant defence have been studied for decades, the exact roles of secondary metabolites in shaping plant-associated microbial and nematode communities remain elusive. We evaluated the effects of benzoxazinoids, a group of secondary metabolites present in several cereals, on root-associated nematodes. We employed 18S rRNA metabarcoding to compare maize root-associated nematode communities in a bx1 knockout maize line impaired in benzoxazinoid synthesis and in its parental wild type. Both genotype and plant age affected the composition of the nematode community in the roots, and the effects of benzoxazinoids on nematode communities were stronger in the roots than in the rhizosphere. Differential abundance analysis and quantitative PCR showed that the root lesion nematode Pratylenchus neglectus was enriched in the bx1 mutant line, while another root lesion nematode, Pratylenchus crenatus, was reduced. Correlation analysis showed that benzoxazinoid concentrations in maize roots mostly correlated negatively with the relative abundance of nematode sequence reads. However, positive correlations between benzoxazinoids and nematode taxa, including several plant-parasitic nematodes, were also identified. Our detailed nematode community analysis suggests differential and selective effects of benzoxazinoids on soil nematodes depending on both the nematode species and the benzoxazinoid compound.

Honey bees are important pollinators and are subject to numerous stressors, such as changing floral resources, parasites, and agrochemical exposure. Pesticide exposure has been linked to the decline in the global honey bee population. We have limited knowledge of the metabolic pathways and synergistic effects of xenobiotics in bees. Quercetin is one of the most abundant phytochemicals in plants and is therefore abundant in the honey bee diet. Quercetin can upregulate the detoxification system in honey bees; however, it is still unknown to what extent quercetin ingestion can reduce the content of absorbed pesticides. In this study, we investigated the effect of dietary quercetin on the contents of three pesticides in honey bees: imidacloprid (insecticide), tebuconazole (fungicide), and tau-fluvalinate (insecticide and acaricide). Bees were divided into two main groups and fed either quercetin-sucrose paste or only sucrose for 72 h. Thereafter, they were orally exposed to ∼10 ng/bee imidacloprid or contact-exposed to ∼ 0.9 μg/bee tau-fluvalinate or ∼ 5.2 μg/bee tebuconazole. After 1 h of oral exposure or 24 h of contact exposure, the bees were anaesthetised with CO2, sacrificed by freezing, and extracted with a validated QuEChERS method. Subsequently, the concentrations of the three pesticides and quercetin in the bees were determined with a triple quadrupole tandem mass spectrometer coupled to an HPLC system. No significant effect on the concentration of tebuconazole or tau-fluvalinate was observed in bees fed quercetin. Intake of quercetin led to a reduction in the concentration of imidacloprid in honey bees. Quercetin-rich plants may be exploited in future beekeeping.

The honey bee pollen/nectar diet is rich in bioactive phytochemicals and recent studies have demonstrated the potential of phytochemicals to influence honey bee disease resistance. To unravel the role of dietary phytochemicals in honey bee health it is essential to understand phytochemical uptake, bioavailability, and metabolism but presently limited knowledge exists. With this study we aim to build a knowledge foundation. For 5 days, we continuously fed honey bees on eight individual phytochemicals and measured the concentrations in whole and dissected bees by HPLC-MS/MS. Ample phytochemical metabolization was observed, and only 6-30% of the consumed quantities were recovered. Clear differences in metabolization rates were evident, with atropine, aucubin, and triptolide displaying significantly slower metabolism. Phytochemical gut uptake was also demonstrated, and oral bioavailability was 4-31%, with the highest percentages observed for amygdalin, triptolide, and aucubin. We conclude that differences in the chemical properties and structure impact phytochemical uptake and metabolism.

Fusarium oxysporum is a destructive root-infecting plant pathogen that causes significant yield losses in many economically important crop species. Hence, a deeper understanding of pathogen infection strategies is needed. With liquid chromatography-tandem mass spectrometry and gas chromatography-time of flight mass spectrometry platforms, we analyzed the metabolic changes in a time-course experiment with Arabidopsis accessions either resistant (Col-0) or susceptible (Ler-0) to isolates of Fusarium oxysporum forma specialis matthioli infection. We showed a concurrent effect of Fusarium-derived polyols and the mycotoxin beauvericin in the suppression of the immune response of susceptible hosts. A significant increase in oxidized glutathione in the resistant host was probably associated with effective reactive oxygen species-mediated resistance responses. Through a combination of targeted and untargeted metabolomics, we demonstrated the concurrent action of several Arabidopsis defense systems as well as the concurrent action of several virulence systems in the fungal attack of susceptible Arabidopsis.

The losses of honey bee colonies and declines of other insect pollinators have been associated with negative effects of pesticides. Honey bees as well as other pollinators are nectar and pollen foragers and thus are exposed to an extensive range of phytochemicals. Understanding the synergistic, additive, and antagonistic effects of plant secondary metabolites and pesticides in honey bees may help to protect honey bee colonies against agrochemicals. In this study, we used untargeted metabolomics to investigate the impact of dietary phytochemical composition on the residual concentration of three pesticides: imidacloprid, tau-fluvalinate and tebuconazole in exposed honey bees. Honey bees were given different diets based on pollen or nectar from four plants: Reseda odorata, Borago officinalis, Phacelia tanacetifolia, and Trifolium repens for two days. Thereafter, they were orally exposed to 10 ng/bee imidacloprid or contact-exposed to 0.9 μg/bee tau-fluvalinate or 5 μg/bee tebuconazole. After 1 h of oral exposure or 24 h of contact exposure, the honey bees were anaesthetised with CO2, sacrificed by freezing, extracted with a validated QuEChERS method, and residual pesticide concentrations were determined by LC-QTRAP-MS/MS. The phytochemical composition in the given diets were profiled with an UHPLC-Q Exactive-MS/MS. The results revealed that the dietary phytochemical composition has a noteworthy influence on the concentration of residual pesticides in honey bees. The correlation coefficient analysis demonstrated that flavonoids have a reducing effect on the residual concentration of imidacloprid and tau-fluvalinate in honey bees. The results also highlighted that exposure to imidacloprid impaired the metabolism of sugars in honey bees. Exploiting flavonoid-rich plants may protect honey bees against pesticides and hold promise as forage plants in future beekeeping.

Plants may utilize bioactive chemical compounds which exude from their root to shape their associated beneficial microorganism in the rhizosphere to acquire resistance against pathogen infection [Fig. 1]. However, the underlying mechanism regulating this interaction has been poorly elucidated. The aim of the present study was to investigate the functional role of root exudate (from different host genotypes) in mediating belowground indirect plant defense. To elucidate the molecular mechanism underlying plant interaction with health-promoting microorganism, Arabidopsis thaliana (Col-0 and Ler-0 accessions) were grown for three weeks in field soil (non-sterile). Subsequently, the roots were exposed to Fusarium oxysporum matthioli (FOM). Sampling was done every five days after the infection for 25 days. The collected samples were subjected to both targeted (aliphatic and indole glucosinolates, plant hormones, and mycotoxins) and untargeted analysis by employing LC-MS/MS and GC-TOF/MS platforms as well as metabarcoding (Illumina MiSeq). Phenotypic characterization of the plants displayed that the Col-0 accessions had developed a resistance to FOM exposure whereas the Ler-0 ecotype was susceptible. The disease progression was also evaluated on a molecular level and resulted in the identification of beauvericin in both root and shoot tissues of the susceptible plants which likely was associated with promoting pathogenicity in FOM. The result from targeted and untargeted metabolomics as well as analysis of the root microbial community composition (bacteria and fungi) revealed the role of novel natural products in shaping the distinct member of the microbial community leading to diseases suppression. Plants may utilize bioactive chemical compounds which exude from their root to shape their associated beneficial microorganism in the rhizosphere to acquire resistance against pathogen infection [Fig. 1]. However, the underlying mechanism regulating this interaction has been poorly elucidated. The aim of the present study was to investigate the functional role of root exudate (from different host genotypes) in mediating belowground indirect plant defense. To elucidate the molecular mechanism underlying plant interaction with health-promoting microorganism, Arabidopsis thaliana (Col-0 and Ler-0 accessions) were grown for three weeks in field soil (non-sterile). Subsequently, the roots were exposed to Fusarium oxysporum matthioli (FOM). Sampling was done every five days after the infection for 25 days. The collected samples were subjected to both targeted (aliphatic and indole glucosinolates, plant hormones, and mycotoxins) and untargeted analysis by employing LC-MS/MS and GC-TOF/MS platforms as well as metabarcoding (Illumina MiSeq). Phenotypic characterization of the plants displayed that the Col-0 accessions had developed a resistance to FOM exposure whereas the Ler-0 ecotype was susceptible. The disease progression was also evaluated on a molecular level and resulted in the identification of beauvericin in both root and shoot tissues of the susceptible plants which likely was associated with promoting pathogenicity in FOM. The result from targeted and untargeted metabolomics as well as analysis of the root microbial community composition (bacteria and fungi) revealed the role of novel natural products in shaping the distinct member of the microbial community leading to diseases suppression.

Honey bees regularly ingest quercetin, one of the most abundant phytochemicals in plants. Past research suggests that honey and pollen feeding upregulates the bees’ detoxification system and that dietary quercetin reduces the toxicity of imidacloprid and tau-fluvalinate [1,2].The aim of this study was to investigate the effect of dietary quercetin on the concentration of three pesticides present in honey bees. Honey bees (n = 600) were divided in five groups and fed either quercetin-sucrose paste or only sucrose for 72 h before being exposed to the neonicotinoid imidacloprid (oral exposure), the fungicide tebuconazole or the acaricide tau-fluvalinate (contact exposure). Bees were anesthetized with CO2, frozen, and extracted with a validated QuEChERS method. Finally, the concentrations of all three above-mentioned pesticides were determined by LC-MSMS. Quantification results of the three pesticides in honey bee samples [Fig. 1] displayed that there was a significant difference in concentration of imidacloprid (P = 2 ͯ 10-5) and tau-fluvalinate (P = 3 ͯ 10-2) between the bees that were fed quercetin-sucrose paste and bees that were fed only sucrose. However, there was no significant difference in the level of tebuconazole in the group of bees that were fed quercetin-sucrose paste and bees that were fed only sucrose (P = 4.2 ͯ 10-1). The results of this study demonstrate that honey bee intake of quercetin leads to a reduction in the concentration of imidacloprid and tau-fluvalinate by up regulating the detoxification systems in bees. Hence, quercetin may be a promising bioactive compound in chemo-ecological aspects to counteract the hazard of pesticides. Honey bees regularly ingest quercetin, one of the most abundant phytochemicals in plants. Past research suggests that honey and pollen feeding upregulates the bees’ detoxification system and that dietary quercetin reduces the toxicity of imidacloprid and tau-fluvalinate [1,2].The aim of this study was to investigate the effect of dietary quercetin on the concentration of three pesticides present in honey bees. Honey bees (n = 600) were divided in five groups and fed either quercetin-sucrose paste or only sucrose for 72 h before being exposed to the neonicotinoid imidacloprid (oral exposure), the fungicide tebuconazole or the acaricide tau-fluvalinate (contact exposure). Bees were anesthetized with CO2, frozen, and extracted with a validated QuEChERS method. Finally, the concentrations of all three above-mentioned pesticides were determined by LC-MSMS. Quantification results of the three pesticides in honey bee samples [Fig. 1] displayed that there was a significant difference in concentration of imidacloprid (P = 2 ͯ 10-5) and tau-fluvalinate (P = 3 ͯ 10-2) between the bees that were fed quercetin-sucrose paste and bees that were fed only sucrose. However, there was no significant difference in the level of tebuconazole in the group of bees that were fed quercetin-sucrose paste and bees that were fed only sucrose (P = 4.2 ͯ 10-1). The results of this study demonstrate that honey bee intake of quercetin leads to a reduction in the concentration of imidacloprid and tau-fluvalinate by up regulating the detoxification systems in bees. Hence, quercetin may be a promising bioactive compound in chemo-ecological aspects to counteract the hazard of pesticides.

Plants are consistently releasing root exudates into the rhizosphere which have the potential to influence the growth and development of neighboring plant species. Root exudates contain low and high molecular weight compounds such as sugars, amino acids, secondary metabolites, phytohormones, proteins, enzymes, and polysaccharides. Although aboveground plant interactions through volatile compounds has been extensively studied, only few studies investigated role of the belowground chemical interactions between plant species. The aim of this study is to elucidate the belowground chemical interactions between rye and hairy vetch grown together as cover crop mixture. Rye and hairy vetch were cultivated alone and together in pots filled with micro glasssbeads. Plant were grown for three weeks in climate chamber and their root exudates were collected for the chemical analysis. Targeted analysis with LC-MS/MS was done to identify the changes occurring in secondary metabolite profile of the rye and hairy vetch’s root exudates as result of co-cultivation. Quantification results displayed that both hairy vetch and rye are altering their root exudation in response to rye-hairy vetch co cultivation. Hairy vetch significantly increased concentration of root exuded flavonoids such as kaempferol and pratensein in response to presence of rye. Concentrations of DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and BOA (1,3-benzoxazol-2-one) in rye’s root exudate were increased significantly as result of co cultivation with hairy vetch. The results from this study will increase our understanding about root exuded natural products with growth suppressive effects, which finally leads us to more sustainable plant protection system. Plants are consistently releasing root exudates into the rhizosphere which have the potential to influence the growth and development of neighboring plant species. Root exudates contain low and high molecular weight compounds such as sugars, amino acids, secondary metabolites, phytohormones, proteins, enzymes, and polysaccharides. Although aboveground plant interactions through volatile compounds has been extensively studied, only few studies investigated role of the belowground chemical interactions between plant species. The aim of this study is to elucidate the belowground chemical interactions between rye and hairy vetch grown together as cover crop mixture. Rye and hairy vetch were cultivated alone and together in pots filled with micro glasssbeads. Plant were grown for three weeks in climate chamber and their root exudates were collected for the chemical analysis. Targeted analysis with LC-MS/MS was done to identify the changes occurring in secondary metabolite profile of the rye and hairy vetch’s root exudates as result of co-cultivation. Quantification results displayed that both hairy vetch and rye are altering their root exudation in response to rye-hairy vetch co cultivation. Hairy vetch significantly increased concentration of root exuded flavonoids such as kaempferol and pratensein in response to presence of rye. Concentrations of DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and BOA (1,3-benzoxazol-2-one) in rye’s root exudate were increased significantly as result of co cultivation with hairy vetch. The results from this study will increase our understanding about root exuded natural products with growth suppressive effects, which finally leads us to more sustainable plant protection system.

The presence of bioactive benzoxazinoids (BXs) in mature whole grain and experimental bread products was recently shown. The aim of this study was to reveal the level of BXs in a broad range of commercial cereal products and discuss these findings in relation to ingredients and preparation processes. The concentrations of 11 BX compounds were determined by liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) in 25 supermarket-available cereal food products. The total BX content varied from below the limit of detection to >500 μg/g dry matter (DM) with some degree of variation among different lots. The products W (Rugsandwich), H (Gulerodsrugbrod), L (Morke Multikerneboller) and AA (Solsikkeklapper), which were all rich in rye-based ingredients, had a total BX concentration >300 μg/g DM. Double hexose derivatives of 2-hydroxy-1,4-benzoxazin-3-one (HBOA), 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2-β- d -glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-glc) were the major BXs in most products. Further studies into the health effects of BXs should be conducted, and future exploitation of these compounds seems possible.

Crop management practices such as tillage can influence the dissipation of herbicides in soil. This study aimed to determine the effects of tillage systems on soil dissipation of prosulfocarb (PSC) using two assessment methods: bioassay and high-performance liquid chromatography (HPLC) analysis. PSC was applied on plots cultivated under three tillage systems (moldboard plowing, tine tillage at 8- to 10-cm soil depth, and direct drilling) at different rates (0, 500, 1,000, 2,000, 4,000 and 8,000 g ai ha−1) and two spraying times, representing early and late sowing time of winter cereals in Denmark. The experiment was conducted over 2 yr. The soil was analyzed for PSC residues by HPLC and a bioassay, using silky windgrass as the indicator plant. Neither technique revealed an effect of tillage systems on PSC dissipation, but the LD50values estimated based on the bioassays were generally lower under direct drilling (11.7 d) than with plowing (17.5 d). Moreover, LD50estimates based on bioassay results were generally lower than those estimated with HPLC analyses. Half-life values estimated with HPLC were low and not within the range of values reported in the literature (ca. 20 d), suggesting enhanced degradation of PSC. In addition to influencing the performance of PSC against problematic weed species, an enhanced dissipation rate could also hamper the benefits of PSC in an antiresistance strategy.

OBJECTIVES: Prostate cancer is a common cancer diagnosis among men, but the role of diet and food components remains controversial. Benzoxazinoids (BXs) is a potent group of bioactive compounds found in rye foods that seem to have the potential to inhibit prostate cancer growth in vitro but studies in humans are lacking. We therefore aimed to investigate the concentrations of different BXs and their metabolites after whole grain rye consumption in comparison to refined wheat and to correlate these compounds to PSA levels in men with indolent prostate cancer. METHODS: In a randomized controlled crossover study, men with prostate cancer (n = 24) consumed 485 gram of whole grain/bran rye and refined wheat foods for six weeks, with two weeks of washout in-between. The diets were isocaloric and contained the same amount of fiber by addition of cellulose in the refined wheat intervention. BXs in plasma samples were analyzed with HPLC- QTRAP/MS-ESI. RESULTS: Seventeen men finished the trial and were included in data analysis. For most BXs, the concentrations were significantly higher after the whole grain rye compared to the refined wheat intervention. The BXs or BXs metabolites HBOA-glc, HHPAA, HBOA-glcA, HPAA-glcA (∼19.4–0.4 nmol/L in plasma after interventions) were significantly inversely correlated to PSA level. CONCLUSIONS: In men with indolent prostate cancer, most BXs were significantly higher after an intervention with whole grain rye compared to refined wheat. The inverse correlation between four BXs or BXs metabolites and PSA may indicate on an inhibitory effect on prostate cancer cell growth. This could open for new preventive food-based strategies, but results would need to be replicated. FUNDING SOURCES: Independent Research Fund Denmark - Technology and Production Formas Swedish Research Council.

Pesticide metabolite identification is gaining increased attention because of the interest in potential metabolite toxicity. Azoxystrobin is one of the most prevalent pesticide residues in foods in Europe. The majority of azoxystrobin metabolites have been identified using radiolabelled standards, which are either expensive or not readily available. Thus, alternative approaches for metabolite identification are desirable. Here, an LC-MS/MS method for quantifying azoxystrobin and identifying its metabolites using quadrupole-linear ion trap mass spectrometry is reported. Seven metabolites of azoxystrobin were identified 2 and 4 weeks after spraying lettuce with azoxystrobin. Among them, two metabolites are reported for the first time. The hydrolysis, reduction, hydroxylation, photoisomerisation and hydrolytic cleavage of ether bonds are identified as biotransformation processes involved in azoxystrobin metabolism in lettuce.

Benzoxanoids (BXs) were identified for the first time in young cereal plants in 1957 and in medicinal plants in 1969. In 2008 our lab discovered that BXs are present in mature cereal grains and in food/feed based on cereal whole grains [1]. We showed that BXs are taken up in pigs, rats and humans [2]–[4], and that they appear in prostate tissue of humans consuming rye bread [5]. The aim of this study was to develop a method for identification and quantification of human phase 1 and phase 2 metabolites in plasma. The method included an initial protein precipitation step of the plasma with the purpose of reducing the matrix interference. The chromatographic analysis of BXs and their metabolites in the remaining extract was performed in LC-MS/MS. Pure standards were unavailable for most of the phase 2 metabolites. Phase 2 metabolites are present in much higher amounts in urine than in plasma. Thus urine metabolites of BXs for which structures could be assigned using data dependent acquisition were isolated and used for verification of MS/MS signals in plasma extracts. The isolation of approx 10 µg of each compound was done with consecutive HLB and mixed mode SPE’s (Oasis, Waters) exploiting the variety of polarity and acidity of the metabolites. Glucuronide and sulphate metabolites of paracetamol were used as standards for semi-quantification due to their structural similarity and commercial availability. Benzoxanoids (BXs) were identified for the first time in young cereal plants in 1957 and in medicinal plants in 1969. In 2008 our lab discovered that BXs are present in mature cereal grains and in food/feed based on cereal whole grains [1]. We showed that BXs are taken up in pigs, rats and humans [2]–[4], and that they appear in prostate tissue of humans consuming rye bread [5]. The aim of this study was to develop a method for identification and quantification of human phase 1 and phase 2 metabolites in plasma. The method included an initial protein precipitation step of the plasma with the purpose of reducing the matrix interference. The chromatographic analysis of BXs and their metabolites in the remaining extract was performed in LC-MS/MS. Pure standards were unavailable for most of the phase 2 metabolites. Phase 2 metabolites are present in much higher amounts in urine than in plasma. Thus urine metabolites of BXs for which structures could be assigned using data dependent acquisition were isolated and used for verification of MS/MS signals in plasma extracts. The isolation of approx 10 µg of each compound was done with consecutive HLB and mixed mode SPE’s (Oasis, Waters) exploiting the variety of polarity and acidity of the metabolites. Glucuronide and sulphate metabolites of paracetamol were used as standards for semi-quantification due to their structural similarity and commercial availability.

Benzoxazinoids (BXs) are phytochemicals present in selected cereal crops and medicinal plants. Effects of BXs in plants as plant defense and allelopathic agents has been widely studied [1, 2], while the knowledge into effects in mammals are limited. Potential therapeutic effects of BXs in humans [3], and presence of BXs in prostate cancer tissue in prostate cancer patients fed with a diet of rye, which is rich in BXs, [4], motivated us towards exploring the effects of BXs in prostate cancer cells. The effects of these compounds were studied in experiment exposing prostate cancer cell lines LNCap, DU145, PC3, and C4, and primary prostate cancer cells to selected BXs. Agluconic forms of BXs, which were reported to be the most active form of the compounds, were tested. BXs, DIBOA (2, 4-dihydroxy-1,4-benzoxazin-3-one) and DIMBOA (2, 4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) caused a decrease in cell viability across all the cell lines tested. The IC50 of the compounds ranged from 40 to 200 μM across different cell lines. The results are encouraging and further experiments will be carried out using RNA sequencing. The ultimate goal is to understand the mechanism behind the potential anti-cancer effect of BXs and to elucidate whether they can be therapeutic agents. Benzoxazinoids (BXs) are phytochemicals present in selected cereal crops and medicinal plants. Effects of BXs in plants as plant defense and allelopathic agents has been widely studied [1, 2], while the knowledge into effects in mammals are limited. Potential therapeutic effects of BXs in humans [3], and presence of BXs in prostate cancer tissue in prostate cancer patients fed with a diet of rye, which is rich in BXs, [4], motivated us towards exploring the effects of BXs in prostate cancer cells. The effects of these compounds were studied in experiment exposing prostate cancer cell lines LNCap, DU145, PC3, and C4, and primary prostate cancer cells to selected BXs. Agluconic forms of BXs, which were reported to be the most active form of the compounds, were tested. BXs, DIBOA (2, 4-dihydroxy-1,4-benzoxazin-3-one) and DIMBOA (2, 4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) caused a decrease in cell viability across all the cell lines tested. The IC50 of the compounds ranged from 40 to 200 μM across different cell lines. The results are encouraging and further experiments will be carried out using RNA sequencing. The ultimate goal is to understand the mechanism behind the potential anti-cancer effect of BXs and to elucidate whether they can be therapeutic agents.

Xenobiotic detoxification in plant as well as in animals has mostly been studied in relationship to the deactivation of the toxic residues of the compound that, surely for azoxystrobin, is represented by its &beta, methoxyacrylate portion. In maize roots treated for 96 h with azoxystrobin, the fungicide accumulated over time and detoxification compounds or conjugates appeared timewise. The main detoxified compound was the methyl ester hydrolysis product (azoxystrobin free acid, 390.14 m/z) thought to be inactive followed by the glutathione conjugated compounds identified as glutathione conjugate (711.21 m/z) and its derivative lacking the glycine residue from the GSH (654.19 m/z). The glycosylated form of azoxystrobin was also found (552.19 m/z) in a minor amount. The identification of these analytes was done by differential untargeted metabolomics analysis using Progenesis QI for label free spectral counting quantification and MS/MS confirmation of the compounds was carried out by either Data Independent Acquisition (DIA) and Data Dependent Acquisition (DDA) using high resolution LC-MS methods. Neutral loss scanning and comparison with MS/MS spectra of azoxystrobin by DDA and MSe confirmed the structures of these new azoxystrobin GSH conjugates.

Background Plants actively shape their associated microbial communities by synthesizing bio-active substances. Plant secondary metabolites are known for their signaling and plant defense functions, yet little is known about their overall effect on the plant microbiome. In this work, we studied the effects of benzoxazinoids (BXs), a group of secondary metabolites present in maize, on the host-associated microbial structure. Using BX knock-out mutants and their W22 parental lines, we employed 16S and ITS2 rRNA gene amplicon analysis to characterize the maize microbiome at early growth stages. Results Rhizo-box experiment showed that BXs affected microbial communities not only in roots and shoots, but also in the rhizosphere. Fungal richness in roots was more affected by BXs than root bacterial richness. Maize genotype (BX mutants and their parental lines) as well as plant age explained both fungal and bacterial community structure. Genotypic effect on microbial communities was stronger in roots than in rhizosphere. Diverse, but specific, microbial taxa were affected by BX in both roots and shoots, for instance, many plant pathogens were negatively correlated to BX content. In addition, a co-occurrence analysis of the root microbiome revealed that BXs affected specific groups of the microbiome. Conclusions This study provides insights into the role of BXs for microbial community assembly in the rhizosphere and in roots and shoots. Coupling the quantification of BX metabolites with bacterial and fungal communities, we were able to suggest a gatekeeper role of BX by showing its correlation with specific microbial taxa and thus providing insights into effects on specific fungal and bacterial taxa in maize roots and shoots. Root microbial co-occurrence networks revealed that BXs affect specific microbial clusters. Electronic supplementary material The online version of this article (10.1186/s40168-019-0677-7) contains supplementary material, which is available to authorized users.

Integrated and more sustainable weed management practices are in great demand all around the globe. The adoption of more weed-suppressive cereal cultivars could be part of an integrated weed management strategy. Recently, a study was published analysing the relative contribution of above-ground competition and allelopathy to weed suppression of Scandinavian winter cereals at the field level. The present study used the same approach with Canadian spring wheat and triticale cultivars, and confirmed the results of the previous study. The competitive traits such as leaf area index, crop height and early vigour, and the allelochemicals belonging to the chemical group of benzoxazinoids measured in the root zone were of equal importance to explain the variance of weed biomass at the field level. In addition, a dendogram showed large genetic variability for competitive and allelopathic traits in the Canadian spring cereals, providing the genetic basis for the initiation of breeding programmes for more weed-suppressive cultivars.

Fusarium head blight (FHB) is a devastating disease of wheat (Triticum aestivum L.) caused by a mycotoxigenic fungus Fusarium graminearum resulting in significantly decreased yields and accumulation of toxic trichothecenes in grains. We tested 7 major secondary metabolites from wheat for their effect on trichothecene production in liquid cultures of F. graminearum producing trichothecene 15-acetyldeoxynivalenol (15-ADON). 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) benzoxazinoid completely abolished toxin production without any apparent effect on fungal growth. DIMBOA strongly affected the expression of Tri6, encoding a major transcriptional regulator of several genes of the trichothecene biosynthesis pathway. DIMBOA also repressed expression of Tri5, encoding trichodiene synthase, the first enzyme in the trichothecene biosynthesis pathway. Thus, DIMBOA could play an important role against the accumulation of trichothecenes in wheat grain. Breeding or engineering of wheat with increased levels of benzoxazinoids could provide varieties with increased resistance against trichothecene contamination of grain and lower susceptibility to FHB.

Fusarium head blight (FHB) causes substantial losses of yield and quality in grains, both in the field and in post-harvest storage. To date, adequate natural genetic resistance is not available for the control of FHB. This study reports the cloning and overexpression of a barley (Hordeum vulgare L.) antifungal gene, nepenthesin 1 (HvNEP-1), in the endosperm of barley grains. Transgenic barley lines overexpressing HvNEP-1 substantially reduced FHB severity and disease progression after inoculation with Fusarium graminearum or Fusarium culmorum. The transgenic barley also showed reduced accumulation of the mycotoxin deoxynivalenol (DON) in grain, far below the minimum value allowable for food. Semi-field evaluation of four HvNEP-1 transgenic lines revealed substantial reduction of FHB severity and progression as compared with the control H. vulgare cultivar Golden promise (GP) plants. Our study demonstrated the utility of HvNEP-1 for the control of FHB in barley, and possibly other grains such as wheat and maize.

Secondary metabolites secreted by microbes and plants act as mediators in plant-microbe interactions including nutrient uptake. However, until now very little is known about their role in nutrient assimilation, particularly amino acids, which are important compounds due to their high N content. Here we show that the addition of flavonoid secondary metabolites, derived from clover, to soil changed the bacterial diversity, enhanced the flux of asparagine, and increased the pools of glutamine/glutamate in the soil. This indicates that flavonoids are functionally important qualitative and quantitative components of clover root exudates. Furthermore, the addition of microbial secondary metabolites negatively affected clover uptake of asparagine and plant performance, which demonstrates that microbial competition for nutrients may have multiple physiological targets in the plant. Finally, the detection of intact asparagine in clover roots confirms that amino acid uptake is significant to the plant in agricultural soil. In conclusion, amino-acid flow in the clover rhizosphere can be modified by the effects of clover-derived flavonoids on the bacterial community structure, which affects the flux and pools of amino acids; microbial secondary metabolites, which reduce clover uptake of asparagine; and direct recapture of amino acids by clover.

Neonicotinoids are the most widely applied class of insecticides in cocoa farming in Ghana. Despite the intensive application of these insecticides, knowledge of their fate in the Ghanaian and sub-Saharan African environment remains low. This study examined the behavior of neonicotinoids in soils from cocoa plantations in Ghana by estimating their sorption and degradation using established kinetic models and isotherms. Studies of sorption were conducted using the batch equilibrium method on imidacloprid, thiamethoxam, clothianidin, acetamiprid and thiacloprid, while degradation of imidacloprid, thiamethoxam and their respective deuterated counterparts was studied using models proposed by the European forum for coordination of pesticide fate and their use (FOCUS). Analytes were extracted using the quick, easy, cheap, effective, rugged and safe (QuEChERS) procedure and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Average recoveries were high (≥ 85%) for all analytes. The findings from the study suggest that neonicotinoid insecticides may be persistent in the soils studied based on estimated half-lives150 days. The study also revealed generally low-sorption coefficients for neonicotinoids in soils, largely influenced by soil organic carbon.

Hairy roots induced by Agrobacterium rhizogenes are well established models to study the metabolism of xenobiotics in plants for phytoremediation purposes. However, the model requires special skills and resources for growing and is a time-consuming process. The roots induction process alters the genetic construct of a plant and is known to express genes that are normally absent from the non-transgenic plants. In this study, we propose and establish a non-transgenic maize root model to study xenobiotic metabolism in plants for phytoremediation purpose using azoxystrobin as a xenobiotic compound. Maize roots were grown aseptically in Murashige and Skoog medium for two weeks and were incubated in 100 μM azoxystrobin solution. Azoxystrobin was taken up by the roots to the highest concentration within 15 min of treatment and its phase I metabolites were also detected at the same time. Conjugated metabolites of azoxystrobin were detected and their identities were confirmed by enzymatic and mass spectrometric methods. Further, azoxystrobin metabolites identified in maize root culture were compared against azoxystrobin metabolites in azoxystrobin sprayed lettuce grown in green house. A very close similarity between metabolites identified in maize root culture and lettuce plant was obtained. The results from this study establish that non-transgenic maize roots can be used for xenobiotic metabolism studies instead of genetically transformed hairy roots due to the ease of growing and handling.

Weed suppressive potential of 33 winter wheat, 24 winter rye and 11 winter triticale cultivars recently introduced to the Scandinavian market was investigated. Competitive traits recorded were early vigor, leaf area index and crop height. Allelopathic potential was assessed by analyzing plant root and shoot material by LC-MS/MS for their content of 12 phytotoxic metabolites of the chemical group of benzoxazinoids (BX). Total BX content was highest in rye, followed by triticale and wheat. Benzoxazinoid composition varied between species with non-methoxy substituted BX dominating in rye and methoxy substituted BX dominating in wheat and triticale. Principal component analysis exhibited a clear relationship of allelopathic and competitive traits, together explaining 62% of the variance in the data set. This result underlines the need for further investigations of the relative contribution of allelopathy and competition to weed suppression in future studies.

There is an urgent need for more integrated weed management in commercial cereal production. However, there is gap in the general understanding of crop–weed interactions and the traits contributing to weed suppression by winter cereals. In this research, we measured the above-ground competitive traits crop height, leaf area index and early ground cover and below-ground allelopathic traits by quantification of benzoxazinoid (BX) content in the root zone by HPLC–MS/MS. A partial least squares regression was applied to identify the relative contribution of each trait to explaining the observed variance in weed biomass. The use of three winter cereals, wheat, triticale, and rye enabled us to cover a broad range of weed suppression strategies. For the first time, we were able to demonstrate a positive correlation between BX in plant material and phenoxazinoids in the soil. The highest concentration of BX in the rhizosphere was found at 3–10 cm. Furthermore, differences in crop weed interactions between wheat, rye, and dicotyledonous and monocotyledonous weeds were identified. Interestingly, there was no significant difference between the contribution of any single competitive or allelopathic trait to explain weed biomass. This is valuable information for the establishment of breeding programs for enhanced weed suppression by the cereal crops examined and suggests that an improvement of any of the competitive or allelopathic traits will lead to increased weed suppression. Furthermore, the transfer of traits from rye to wheat via triticale is promising as it may broaden the weed suppressive abilities of wheat.