The dissipation of organophosphate esters mediated by ryegrass root exudate oxalic acid in soil: Analysis of enzymes activities, microorganism.

Complex rhizoremediation is the main mechanism of phytoremediation in organic-contaminated soil. Low molecular weight organic acids (LMWOAs) in root exudates have been shown to increase the bioavailability of contaminants and are essential for promoting the dissipation of contaminants. The effects of root exudates on the dissipation of organophosphate esters (OPEs) in soil are unclear. Consequently, we studied the combined effects of root exudates, soil enzymes and microorganisms on OPEs (tri (1-chloro-2-propyl) phosphate (TCPP) and triphenyl phosphate (TPP)) dissipation through pot experiments. Oxalic acid (OA) was confirmed to be the main component of LMWOAs in root exudates of ryegrass. The existence of OA increased the dissipation rate of OPEs by 6.04%-25.50%. Catalase and dehydrogenase activities were firstly activated and then inhibited in soil. While, urease activity was activated and alkaline phosphatase activity was inhibited during the exposure period. More bacteria enrichment (e.g., Sphingomonas, Pseudomonas, Flavisolibacter, Pontibacter, Methylophilus and Massilia) improved the biodegradation of OPEs. In addition, the transformation paths of OPEs hydrolysis and methylation under the action of root exudates were observed. This study provided theoretical insights into reducing the pollution risk of OPEs in the soil.

Plant seedlings of peas, tomatoes, and cucumbers exude compounds that are needed for growth and chemoattraction of Rhizobium leguminosarum bv. viciae 3841 and Azospirillum brasilense Sp7.

This study characterizes seedling exudates of peas, tomatoes, and cucumbers at the level of chemical composition and functionality. A plant experiment confirmed that Rhizobium leguminosarum bv. viciae 3841 enhanced growth of pea shoots, while Azospirillum brasilense Sp7 supported growth of pea, tomato, and cucumber roots. Chemical analysis of exudates after 1 day of seedling incubation in water yielded differences between the exudates of the three plants. Most remarkably, cucumber seedling exudate did not contain detectable sugars. All exudates contained amino acids, nucleobases/nucleosides, and organic acids, among other compounds. Cucumber seedling exudate contained reduced glutathione. Migration on semi solid agar plates containing individual exudate compounds as putative chemoattractants revealed that R. leguminosarum bv. viciae was more selective than A. brasilense, which migrated towards any of the compounds tested. Migration on semi solid agar plates containing 1:1 dilutions of seedling exudate was observed for each of the combinations of bacteria and exudates tested. Likewise, R. leguminosarum bv. viciae and A. brasilense grew on each of the three seedling exudates, though at varying growth rates. We conclude that the seedling exudates of peas, tomatoes, and cucumbers contain everything that is needed for their symbiotic bacteria to migrate and grow on.

Vanillin in Resistant Tomato Plant Root Exudate Suppresses Meloidogyne incognita Parasitism.

Tomato (Solanum lycopersicum) plants are susceptible to infection by root-knot nematodes, which cause severe economic losses. Planting resistant tomato plants can reduce nematode damage; however, the effects of resistant tomato root exudates in suppressing Meloidogyne incognita remain insufficiently understood. Here, we determined that the resistant tomato plant Lycopersicon esculentum cv. Xianke-8 (XK8) alleviates nematode damage by downregulating the expression of the essential parasitic nematode gene Mi-flp-18 to reduce the infection and reproduction of M. incognita. Using gas chromatography-mass spectrometry, we identified vanillin as a unique compound (compared to susceptible tomato cultivars) in XK8 root exudates that acts as a lethal trap and inhibitor of egg hatching. Moreover, the soil application of 0.4-4.0 mmol/kg vanillin significantly reduced galls and egg masses. The parasite gene Mi-flp-18 was downregulated upon treatment with vanillin, both in vitro and in pot experiments. Collectively, our results reveal an effective nematicidal compound that can use in feasible and economical strategies to control RKNs.

Development of a root exudate collection protocol for metabolomics analysis using Nuclear Magnetic Resonance.

Large amounts of root exudates are released by plant roots into the soil. Due to their importance in regulating the rhizosphere properties, it is necessary to unravel the precise composition and function of exudates at the root-soil interface. However, obtaining root exudates without inducing artefacts is a difficult task. To analyse the low molecular weight molecules secreted by pea roots, a protocol of root exudate collection was developed to perform a metabolomics analysis using Nuclear Magnetic Resonance (NMR). To date a few NMR studies are dedicated to root exudates. Plant culture, exudates collection and sample preparation methods had thus to be adapted to the NMR approach. Here, pea seedlings were hydroponically grown. The obtained NMR fingerprints show that osmotic stress increases the quantity of the exudates but not their diversity. We therefore selected a protocol reducing the harvest time and using an ionic solvent and applied it to the analysis of faba bean exudates. NMR analysis of the metabolic profiles allowed to discriminate between pea and faba bean according to their exudate composition. This protocol is therefore very promising for studying the composition of root exudates from different plant species as well as their evolution in response to different environmental conditions or pathophysiological events.

Interspecific plant interaction via root exudates structures the disease suppressiveness of rhizosphere microbiomes.

Terrestrial plants can affect the growth and health of adjacent plants via interspecific interaction. Here, we studied the mechanism by which plant root exudates affect the recruitment of the rhizosphere microbiome in adjacent plants-with implications for plant protection-using a tomato (Solanum lycopersicum)-potatoonion (Allium cepa var. agrogatum) intercropping system. First, we showed that the intercropping system results in a disease-suppressive rhizosphere microbiome that protects tomato plants against Verticillium wilt disease caused by the soilborne pathogen Verticillium dahliae. Second, 16S rRNA gene sequencing revealed that intercropping with potatoonion altered the composition of the tomato rhizosphere microbiome by promoting the colonization of specific Bacillus sp. This taxon was isolated and shown to inhibit V. dahliae growth and induce systemic resistance in tomato plants. Third, a belowground segregation experiment found that root exudates mediated the interspecific interaction between potatoonion and tomato. Moreover, experiments using split-root tomato plants found that root exudates from potatoonion, especially taxifolin-a flavonoid compound-stimulate tomato plants to recruit plant-beneficial bacteria, such as Bacillus sp. Lastly, ultra-high-pressure liquid chromatography-mass spectrometry analysis found that taxifolin alters tomato root exudate chemistry; thus, this compound acts indirectly in modulating root colonization by Bacillus sp. Our results revealed that this intercropping system can improve tomato plant fitness by changing rhizosphere microbiome recruitment via the use of signaling chemicals released by root exudates of potatoonion. This study revealed a novel mechanism by which interspecific plant interaction modulates the establishment of a disease-suppressive microbiome, thus opening up new avenues of research for precision plant microbiome manipulations.

Disentangling the chemistry of Australian plant exudates from a unique historical collection.

For thousands of years, the unique physicochemical properties of plant exudates have defined uses in material culture and practical applications. Native Australian plant exudates, including resins, kinos, and gums, have been used and continue to be used by Aboriginal Australians for numerous technical and cultural purposes. A historic collection of well-preserved native Australian plant exudates, assembled a century ago by plant naturalists, gives a rare window into the history and chemical composition of these materials. Here we report the full hierarchical characterization of four genera from this collection, Xanthorrhoea, Callitris, Eucalyptus, and Acacia, from the local elemental speciation, to functional groups and main molecular markers. We use high-resolution X-ray Raman spectroscopy (XRS) to achieve bulk-sensitive chemical speciation of these plant exudates, including insoluble, amorphous, and cross-linked fractions, without the limitation of invasive and/or surface specific methods. Combinatorial testing of the XRS data allows direct classification of these complex natural species as terpenoid, aromatic, phenolic, and polysaccharide materials. Differences in intragenera chemistry was evidenced by detailed interpretation of the XRS spectral features. We complement XRS with Fourier-transform infrared (FT-IR) spectroscopy, gas chromatography­mass spectrometry (GC-MS), and pyrolysis­GC-MS (Py-GC-MS). This multimodal approach provides a fundamental understanding of the chemistry of these natural materials long used by Aboriginal Australian peoples.

Time point- and plant part-specific changes in phloem exudate metabolites of leaves and ears of wheat in response to drought and effects on aphids.

Alterations in the frequency and intensity of drought events are expected due to climate change and might have consequences for plant metabolism and the development of plant antagonists. In this study, the responses of spring wheat (Triticum aestivum) and one of its major pests, the aphid Sitobion avenae, to different drought regimes were investigated, considering different time points and plant parts. Plants were kept well-watered or subjected to either continuous or pulsed drought. Phloem exudates were collected twice from leaves and once from ears during the growth period and concentrations of amino acids, organic acids and sugars were determined. Population growth and survival of the aphid S. avenae were monitored on these plant parts. Relative concentrations of metabolites in the phloem exudates varied with the time point, the plant part as well as the irrigation regime. Pronounced increases in relative concentrations were found for proline, especially in pulsed drought-stressed plants. Moreover, relative concentrations of sucrose were lower in phloem exudates of ears than in those of leaves. The population growth and survival of aphids were decreased on plants subjected to drought and populations grew twice as large on ears compared to leaves. Our study revealed that changes in irrigation frequency and intensity modulate plant-aphid interactions. These effects may at least partly be mediated by changes in the metabolic composition of the phloem sap.

Characterization of Phenolic Plant Exudates by Nuclear Magnetic Resonance Spectroscopy.

The class of plant exudates that contain the phenol functionality, termed phenolics, is defined, surveyed, and characterized by solid-state 13C NMR spectroscopy and by solution-state 1H NMR spectroscopy. Materials in this group are identified by the phenolic 13C resonance (from the ipso carbon of ArOH) at δ 145-160 (δ 160-167 for ArOR). The resonance patterns define several subclasses based on the collective similarity of their 13C spectra, specifically, aloetics from the genus Aloe, guaiacs from the genus Guaiacum and other eurosid and conifer genera, xanthics from the genus Garcinia, and kinos from the genus Eucalyptus and many other genera. Phenolic exudates often are mixed with terpenoid materials (the building block of exudates known as resins) and carbohydrates (the building block of exudates known as gums) to form hybrid subgroups such as guaiac gums, guaiac resins, and kino resins. There are numerous phenolic exudates not affiliated with any of these groups, both as pure phenolics and as hybrids (phenolic resins, phenolic gum resins, and phenolic waxes).

Evaluation and Quantification of Natural Strigolactones from Root Exudates.

Strigolactones (SLs) in the root exudates can be detected by germination assays with root parasitic weed seeds, but precise and accurate evaluation and quantification are possible only by chemical analysis with the liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here we describe methods for root exudate collection, sample preparation, and LC-MS/MS analysis of SLs.

Isolation and Identification of Naturally Occurring Strigolactones.

The accurate structure determination of strigolactones (SLs) that are produced by plants leads to the precise understanding of the biosynthesis and functions of their molecules. SLs need to be isolated and purified from the plant roots or root exudates in a hydroponic solution using appropriate methods in order to determine the structures. In this chapter, we describe a small-scale extraction method for chromatographic analysis of known SLs and a large-scale purification method for isolation of unknown SLs, together with methods for the hydroponic culture of plants and collection of root exudates. Finally, we present spectroscopic data that are helpful in identifying SLs.

Mass spectrometry-based quantification and spatial localization of small organic acid exudates in plant roots under phosphorus deficiency and aluminum toxicity.

Low-molecular-weight organic acid (OA) extrusion by plant roots is critical for plant nutrition, tolerance to cations toxicity, and plant-microbe interactions. Therefore, methodologies for the rapid and precise quantification of OAs are necessary to be incorporated in the analysis of roots and their exudates. The spatial location of root exudates is also important to understand the molecular mechanisms directing OA production and release into the rhizosphere. Here, we report the development of two complementary methodologies for OA determination, which were employed to evaluate the effect of inorganic ortho-phosphate (Pi) deficiency and aluminum toxicity on OA excretion by Arabidopsis roots. OA exudation by roots is considered a core response to different types of abiotic stress and for the interaction of roots with soil microbes, and for decades has been a target trait to produce plant varieties with increased capacities of Pi uptake and Al tolerance. Using targeted ultra-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UPLC-HRMS/MS), we achieved the quantification of six OAs in root exudates at sub-micromolar detection limits with an analysis time of less than 5 min per sample. We also employed targeted (MS/MS) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to detect the spatial location of citric and malic acid with high specificity in roots and exudates. Using these methods, we studied OA exudation in response to Al toxicity and Pi deficiency in Arabidopsis seedlings overexpressing genes involved in OA excretion. Finally, we show the transferability of the MALDI-MSI method by analyzing OA excretion in Marchantia polymorpha gemmalings subjected to Pi deficiency.

In vitro assessment of the efficacy of protein exudates from seeds against Haemonchus contortus.

Nematodes develop resistance to the most common commercially available drugs. The aim of this study was to identify and evaluate the action of protein exudates from Mimosa caesalpiniifolia, Leucaena leucocephala, Acacia mangium, and Stylosanthes capitata seeds on the gastrointestinal nematode Haemonchus contortus. The exuded proteins were precipitated, dialyzed, lyophilized, and assessed for their effect on egg hatching and artificial larval exsheathment inhibition. Proteome analysis of the protein extracts was also performed. Although no egg-hatching inhibition was observed, all exudates showed efficacy in inhibiting the larval exsheathment of H. contortus larvae with an EC50 varying from 0.61 to 0.26 mg P mL-1. Proteomic analysis revealed the presence of proteases, protease inhibitors, chitinases, and lectins among other proteins in the exudates. Most of the exuded proteins belong to the oxidative stress/plant defense and energy/carbohydrate metabolism functional clusters. This study concluded that the bioactive proteins from different classes exuded by seeds of M. caesalpiniifolia, L. leucocephala, A. mangium, and S. capitata show stage-specific inhibition against H. contortus.

Activity of Adesmia boronioides resinous exudate against phytopathogenic bacteria.

Resinous exudate obtained from the aerial parts of Adesmia boronioides Hook.f. were evaluated to determine anti-phytopathogenic effects. Briefly, resinous exudate was obtained by dipping fresh plant material in dichloromethane; chemical composition was determined by GC-MS; and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated against four phytopathogenic bacteria. Resinous exudate yield was 8.5% (resin/fresh plant), of which esquel-6-en-9-one (14.25%), esquel-7-en-9-one (5.86%), and veratric acid (2.59%) were the effective antibacterial compounds. Tested against Pectobacterium carotovorum subsp. carotovora, Erwinia amylovora, Bacillus subtilis, and Pseudomonas syringae, MICs and MBCs ranged from 16 to 128 µg/mL and 32-256 µg/mL, respectively. These results provide initial evidence that resinous bush A. boronioides is a new and alternative source of substances with agricultural interest.

Effects of root morphology, respiration and carboxylate exudation on carbon economy in two non-mycorrhizal lupines under phosphorus deficiency.

Under phosphorus (P) deficiency, Lupinus albus develops cluster roots that allow efficient P acquisition, while L. angustifolius without cluster roots also grows well. Both species are non-mycorrhizal. We quantitatively examined the carbon budgets to investigate the different strategies of these species. Biomass allocation, respiratory rates, protein amounts and carboxylate exudation rates were examined in hydroponically-grown plants treated with low (1 µM; P1) or high (100 µM; P100) P. At P1, L. albus formed cluster roots, and L. angustifolius increased biomass allocation to the roots. The respiratory rates of the roots were faster in L. albus than in L. angustifolius. The protein amounts of the non-phosphorylating alternative oxidase and uncoupling protein were greater in the cluster roots of L. albus at P1 than in the roots at P100, but similar between the P treatments in L. angustifolius roots. At P1, L. albus exuded carboxylates at a faster rate than L. angustifolius. The carbon budgets at P1 were surprisingly similar between the two species, which is attributed to the contrasting root growth and development strategies. L. albus developed cluster roots with rapid respiratory and carboxylate exudation rates, while L. angustifolius developed a larger root system with slow respiratory and exudation rates.

Root exudates drive soil-microbe-nutrient feedbacks in response to plant growth.

Although interactions between plants and microbes at the plant-soil interface are known to be important for plant nutrient acquisition, relatively little is known about how root exudates contribute to nutrient exchange over the course of plant development. In this study, root exudates from slow- and fast-growing stages of Arabidopsis thaliana plants were collected, chemically analysed and then applied to a sandy nutrient-depleted soil. We then tracked the impacts of these exudates on soil bacterial communities, soil nutrients (ammonium, nitrate, available phosphorus and potassium) and plant growth. Both pools of exudates shifted bacterial community structure. GeoChip analyses revealed increases in the functional gene potential of both exudate-treated soils, with similar responses observed for slow-growing and fast-growing plant exudate treatments. The fast-growing stage root exudates induced higher nutrient mineralization and enhanced plant growth as compared to treatments with slow-growing stage exudates and the control. These results suggest that plants may adjust their exudation patterns over the course of their different growth phases to help tailor microbial recruitment to meet increased nutrient demands during periods demanding faster growth.

The effect of tree age, daily sap volume and date of sap collection on the content of minerals and heavy metals in silver birch (Betula pendula Roth) tree sap.

The aim of this study was to determine the effect of the age of trees, daily sap volume as well as the term of tapping birch sap collected in the forest environment on the content of selected minerals (zinc, copper and manganese) and heavy metals (lead, nickel, chromium and cadmium). The study was performed on material taken from two stands (aged 34 and 84 years) in a moist broadleaved forest habitat with a dominant share of silver birch (Betula pendula Roth). The research results confirmed the presence of both nutritional essential minerals and hazardous heavy metals in the birch sap. At the same time, the content of minerals and heavy metals was found to be very variable and the differences between their concentrations, recorded on the same day of collecting in several trees of the same age group, can be even several dozen times higher. Depending on the examined elements, the factors influencing their content vary. The age of the trees determines only the manganese content; daily sap volume significantly affects the content of manganese and copper, and date of collection differentiates the content of zinc, lead, nickel and cadmium. The results may be interesting in the context of developing procedures for collecting birch sap for the purpose of obtaining raw material with beneficial nutritional values and a high level of health safety. For this reason, our recommendation for guaranteeing the health safety and high nutritional value of birch sap is to combine batches of raw material taken from as many trees as possible, and at the same time to publicize the fact that collecting birch sap from just one single tree may result in a raw material that is both dangerous and has no nutritional benefits.

Root-Exuded Benzoxazinoids: Uptake and Translocation in Neighboring Plants.

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-ß-d-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-glc) and 2-ß-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.

Constituents Leached by Tomato Seeds Regulate the Behavior of Root-Knot Nematodes and Their Antifungal Effects against Seed-Borne Fungi.

Germinating seeds can release diverse phytochemicals that repel, inhibit, or kill pathogens such as root-knot nematodes and seed-borne fungi. However, little is known about the composition of these phytochemicals and their effects on pathogens. In this study, we demonstrated that tomato seed exudates can attract the nematode Meloidogyne incognita using a dual-choice assay. Eighteen compounds were then isolated and identified from the exudates. Of these, esters (1-3), fatty acids (4-6), and phenolic acids (10-12) were proven to be the signaling molecules that facilitated the host-seeking process of second-stage juveniles (J2s) of nematodes, while alkaloids (17 and 18) disrupted J2s in locating their host. Furthermore, some phenolic acids and alkaloids showed antifungal effects against seed-borne fungi. In particular, ferulic acid (12) showed obvious activity against Aspergillus flavus (minimum inhibitory concentration (MIC), 32 µg/mL), while dihydrocapsaicin (17) showed noticeable activity against Fusarium oxysporum (MIC, 16 µg/mL). Overall, this study presents the first evidence that M. incognita can be attracted to or deterred by various compounds in seed exudates through identification of the structures of the compounds in the exudates and analysis of their effects on nematodes. Furthermore, some antifungal compounds were also found. The findings of this work suggest that seed exudates are new source for finding insights into the development of plant protective substances with nematocidal and antifungal effects.

Metabolomics reveals chemical changes in Acer saccharum sap over a maple syrup production season.

Maple syrup, made by boiling the sap of Acer saccharum, is an important agriculture commodity in eastern Canada and New England. Although the collection season is relatively short, a rich progression in the sensory qualities of maple syrup can occur throughout the season. A risk associated with maple syrup production at the end of a season is the development of off-flavors that result in syrup with little to no commercial value. Maple syrup producers in Canada and the USA call this 'buddy syrup'. In this study, sugar maple (Acer saccharum) sap was collected in sequential samples through the harvest season from stands across Ontario. Metabolomics analysis of the sap samples was performed by high-resolution mass spectrometry. This revealed an evolution of the chemical composition, mainly occurring 30 days prior to leaf emergence. The major chemical constituent of maple syrup, sucrose, decreased sharply in late season sap, driven by microbial activity. The alditol mannitol increased in late season sap to concentrations ≥2 mg/mL and is likely an indicator of the start of photosynthesis. Amino acids, notably methionine and asparagine were present in higher amounts in late season sap. Non-targeted analysis revealed a series of related compounds that contained quaternary ammonium moieties including choline, hercynine, trigonelline, glycine betaine and carnitine increased in late season sap. These classes of compounds could act as methyl donors during the heating/evaporation of sap into syrup, affecting taste. Based on descriptions of the nature of buddy syrup and an extensive literature on flavors in foods, the amino acids methionine and asparagine were found as likely precursors to the compounds responsible for buddy syrup.