Combined Effect of Salt Stress and Nitrogen Level on the Primary Metabolism of Two Contrasting Hydroponically Grown Cichorium spinosum L. Ecotypes.

Stamnagathi (Cichorium spinosum L.) is an indigenous plant species well-known for its health-promoting properties. Salinity is a long-term issue with devastating consequences on land and farmers. Nitrogen (N) constitutes a crucial element for plant growth and development (chlorophyll, primary metabolites, etc.). Thus, it is of paramount importance to investigate the impact of salinity and N supply on plants' metabolism. Within this context, a study was conducted aiming to assess the impact of salinity and N stress on the primary metabolism of two contrasting ecotypes of stamnagathi (montane and seaside). Both ecotypes were exposed to three different salinity levels (0.3 mM-non-saline treatment, 20 mM-medium, and 40 mM-high salinity level) combined with two different total-N supply levels: a low-N at 4 mM and a high-N at 16 mM, respectively. The differences between the two ecotypes revealed the variable responses of the plant under the applied treatments. Fluctuations were observed at the level of TCA cycle intermediates (fumarate, malate, and succinate) of the montane ecotype, while the seaside ecotype was not affected. In addition, the results showed that proline (Pro) levels increased in both ecotypes grown under a low N-supply and high salt stress, while other osmoprotectant metabolites such as γ-aminobutyric acid (GABA) exhibited variable responses under the different N supply levels. Fatty acids such as α-linolenate and linoleate also displayed variable fluctuations following plant treatments. The carbohydrate content of the plants, as indicated by the levels of glucose, fructose, α,α-trehalose, and myo-inositol, was significantly affected by the applied treatments. These findings suggest that the different adaptation mechanisms among the two contrasting ecotypes could be strongly correlated with the observed changes in their primary metabolism. This study also suggests that the seaside ecotype may have developed unique adaptation mechanisms to cope with high N supply and salinity stress, making it a promising candidate for future breeding programs aimed at developing stress tolerant varieties of C. spinosum L.

GC/EI/MS and 1H NMR Metabolomics Reveal the Effect of an Olive Tree Endophytic Bacillus sp. Lipopeptide Extract on the Metabolism of Colletotrichum acutatum.

The transition to the Green Deal era requires the discovery of alternative sources of bioactivity and an in-depth understanding of their toxicity to target and non-target organisms. Endophytes have recently emerged as a source of bioactivity of high potential for applications in plant protection, used either per se as biological control agents or their metabolites as bioactive compounds. The olive tree endophytic isolate Bacillus sp. PTA13 produces an array of bioactive lipopeptides (LPs), which additionally exhibit reduced phytotoxicity, features that make them candidates for further research focusing on olive tree plant protection. Here, GC/EI/MS and 1H NMR metabolomics were employed to study the toxicity of a Bacillus sp. PTA13 LP extract on the olive tree pathogen Colletotrichum acutatum, which causes the devastating disease olive anthracnose. The discovery of resistant isolates of the pathogen to the applied fungicides makes the research on the development of improved sources of bioactivity of paramount importance. Analyses revealed that the applied extract affects the metabolism of the fungus by interfering with the biosynthesis of various metabolites and its energy production. LPs had a great impact on the aromatic amino acid metabolism, the energy equilibrium of the fungus and its fatty acid content. Additionally, the applied LPs affected the levels of pathogenesis-related metabolites, a finding that supports their potential for further research as plant protection agents.

Genetic and Metabolite Variability among Commercial Varieties and Advanced Lines of Vicia faba L.

Vicia faba L. (faba bean) is one of the most promising pulse crops due to its nutritional value and high nitrogen fixation capacity. The aim of the present study was to compare the genetic diversity and the seed metabolite profiles of five genetic materials of faba bean. Specifically, three newly developed advanced lines (KK18, KK14 and KK10) and two commercial cultivars (POLIKARPI and TANAGRA), were evaluated for this purpose. Genetic diversity among populations was assessed by SCoT molecular markers. Through UPGMA dendrogram, genetic distances between populations were estimated. Untargeted metabolomics analysis of the seeds was performed employing GC/EI/MS. The cultivar POLYKARPI exhibited the highest polymorphism. All varieties showed a higher within-cultivars and advanced lines variability than between. POLYKARPI and KK14 had the lowest genetic distances, while KK18 and TANAGRA presented the highest ones. The advanced line KK18 displayed the best nutritional profile, the highest concentration of desirable metabolites (lactic acid and trehalose), the lowest concentration of anti-nutritional factors (oxalic acid) and the lowest concentration of saturated fatty acids (palmitic and stearic acid). According to the results of the present study, KK18 line is a very promising material for further exploration and utilization in breeding programs.

A Metabolomic Approach to Assess the Toxicity of the Olive Tree Endophyte Bacillus sp. PTA13 Lipopeptides to the Aquatic Macrophyte Lemna minor L.

Pesticides represent a major human input into the ecosystem, posing a serious risk to non-target organisms. Therefore, there is pressure toward the reduction in their use and the discovery of alternative sources of bioactivity. Endophytic microorganisms represent a source of bioactivity, whose potential for plant protection has been recently established. In this context, an olive tree endophytic Bacillus sp. was isolated, exhibiting superior antifungal activity, mainly attributed to its major surfactin, iturin, and fengycin and the minor gageotetrin and bacilotetrin groups of lipopeptides (LP). Based on the potential of LP and the lack of information on their toxicity to aquatic organisms, we have investigated the toxicity of an LP extract to the model macrophyte Lemna minor L. The extract exhibited low phytotoxicity (EC50 = 419 µg·mL-1), and for the investigation of its effect on the plant, GC/EI/MS metabolomics was applied following exposure to sub-lethal doses (EC25 and EC50). Results revealed a general disturbance of plants' biosynthetic capacity in response to LP treatments, with substantial effect on the amino acid pool and the defense mechanism regulated by jasmonate. There are no previous reports on the phytotoxicity of LP to L. minor, with evidence supporting their improved toxicological profile and potential in plant protection.

Discovering the Next-Generation Plant Protection Products: A Proof-of-Concept via the Isolation and Bioactivity Assessment of the Olive Tree Endophyte Bacillus sp. PTA13 Lipopeptides.

Endophytic microorganisms (EMs) have recently attracted interest for applications in plant protection, mainly due to their bioactive compound-producing capacity. Therefore, we underwent the task of isolating olive tree EMs and investigating their bioactivity against the devastating pathogen Colletotrichum acutatum. Several EMs were isolated; however, the Bacillus sp. PTA13 isolate exhibited the highest toxicity to the phytopathogen. Bacteria of the genus Bacillus exhibit superior bioactive metabolite-producing capacity, with the lipopeptides (LPs) of surfactin, iturin, and fengycin groups being the most studied. A total LP extract and several fractions were obtained, and their bioactivity was assessed against C. acutatum strains. LPs of the major surfactin, iturin, and fengycin groups and the minor gageotetrin and bacilotetrin groups were annotated. The results confirmed the bioactivity of the major LPs, with fengycins being the most fungitoxic. Interestingly, the minor LP fraction exhibited selective toxicity to the fungicide-resistant C. acutatum isolate, an observation that highlights the significance of our approach to comprehensively mine the total LP extract. This work represents a proof of concept of the exploitation of EMs in customized olive tree plant protection and aligns well with strategies that focus on the sustainability and safety of food production via the development of next-generation plant protection products.

Metabolomics reveals differential mechanisms of toxicity of hyperbranched poly(ethyleneimine)-derived nanoparticles to the soil-borne fungus Verticillium dahliae Kleb.

There is a consensus on the urge for the discovery and assessment of alternative, improved sources of bioactivity that could be developed as plant protection products (PPPs), in order to combat issues that the agrochemical sector is facing. Based on the recent advances in nanotechnology, nanoparticles seem to have a great potential towards the development of the next generation nano-PPPs used as active ingredients (a.i.) per se or as nanocarriers in their formulation. Nonetheless, information on their mode(s)-of-action (MoA) and mechanisms of toxicity is yet largely unknown, representing a bottleneck in their further assessment and development. Therefore, we have undertaken the task to assess the fungitoxicity of hyperbranched poly(ethyleneimine) (HPEI), quaternized hyperbranched poly(ethyleneimine) (QPEI), and guanidinylated hyperbranched poly(ethyleneimine) (GPEI) nanoparticles to the soil-born plant pathogenic fungus Verticillium dahliae Kleb, and dissect their effects on its metabolism applying GC/EI/MS metabolomics. Results revealed that functionalization of HPEI nanoparticles with guanidinium end groups (GPEI) increases their toxicity to V. dahliae, while functionalization with quaternary ammonium end groups (QPEI) decreases it. The treatments with the nanoparticles affected the chemical homeostasis of the fungus, altering substantially its amino acid pool, energy production, and fatty acid content, causing additionally oxidative and osmotic stresses. To the best of our knowledge, this is the first report on the comparative toxicity of HPEI, QPEI, and GPEI to filamentous fungi applying metabolomics. The findings could be exploited in the study of the quantitative structure-activity relationship (QSAR) of HPEI-derived nanoparticles and their further development as nano-PPPs.