Untargeted metabolomics as a hypothesis-generation tool in plant protection product discovery: Highlighting the potential of trehalose and glycerol metabolism of fungal conidiospores as novel targets.

INTRODUCTION: The production of high quality and safe food represents a main priority for the agri-food sector in the effort to sustain the exponentially growing human population. Nonetheless, there are major challenges that require the discovery of new, alternative, and improved plant protection products (PPPs). Focusing on fungal plant pathogens, the dissection of mechanisms that are essential for their survival provides insights that could be exploited towards the achievement of the aforementioned aim. In this context, the germination of fungal spores, which are essential structures for their dispersal, survival, and pathogenesis, represents a target of high potential for PPPs. To the best of our knowledge, no PPPs that target the germination of fungal spores currently exist. OBJECTIVES: Within this context, we have mined for changes in the metabolite profiles of the model fungus Aspergillus nidulans FGSC A4 conidiospores during germination, in an effort to discover key metabolites and reactions that could potentially become targets of PPPs. METHODS: Untargeted GC/EI-TOF/MS metabolomics and multivariate analyses were employed to monitor time-resolved changes in the metabolomes of germinating A. nidulans conidiospores. RESULTS: Analyses revealed that trehalose hydrolysis plays a pivotal role in conidiospore germination and highlighted the osmoregulating role of the sugar alcohols, glycerol, and mannitol. CONCLUSION: The ineffectiveness to introduce active ingredients that exhibit new mode(s)-of-action as fungicides, dictates the urge for the discovery of PPPs, which could be exploited to combat major plant protection issues. Based on the crucial role of trehalose hydrolysis in conidiospore dormancy breakage, and the subsequent involvement of glycerol in their germination, it is plausible to suggest their biosynthesis pathways as potential novel targets for the next-generation antifungal PPPs. Our study confirmed the applicability of untargeted metabolomics as a hypothesis-generation tool in PPPs' research and discovery.

The yeast (Saccharomyces cerevisiae) YCF1 vacuole transporter: Evidence on its implication into the yeast resistance to flusilazole as revealed by GC/EI/MS metabolomics.

The development of plant protection product (PPPs)-resistant populations of plant pathogens, pests, and weeds, represents a major challenge that the crop protection sector is facing. Focusing on plant pathogenic fungi, the increased efflux of the active ingredients (a.i.) from the cytoplasm is highly correlated to elevated resistance levels to the applied fungicides. Such mechanism is regulated by ATP-binding cassette transporters (ABC transporters), and although it has been investigated for the past two decades, the latest developments in "omics" technologies could provide new insights with potential applications in crop protection. Within this context, and based on results from preliminary experiments, we have undertaken the task of mining the involvement of the ABC transporter YCF1, which is located in the vacuole membrane, in the fungicide resistance development, applying a functional genomics approach and using yeast (Saccharomyces cerevisiae) as the model organism. Among the fungicides being assessed, flusilazole, which belongs to the azole group of dimethylation inhibitors (DMIs), was discovered as a possible substrate of the YCF1. GC/EI/MS metabolomics analysis revealed the effect of the fungicide's toxicity and that of genotype on yeast's metabolism, confirming the role of this transporter. Fluctuations in the activity of various yeast biosynthetic pathways associated with stress responses were recorded, and corresponding metabolites-biomarkers of flusilazole toxicity were discovered. The metabolites α,α-trehalose, glycerol, myo-inositol-1-phosphate, GABA, l-glutamine, l-tryptophan, l-phenylalanine, l-tyrosine, and phosphate, were the major identified biomarkers of toxicity. Among these, are metabolites that play important roles in fungal metabolism (e.g., cell responses to osmotic stress) or serve as signaling molecules. To the best of our knowledge, this is the first report on the implication of YCF1 in fungal resistance to PPPs. Additionally, the results of GC/EI/MS yeast metabolomics confirmed the robustness of the method and its applicability in the high-throughput study of fungal resistance to fungicides.

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.

Multiple nucleobase transporters contribute to boscalid sensitivity in Aspergillus nidulans.

The development of fungicide-resistant fungal populations represents a major challenge for the agrochemical and agri-food sectors, which threatens food supply and security. The issue becomes complex for fungi that cause quantitative and qualitative losses due to mycotoxin biosynthesis. Nonetheless, currently, the molecular details underlying fungicide action and fungal resistance mechanisms are partially known. Here, we have investigated whether plasma membrane transporters contribute to specific fungicide uptake in the model fungus Aspergillus nidulans. Independent physiological tests and toxicity screening of selected fungicides provided evidence that the antifungal activity of Succinate Dehydrogenase Inhibitors (SDHIs) is associated with the expression of several nucleobase-related transporters. In particular, it was shown that a strain genetically inactivated in all seven nucleobase-related transporters is resistant to the fungicide boscalid, whereas none of the single null mutants exhibited significant resistance level. By constructing and testing isogenic strains that over-express each one of the seven transporters, we confirmed that five of them, namely, UapC, AzgA, FycB, CntA, and FurA, contribute to boscalid uptake. Additionally, by employing metabolomics we have examined the effect of boscalid on the metabolism of isogenic strains expressing or genetically lacking boscalid-related nucleobase transporters. The results confirmed the involvement of specific nucleobase transporters in fungicide uptake, leading to the discovery of corresponding metabolites-biomarkers. This work is the first report on the involvement of specific transporters in fungicide uptake and toxicity and their impact on fungal metabolism regulation and results might be further exploited towards the deeper understanding of fungal resistance to fungicides.

An integrated RNAseq-1H NMR metabolomics approach to understand soybean primary metabolism regulation in response to Rhizoctonia foliar blight disease.

BACKGROUND: Rhizoctonia solani AG1-IA is a devastating phytopathogen causing Rhizoctonia foliar blight (RFB) of soybean worldwide with yield losses reaching 60%. Plant defense mechanisms are complex and information from different metabolic pathways is required to thoroughly understand plant defense regulation and function. Combining information from different "omics" levels such as transcriptomics, metabolomics, and proteomics is required to gain insights into plant metabolism and its regulation. As such, we studied fluctuations in soybean metabolism in response to R. solani infection at early and late disease stages using an integrated transcriptomics-metabolomics approach, focusing on the regulation of soybean primary metabolism and oxidative stress tolerance. RESULTS: Transcriptomics (RNAseq) and metabolomics (1H NMR) data were analyzed individually and by integration using bidirectional orthogonal projections to latent structures (O2PLS) to reveal possible links between the metabolome and transcriptome during early and late infection stages. O2PLS analysis detected 516 significant transcripts, double that reported in the univariate analysis, and more significant metabolites than detected in partial least squares discriminant analysis. Strong separation of treatments based on integration of the metabolomes and transcriptomes of the analyzed soybean leaves was revealed, similar trends as those seen in analyses done on individual datasets, validating the integration method being applied. Strong fluctuations of soybean primary metabolism occurred in glycolysis, the TCA cycle, photosynthesis and photosynthates in response to R. solani infection. Data were validated using quantitative real-time PCR on a set of specific markers as well as randomly selected genes. Significant increases in transcript and metabolite levels involved in redox reactions and ROS signaling, such as peroxidases, thiamine, tocopherol, proline, L-alanine and GABA were also recorded. Levels of ethanol increased 24 h post-infection in soybean leaves, and alcohol dehydrogenase (ADH) loss-of-function mutants of Arabidopsis thaliana had higher necrosis than wild type plants. CONCLUSIONS: As a proof-of-concept, this study offers novel insights into the biological correlations and identification of candidate genes and metabolites that can be used in soybean breeding for resistance to R. solani AG1-IA infection. Additionally, these findings imply that alcohol and its associated gene product ADH may have important roles in plant resistance to R. solani AG1-IA causing foliar blight.

Maple Bark Biochar Affects Rhizoctonia solani Metabolism and Increases Damping-Off Severity.

Many studies have investigated the effect of biochar on plant yield, nutrient uptake, and soil microbial populations; however, little work has been done on its effect on soilborne plant diseases. To determine the effect of maple bark biochar on Rhizoctonia damping-off, 11 plant species were grown in a soilless potting substrate amended with different concentrations of biochar and inoculated or not with Rhizoctonia solani anastomosis group 4. Additionally, the effect of biochar amendment on R. solani growth and metabolism in vitro was evaluated. Increasing concentrations of maple bark biochar increased Rhizoctonia damping-off of all 11 plant species. Using multivariate analyses, we observed positive correlations between biochar amendments, disease severity and incidence, abundance of culturable bacterial communities, and physicochemical parameters. Additionally, biochar amendment significantly increased R. solani growth and hyphal extension in vitro, and altered its primary metabolism, notably the mannitol and tricarboxylic acid cycles and the glycolysis pathway. One or several organic compounds present in the biochar, as identified by gas chromatography-mass spectrometry analysis, may be metabolized by R. solani. Taken together, these results indicate that future studies on biochar should focus on the effect of its use as an amendment on soilborne plant pathogens before applying it to soils.

Diagnostic exome sequencing to elucidate the genetic basis of likely recessive disorders in consanguineous families.

Rare, atypical, and undiagnosed autosomal-recessive disorders frequently occur in the offspring of consanguineous couples. Current routine diagnostic genetic tests fail to establish a diagnosis in many cases. We employed exome sequencing to identify the underlying molecular defects in patients with unresolved but putatively autosomal-recessive disorders in consanguineous families and postulated that the pathogenic variants would reside within homozygous regions. Fifty consanguineous families participated in the study, with a wide spectrum of clinical phenotypes suggestive of autosomal-recessive inheritance, but with no definitive molecular diagnosis. DNA samples from the patient(s), unaffected sibling(s), and the parents were genotyped with a 720K SNP array. Exome sequencing and array CGH (comparative genomic hybridization) were then performed on one affected individual per family. High-confidence pathogenic variants were found in homozygosity in known disease-causing genes in 18 families (36%) (one by array CGH and 17 by exome sequencing), accounting for the clinical phenotype in whole or in part. In the remainder of the families, no causative variant in a known pathogenic gene was identified. Our study shows that exome sequencing, in addition to being a powerful diagnostic tool, promises to rapidly expand our knowledge of rare genetic Mendelian disorders and can be used to establish more detailed causative links between mutant genotypes and clinical phenotypes.

Plant Biostimulants Enhance Tomato Resilience to Salinity Stress: Insights from Two Greek Landraces.

Salinity, one of the major abiotic stresses in plants, significantly hampers germination, photosynthesis, biomass production, nutrient balance, and yield of staple crops. To mitigate the impact of such stress without compromising yield and quality, sustainable agronomic practices are required. Among these practices, seaweed extracts (SWEs) and microbial biostimulants (PGRBs) have emerged as important categories of plant biostimulants (PBs). This research aimed at elucidating the effects on growth, yield, quality, and nutrient status of two Greek tomato landraces ('Tomataki' and 'Thessaloniki') following treatments with the Ascophyllum nodosum seaweed extract 'Algastar' and the PGPB 'Nitrostim' formulation. Plants were subjected to bi-weekly applications of biostimulants and supplied with two nutrient solutions: 0.5 mM (control) and 30 mM NaCl. The results revealed that the different mode(s) of action of the two PBs impacted the tolerance of the different landraces, since 'Tomataki' was benefited only from the SWE application while 'Thessaloniki' showed significant increase in fruit numbers and average fruit weight with the application of both PBs at 0.5 and 30 mM NaCl in the root zone. In conclusion, the stress induced by salinity can be mitigated by increasing tomato tolerance through the application of PBs, a sustainable tool for productivity enhancement, which aligns well with the strategy of the European Green Deal.

Characterization and transcriptional regulation of Stachybotrys elegans mitogen-activated-protein kinase gene smkA following mycoparasitism and starvation conditions.

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in the development and conidiation of fungal pathogens on their hosts and the sensing of host-derived cues. Mycoparasitism is a fungus-fungus interaction comprising host-pathogen cross talk. Until now, only little information is available on the role of the MAPK signaling pathway during this interaction. Here, we report on the differential expression of a MAPK/ERK gene in the mycoparasite Stachybotrys elegans in response to direct parasitism of different vegetative structures of the plant pathogen Rhizoctonia solani (i.e., carbon-rich condition) and to nutrient starvation (i.e., carbon-poor condition). Western blot analysis against ERK1/2 highlighted an increase in their phosphorylated forms when S. elegans was grown under starvation condition compared to that detected in response to mycoparasitism. A higher abundance of phosphorylated ERK1/2 at the third day of interaction compared to that estimated under starvation condition was detected applying LC-MS/MS. At the transcriptional level, smkA, a YERK1 class member, was significantly induced in response to hyphal parasitism compared to parasitized sclerotia at 3, 4, and 5 days of interaction. However, under starvation condition, smkA levels were significantly induced after 7 days of growth. Southern blot analysis revealed that smkA is member of a small gene family. Collectively, these results suggest that smkA could be implicated in the mycoparasitic process in S. elegans as well as in stress-activated pathways. These results may be of wider significance in other fungus-fungus interactions.

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.

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.

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.

Combined Transcriptomic and Metabolomic Analysis Reveals Insights into Resistance of Arabidopsis bam3 Mutant against the Phytopathogenic Fungus Fusarium oxysporum.

The wilt-inducing strains of Fusarium oxysporum are responsible for severe damage to many economically important plant species. The most cost-effective and environmentally safe method for the management of Fusarium wilt is the use of resistant cultivars when they are available. In the present study, the Arabidopsis genotype with disruptions in the ß-amylase 3 (BAM3) gene, which encodes the major hydrolytic enzyme that degrades starch to maltose, had significantly lower susceptibility to Fusarium oxysporum f. sp. raphani (For) compared to wild-type (wt) plants. It showed the lowest disease severity and contained reduced quantities of fungal DNA in the plant vascular tissues when analyzed with real-time PCR. Through metabolomic analysis using gas chromatography (GC)-mass spectrometry (MS) and gene-expression analysis by reverse-transcription quantitative PCR (RT-qPCR), we observed that defense responses of Arabidopsis bam3 mutants are associated with starch-degradation enzymes, the corresponding modification of the carbohydrate balance, and alterations in sugar (glucose, sucrose, trehalose, and myo-inositol) and auxin metabolism.

Metabolomic and Genomic Approach to Study Defense Induction by Nesidiocoris tenuis against Tuta absoluta and Tetranychus urticae in Tomato Plants.

The phytophagy of the predator Nesidiocoris tenuis (Hemiptera: Miridae) can trigger defense responses in tomato plants against pests, such as two spotted spider mite Tetranychus urticae (Acari: Tetranychidae) and South American leaf miner Tuta absoluta (Lepidoptera: Gelechiidae). The expression of genes governing Jasmonic Acid (JA) biosynthesis pathway and fluctuations in the levels of underlying metabolites have been rarely studied in mirid-infested plants. In the present study, fifteen 3rd instar nymphs of N.tenuis were caged on each top and lower leaf of tomato plants for 4 d to induce plant defense; after this period the predators were removed. With regard to T. absoluta, oviposition preference; larval period; and pupal weight were significantly reduced in N. tenuis-punctured plants. T. urticae adults exhibited a significantly higher escape tendency and reduced survival on punctured plants. Metabolomics confirmed such observations revealing substantial differences between N. tenuis-punctured and unpunctured (control) plants. Metabolites directly associated with the activation of the JA defense pathway, such as the precursor α-linolenic acid, had increased concentrations. The expression of the defense-related genes PI-II, MYC2, VSP2, and HEL was increased in the top leaves and only VSP2 and MBP2 in the lower leaves; interestingly, in the middle (unpunctured) leaves VSP2, HEL, and MBP2 were also upregulated, indicating systemic signaling. Collectively, phytophagy of N. tenuis caused adverse effects on T. absoluta and T. urticae, whereas the multi-omics approach (phenomics, metabolomics, and genomics) offered valuable insights into the nature of the plant defense responses and provided useful evidence for future applications in integrated pest management, plausibly resulting in the reduction in the required pesticide volumes.

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.

Do pesticides promote or hinder sustainability in agriculture? The challenge of sustainable use of pesticides in modern agriculture.

Sustainable agriculture aims to meet the food needs of the growing world population while ensuring minimal impact on the environment and humans as well as productivity. Although pesticides represent the backbone of the agri-food sector in its endeavor to secure food production their application is perceived by many as an obstacle towards the achievement of sustainability; the main concerns are linked with their adverse effects on human health and the environment. Τhis review aims to present the status of chemical plant protection and provide insights into the use of pesticides within the context of sustainable agriculture. It mainly focuses on the strengthened legislation frameworks, which especially in the European Union and the United States of America ensure the placement in the market of pesticides with acceptable toxicological and environmental profiles without compromising crop production. Furthermore, the implementation of Integrated Pest Management principles plays a key role in the sustainable use of pesticides. The stringent regulatory requirements have resulted in the dramatic increase of the associated effort and costs in pesticide research and development (R&D) of improved products. Nevertheless, the investment of leading agrochemical companies in the R&D of new pesticides remains high. All the above set the ground for the sustainable use of pesticides in crop production while their successful application remains a challenge.

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.

Impact of Plant Growth-Promoting Rhizobacteria Inoculation and Grafting on Tolerance of Tomato to Combined Water and Nutrient Stress Assessed via Metabolomics Analysis.

In the current study, inoculation with plant growth-promoting rhizobacteria (PGPR) and grafting were tested as possible cultural practices that may enhance resilience of tomato to stress induced by combined water and nutrient shortage. The roots of tomato grown on perlite were either inoculated or not with PGPR, applying four different treatments. These were PGPR-T1, a mix of two Enterobacter sp. strains (C1.2 and C1.5); PGPR-T2, Paenibacillus sp. strain DN1.2; PGPR-T3, Enterobacter mori strain C3.1; and PGPR-T4, Lelliottia sp. strain D2.4. PGPR-treated plants were either self-grafted or grafted onto Solanum lycopersicum cv. M82 and received either full or 50% of their standard water, nitrogen, and phosphorus needs. The vegetative biomass of plants subjected to PGPR-T1 was not reduced when plants were cultivated under combined stress, while it was reduced by stress to the rest of the PGPR treatments. However, PGPR-T3 increased considerably plant biomass of non-stressed tomato plants than did all other treatments. PGPR application had no impact on fruit biomass, while grafting onto 'M82' increased fruit production than did self-grafting. Metabolomics analysis in tomato leaves revealed that combined stress affects several metabolites, most of them already described as stress-related, including trehalose, myo-inositol, and monopalmitin. PGPR inoculation with E. mori strain C3.1 affected metabolites, which are important for plant/microbe symbiosis (myo-inositol and monopalmitin). The rootstock M82 did not affect many metabolites in plant leaves, but it clearly decreased the levels of malate and D-fructose and imposed an accumulation of oleic acid. In conclusion, PGPR are capable of increasing tomato tolerance to combined stress. However, further research is required to evaluate more strains and refine protocols for their application. Metabolites that were discovered as biomarkers could be used to accelerate the screening process for traits such as stress tolerance to abiotic and/or abiotic stresses. Finally, 'M82' is a suitable rootstock for tomato, as it is capable of increasing fruit biomass production.

Yeast (Saccharomyces cerevisiae) GC/EI/MS metabolomics dataset.

Gas chromatography-electron impact-mass spectrometry (GC/EI/MS) global profiling of the endo-metabolome of wild and genetically engineered yeast (Saccharomyces cerevisiae) strains was performed. The strains were treated or not with sub-lethal doses of the fungicide flusilazole, in order to mine the involvement of the ABC transporter YCF1, which is located in the yeast vacuole membrane, in its resistance to fungicides. Raw GC/EI/MS total ion chromatograms (*.cdf format) of the yeast endo-metabolome were recoded, which are included in this article. Since yeast is a model organism, the dataset could serve as a reference for yeast metabolomics studies related to the investigation of the effects of bioactive ingredients on its metabolism. The dataset support the research article "Karamanou D. and Aliferis K.A, 2019. The yeast (Saccharomyces cerevisiae) YCF1 vacuole transporter: evidence on its implication into the yeast resistance to flusilazole as revealed by GC/EI/MS metabolomics. Pest. Biochem. Physiol. doi: https://doi.org/10.1016/j.pestbp.2019.09.013". 10.1016/j.pestbp.2019.09.013.

Cannabinomics: Application of Metabolomics in Cannabis (Cannabis sativa L.) Research and Development.

Cannabis (Cannabis sativa L.) is a complex, polymorphic plant species, which produces a vast array of bioactive metabolites, the two major chemical groups being cannabinoids and terpenoids. Nonetheless, the psychoactive cannabinoid tetrahydrocannabinol (Δ 9 -THC) and the non-psychoactive cannabidiol (CBD), are the two major cannabinoids that have monopolized the research interest. Currently, more than 600 Cannabis varieties are commercially available, providing access to a multitude of potent extracts with complex compositions, whose genetics are largely inconclusive. Recently introduced legislation on Cannabis cultivation in many countries represents a great opportunity, but at the same time, a great challenge for Cannabis research and development (R&D) toward applications in the pharmaceutical, food, cosmetics, and agrochemical industries. Based on its versatility and unique capabilities in the deconvolution of the metabolite composition of complex matrices, metabolomics represents an ideal bioanalytical tool that could greatly assist and accelerate Cannabis R&D. Among others, Cannabis metabolomics or cannabinomics can be applied in the taxonomy of Cannabis varieties in chemovars, the research on the discovery and assessment of new Cannabis-based sources of bioactivity in medicine, the development of new food products, and the optimization of its cultivation, aiming for improvements in yield and potency. Although Cannabis research is still in its infancy, it is highly foreseen that the employment of advanced metabolomics will provide insights that could assist the sector to face the aforementioned challenges. Within this context, here, the current state-of-the-art and conceptual aspects of cannabinomics are presented.