Root Exudates Metabolic Profiling Confirms Distinct Defense Mechanisms Between Cultivars and Treatments with Beneficial Microorganisms and Phosphonate Salts Against Verticillium Wilt in Olive Trees.

Root exudates play a key role in the life cycle of Verticillium dahliae, the causal agent of Verticillium wilt diseases, since they induce microsclerotia germination to initiate plant infection through the roots. In olive plants, the genotype and the application of biological control agents (BCAs) or phosphonate salts influence the ability of root exudates to decrease V. dahliae viability. Understanding the chemical composition of root exudates could provide new insights into the mechanisms of olive plant defense against V. dahliae. Therefore, the main goal of this study was to analyze the metabolomic profiles of root exudates collected from the olive cultivars Arbequina, Frantoio and Picual subjected to treatment with BCAs (Aureobasidium pullulans AP08, Bacillus amyloliquefaciens PAB-024) or phosphonate salts (copper phosphite, potassium phosphite). These treatments were selected due to their effectiveness as inducers of resistance against Verticillium wilt in olive plants. Our metabolomic analysis revealed that the olive cultivars exhibited differences in root exudates, which could be related to the different degrees of susceptibility to V. dahliae. The composition of root exudates also changed with the application of BCAs or phosphonate fertilizer, highlighting the complex and dynamic nature of the interactions between olive cultivars and treatments preventing V. dahliae infections. Thus, the identification of genotype-specific metabolic changes and specific metabolites induced by these treatments emphasizes the potential of resistance inducers for enhancing plant defense and promoting the growth of beneficial microorganisms.

Solanum lycopersicum heme-binding protein 2 as a potent antimicrobial weapon against plant pathogens.

The rise in antibiotic-resistant bacteria caused by the excessive use of antibiotics has led to the urgent exploration of alternative antimicrobial solutions. Among these alternatives, antimicrobial proteins, and peptides (Apps) have garnered attention due to their wide-ranging antimicrobial effects. This study focuses on evaluating the antimicrobial properties of Solanum lycopersicum heme-binding protein 2 (SlHBP2), an apoplastic protein extracted from tomato plants treated with 1-Methyl tryptophan (1-MT), against Pseudomonas syringae pv. tomato DC3000 (Pst). Computational studies indicate that SlHBP2 is annotated as a SOUL heme-binding family protein. Remarkably, recombinant SlHBP2 demonstrated significant efficacy in inhibiting the growth of Pst within a concentration range of 3-25 µg/mL. Moreover, SlHBP2 exhibited potent antimicrobial effects against other microorganisms, including Xanthomonas vesicatoria (Xv), Clavibacter michiganensis subsp. michiganensis (Cmm), and Botrytis cinerea. To understand the mechanism of action employed by SlHBP2 against Pst, various techniques such as microscopy and fluorescence assays were employed. The results revealed that SlHBP2 disrupts the bacterial cell wall and causes leakage of intracellular contents. To summarize, the findings suggest that SlHBP2 has significant antimicrobial properties, making it a potential antimicrobial agent against a wide range of pathogens. Although further studies are warranted to explore the full potential of SlHBP2 and its suitability in various applications.

Biocontrol of Plant Diseases by Means of Antagonist Microorganisms, Biostimulants and Induced Resistance as Alternatives to Chemicals.

Plant diseases are one of the biggest problems in conventional agriculture as they reduce both yield and crop value [...].

Exploiting Tomato Genotypes to Understand Heat Stress Tolerance.

Increased temperatures caused by climate change constitute a significant threat to agriculture and food security. The selection of improved crop varieties with greater tolerance to heat stress is crucial for the future of agriculture. To overcome this challenge, four traditional tomato varieties from the Mediterranean basin and two commercial genotypes were selected to characterize their responses at high temperatures. The screening of phenotypes under heat shock conditions allowed to classify the tomato genotypes as: heat-sensitive: TH-30, ADX2; intermediate: ISR-10 and Ailsa Craig; heat-tolerant: MM and MO-10. These results reveal the intra-genetical variation of heat stress responses, which can be exploited as promising sources of tolerance to climate change conditions. Two different thermotolerance strategies were observed. The MO-10 plants tolerance was based on the control of the leaf cooling mechanism and the rapid RBOHB activation and ABA signaling pathways. The variety MM displayed a different strategy based on the activation of HSP70 and 90, as well as accumulation of phenolic compounds correlated with early induction of PAL expression. The importance of secondary metabolism in the recovery phase has been also revealed. Understanding the molecular events allowing plants to overcome heat stress constitutes a promising approach for selecting climate resilient tomato varieties.

Resistance Induction in Olive Tree (Olea europaea) Against Verticillium Wilt by Two Beneficial Microorganisms and a Copper Phosphite Fertilizer.

Enhancement of the natural defenses of a plant by beneficial microorganisms, i.e., endophytic bacteria and fungi or fertilizers such as copper phosphonates, could result in a potential alternative strategy against verticillium wilt of olive tree (Olea europaea). In this study, two beneficial microorganisms (the fungus Aureobasidium pullulans AP08 and the bacterium Bacillus amyloliquefaciens PAB-024) and a phosphonate salt copper phosphite (CuPh) were evaluated for their effectiveness as host resistance inducers against Verticillium dahliae in olive. To this end, 6-month-old healthy olive plants of the susceptible cultivar Picual were treated by foliar or root applications by spraying 15 ml per plant or by irrigation with 350 ml per plant of the dilutions of each product (CuPh: 3 or 10 ml l-1, respectively; PAB-024: 108 UFC ml-1; AP08: 106 UFC ml-1). Treatments were conducted weekly from 2 weeks before inoculation to 10 days after inoculation. A cornmeal-water-sand mixture (1:2:9; w:v:w) colonized by V. dahliae was used for plant inoculation. Additionally, treated and noninoculated, nontreated and inoculated, and nontreated and noninoculated plants were included for comparative purposes. Disease severity progress and shoot fresh weight were assessed. Parameters involved in plant resistance were monitored through determination and quantification of reactive oxygen species (ROS) response (H2O2), and evaluation of hormones was done by gene expression analysis. Aureobasidium pullulans and CuPh were the most effective in disease reduction in planta by foliar or root application, respectively. Plants treated with CuPh showed significantly higher shoot fresh weight compared to the other treatments. ROS was significantly enhanced in plants treated with B. amyloliquefaciens PAB-024 compared to the rest of treatments and control. With regard to the evaluation of hormones, high levels of salicylic acid were detected on leaves from all treatment combinations, but without significant enhancements compared to the nontreated control. Regarding the gene expression related to salicylic acid, only the WRKY5 gene has shown a strong enhancement in the treatment with B. amyloliquefaciens. On the other hand, a strong accumulation of jasmonic acid and jasmonic acid-isoleucine in plants treated with A. pullulans was observed in all the tissues analyzed and also in the roots of plants treated with B. amyloliquefaciens and CuPh.

Jasmonic acid pathway is required in the resistance induced by Acremonium sclerotigenum in tomato against Pseudomonas syringae.

The use of fungal endophytes is considered as a new tool to confer resistance in plants against stresses. However, the mechanisms involved in colonization as well as in the induction of resistance by the endophytes are usually unclear. In this work, we tested whether a fungal endophyte isolated from an ancestor of wheat could induce resistance in plants of a different class from the ones that were isolated from the beginning. Seeds of Solanum lycopersicum were inoculated with Acremonium sclerotigenum and after four weeks, seedlings were inoculated with the bacterium Pseudomonas syringae pv tomato. Plants inoculated with endophytes showed significantly lower symptoms of infection as well as lower levels of colony forming units compared with control plants. Moreover, the presence of the endophytes induced an enhancement of Jasmonic acid (JA) upon inoculation with P. syringae compared with endophyte free plants. To ascertain the implication of JA in the resistance induced by A. sclerotigenum, two mutants defective in JA were tested. Results showed that the endophyte is not able to induce resistance in the mutant spr2, which is truncated in the first step of JA biosynthesis. On the contrary, acx1 mutant plants, which are unable to synthesize JA from OPC8, show a phenotype similar to wild type plants. Moreover, experiments with GFP-tagged endophytes showed no differences in the colonization in both mutants. In conclusion, the jasmonic acid pathway is required for the resistance mediated by the endophyte A. sclerotigenum in tomato against the biotrophic bacterium P. syringae but is not necessary for the colonization.

1-Methyltryptophan Treatment Increases Defense-Related Proteins in the Apoplast of Tomato Plants.

The activation of induced resistance in plants may enhance the production of defensive proteins to avoid the invasion of pathogens. In this way, the composition of the apoplastic fluid could represent an important layer of defense that plants can modify to avoid the attack. In this study, we performed a proteomic study of the apoplastic fluid from plants treated with the resistance inducer 1-methyltryptophan (1-MT) as well as infected with Pseudomonas syringae pv. tomato (Pst). Our results showed that both the inoculation with Pst and the application of the inducer provoke changes in the proteomic composition in the apoplast enhancing the accumulation of proteins involved in plant defense. Finally, one of the identified proteins that are overaccumulated upon the treatment have been expressed in Escherichia coli and purified in order to test their antimicrobial effect. The result showed that the tested protein is able to reduce the growth of Pst in vitro. Taken together, in this work, we described the proteomic changes in the apoplast induced by the treatment and by the inoculation, as well as demonstrated that the proteins identified have a role in the plant protection.

Botrytis cinerea methyl isocitrate lyase mediates oxidative stress tolerance and programmed cell death by modulating cellular succinate levels.

Fungi lack the entire animal core apoptotic machinery. Nevertheless, regulated cell death with apoptotic markers occurs in multicellular as well as in unicellular fungi and is essential for proper fungal development and stress adaptation. The discrepancy between appearance of an apoptotic-like regulated cell death (RCD) in the absence of core apoptotic machinery is further complicated by the fact that heterologous expression of animal apoptotic genes in fungi affects fungal RCD. Here we describe the role of BcMcl, a methyl isocitrate lyase from the plant pathogenic fungus Botrytis cinerea, in succinate metabolism, and the connection of succinate with stress responses and cell death. Over expression of bcmcl resulted in elevated tolerance to oxidative stress and reduced levels of RCD, which were associated with accumulation of elevated levels of succinate. Deletion of bcmcl had almost no effect on fungal development or stress sensitivity, and succinate levels were unchanged in the deletion strain. Gene expression experiments showed co-regulation of bcmcl and bcicl (isocitrate lyase); expression of the bcicl gene was enhanced in bcmcl deletion and suppressed in bcmcl over expression strains. External addition of succinate reproduced the phenotypes of the bcmcl over expression strains, including developmental defects, reduced virulence, and improved oxidative stress tolerance. Collectively, our results implicate mitochondria metabolic pathways, and in particular succinate metabolism, in regulation of fungal stress tolerance, and highlight the role of this onco-metabolite as potential mediator of fungal RCD.

The Apoplast: A Key Player in Plant Survival.

The apoplast comprises the intercellular space, the cell walls, and the xylem. Important functions for the plant, such as nutrient and water transport, cellulose synthesis, and the synthesis of molecules involved in plant defense against both biotic and abiotic stresses, take place in it. The most important molecules are ROS, antioxidants, proteins, and hormones. Even though only a small quantity of ROS is localized within the apoplast, apoplastic ROS have an important role in plant development and plant responses to various stress conditions. In the apoplast, like in the intracellular cell compartments, a specific set of antioxidants can be found that can detoxify the different types of ROS produced in it. These scavenging ROS components confer stress tolerance and avoid cellular damage. Moreover, the production and accumulation of proteins and peptides in the apoplast take place in response to various stresses. Hormones are also present in the apoplast where they perform important functions. In addition, the apoplast is also the space where microbe-associated molecular Patterns (MAMPs) are secreted by pathogens. In summary, the diversity of molecules found in the apoplast highlights its importance in the survival of plant cells.

Role of Jasmonic Acid Pathway in Tomato Plant-Pseudomonas syringae Interaction.

The jasmonic acid pathway has been considered as the backbone of the response against necrotrophic pathogens. However, a hemi-biotrophic pathogen, such as Pseudomonas syringae, has taken advantage of the crosstalk between the different plant hormones in order to manipulate the responses for its own interest. Despite that, the way in which Pseudomonas syringae releases coronatine to activate jasmonic acid-derived responses and block the activation of salicylic acid-mediated responses is widely known. However, the implication of the jasmonic intermediates in the plant-Pseudomonas interaction is not studied yet. In this work, we analyzed the response of both, plant and bacteria using SiOPR3 tomato plants. Interestingly, SiOPR3 plants are more resistant to infection with Pseudomonas. The gene expression of bacteria showed that, in SiOPR3 plants, the activation of pathogenicity is repressed in comparison to wild type plants, suggesting that the jasmonic acid pathway might play a role in the pathogenicity of the bacteria. Moreover, treatments with JA restore the susceptibility as well as activate the expression of bacterial pathogenicity genes. The observed results suggest that a complete jasmonic acid pathway is necessary for the susceptibility of tomato plants to Pseudomonas syringae.

Endophytes from wild cereals protect wheat plants from drought by alteration of physiological responses of the plants to water stress.

Endophytes contribute to plant performance, especially under harsh conditions. We therefore hypothesized that wild plants have retained beneficial endophytes that are less abundant or not present in related crop plants. To test this hypothesis, we selected two endophytes that were found in Sharon goatgrass, an ancestor of wheat, and tested their effect on bread wheat. Both endophytes infected wheat and improved sustainability and performance under water-limited conditions. To determine how the endophytes modify plant development, we measured parameters of plant growth and physiological status and performed a comparative metabolomics analysis. Endophyte-treated wheat plants had reduced levels of stress damage markers and reduced accumulation of stress-adaptation metabolites. Metabolomics profiling revealed significant differences in the response to water stress of endophyte-treated plants compared with untreated plants. Our results demonstrate the potential of endophytes from wild plants for improvement of related crops and show that the beneficial effects of two endophytes are associated with alteration of physiological responses to water-limited conditions.

1-Methyltryptophan Modifies Apoplast Content in Tomato Plants Improving Resistance Against Pseudomonas syringae.

Plants can produce numerous natural products, many of which have been shown to confer protection against microbial attack. In this way, we identified 1-methyltryptophan (1-MT), a natural compound produced by tomato plants in response to Pseudomonas syringae attack, whose application by soil drench provided protection against this pathogen. In the present work, we have studied the mechanisms underlying this protection. The results demonstrated that 1-MT can be considered a new activator of plant defense responses that acts by inhibiting the stomatal opening produced by coronatine (COR) and could thereby, prevent bacteria entering the mesophyll. Besides, 1-MT acts by blocking the jasmonic acid (JA) pathway that, could avoid manipulation of the salicylic acid (SA) pathway by the bacterium, and thus hinder its growth. Although the concentration of 1-MT reached in the plant did not show antimicrobial effects, we cannot rule out a role for 1-MT acting alone because it affects the expression of the fliC gene that is involved in synthesis of the flagellum. These changes would result in reduced bacterium motility and, therefore, infective capacity. The results highlight the effect of a tryptophan derivative on induced resistance in plants.

Elucidating the mechanism of action of copper heptagluconate on the plant immune system against Pseudomonas syringae in tomato (Solanum lycopersicum L).

BACKGROUND: Phytopathogenic problems caused by the bacterial pathogen Pseudomonas syringae in tomato are becoming more serious due to the emergence of strains resistant to classical pesticides. This has led to research into new formulations with lower environmental problems. One of the most promising alternatives to the use of classical pesticides is the induction of natural plant defences. New formulations based on Cu complexed with heptagluconic acid induce plant innate defences and could be an alternative to classical treatments based on inorganic Cu against bacterial speck. To study the efficacy of this compound in tomato against P. syringae, we tested its systemic effect Applying the treatments via radicular. RESULTS: Treated plants showed less infection development and lower number of viable bacteria in leaves. We also observed better performance of parameters involved in plant resistance such as the antioxidant response and the accumulation of phenolic compounds. CONCLUSION: Results showed that soil drench applications can be highly effective for the prevention and control of bacterial speck in tomato plants, showing a reduction in symptoms of ∼ 50%. Moreover, application of Cu heptagluconate induced accumulation of the plant polyphenols caffeic and chlorogenic acids, and reduced the amount of reactive oxygen species in infected plants. © 2018 Society of Chemical Industry.

Bioassimilable sulphur provides effective control of Oidium neolycopersici in tomato, enhancing the plant immune system.

BACKGROUND: Developments of alternatives to the use of chemical pesticides to control pests are focused on the induction of natural plant defences. The study of new compounds based on liquid bioassimilable sulphur and its effect as an inductor of the immune system of plants would provide an alternative option to farmers to enhance plant resistance against pathogen attacks such as powdery mildew. In order to elucidate the efficacy of this compound in tomato against powdery mildew, we tested several treatments: curative foliar, preventive foliar, preventive in soil drench and combining preventive in soil drench and curative foliar. RESULTS: In all cases, treated plants showed lower infection development, better physiological parameters and a higher level of chlorophyll. We also observed better performance in parameters involved in plant resistance such as antioxidant response, callose deposition and hormonal levels. CONCLUSION: The results indicate that preventive and curative treatments can be highly effective for the prevention and control of powdery mildew in tomato plants. Foliar treatments are able to stop the pathogen development when they are applied as curative. Soil drench treatments induce immune response mechanisms of plants, increasing significantly callose deposition and promoting plant development. © 2016 Society of Chemical Industry.

Priming by Hexanoic Acid Induce Activation of Mevalonic and Linolenic Pathways and Promotes the Emission of Plant Volatiles.

Hexanoic acid (Hx) is a short natural monocarboxylic acid present in some fruits and plants. Previous studies reported that soil drench application of this acid induces effective resistance in tomato plants against Botrytis cinerea and Pseudomonas syringae and in citrus against Alternaria alternata and Xanthomonas citri. In this work, we performed an in deep study of the metabolic changes produced in citrus by the application of Hx in response to the challenge pathogen A. alternata, focusing on the response of the plant. Moreover, we used (13)C labeled hexanoic to analyze its behavior inside the plants. Finally, we studied the volatile emission of the treated plants after the challenge inoculation. Drench application of (13)C labeled hexanoic demonstrated that this molecule stays in the roots and is not mobilized to the leaves, suggesting long distance induction of resistance. Moreover, the study of the metabolic profile showed an alteration of more than 200 molecules differentially induced by the application of the compound and the inoculation with the fungus. Bioinformatics analysis of data showed that most of these altered molecules could be related with the mevalonic and linolenic pathways suggesting the implication of these pathways in the induced resistance mediated by Hx. Finally, the application of this compound showed an enhancement of the emission of 17 volatile metabolites. Taken together, this study indicates that after the application of Hx this compound remains in the roots, provoking molecular changes that may trigger the defensive response in the rest of the plant mediated by changes in the mevalonic and linolenic pathways and enhancing the emission of volatile compounds, suggesting for the first time the implication of mevalonic pathway in response to hexanoic application.

NH4+ protects tomato plants against Pseudomonas syringae by activation of systemic acquired acclimation.

NH4 (+) nutrition provokes mild toxicity by enhancing H2O2 accumulation, which acts as a signal activating systemic acquired acclimation (SAA). Until now, induced resistance mechanisms in response to an abiotic stimulus and related to SAA were only reported for exposure to a subsequent abiotic stress. Herein, the first evidence is provided that this acclimation to an abiotic stimulus induces resistance to later pathogen infection, since NH4 (+) nutrition (N-NH4 (+))-induced resistance (NH4 (+)-IR) against Pseudomonas syringae pv tomato DC3000 (Pst) in tomato plants was demonstrated. N-NH4 (+) plants displayed basal H2O2, abscisic acid (ABA), and putrescine (Put) accumulation. H2O2 accumulation acted as a signal to induce ABA-dependent signalling pathways required to prevent NH4 (+) toxicity. This acclimatory event provoked an increase in resistance against later pathogen infection. N-NH4 (+) plants displayed basal stomatal closure produced by H2O2 derived from enhanced CuAO and rboh1 activity that may reduce the entry of bacteria into the mesophyll, diminishing the disease symptoms as well as strongly inducing the oxidative burst upon Pst infection, favouring NH4 (+)-IR. Experiments with inhibitors of Put accumulation and the ABA-deficient mutant flacca demonstrated that Put and ABA downstream signalling pathways are required to complete NH4 (+)-IR. The metabolic profile revealed that infected N-NH4 (+) plants showed greater ferulic acid accumulation compared with control plants. Although classical salicylic acid (SA)-dependent responses against biotrophic pathogens were not found, the important role of Put in the resistance of tomato against Pst was demonstrated. Moreover, this work revealed the cross-talk between abiotic stress acclimation (NH4 (+) nutrition) and resistance to subsequent Pst infection.

Resistance inducers modulate Pseudomonas syringae pv. tomato strain DC3000 response in tomato plants.

The efficacy of hexanoic acid (Hx) as an inducer of resistance in tomato plants against Pseudomonas syringae pv. tomato DC3000 was previously demonstrated, and the plant response was characterized. Because little is known about the reaction of the pathogen to this effect, the goal of the present work was to determine whether the changes in the plant defence system affect the pathogen behaviour. This work provides the first demonstration of the response of the pathogen to the changes observed in plants after Hx application in terms of not only the population size but also the transcriptional levels of genes involved in quorum sensing establishment and pathogenesis. Therefore, it is possible that Hx treatment attenuates the virulence and survival of bacteria by preventing or diminishing the appearance of symptoms and controlling the growth of the bacteria in the mesophyll. It is interesting to note that the gene transcriptional changes in the bacteria from the treated plants occur at the same time as the changes in the plants. Hx is able to alter bacteria pathogenesis and survival only when it is applied as a resistance inducer because the changes that it promotes in plants affect the bacteria.

Enhancement of the citrus immune system provides effective resistance against Alternaria brown spot disease.

In addition to basal defense mechanisms, plants are able to develop enhanced defense mechanisms such as induced resistance (IR) upon appropriate stimulation. We recently described the means by which several carboxylic acids protect Arabidopsis and tomato plants against fungi. In this work, we demonstrate the effectiveness of hexanoic acid (Hx) in the control of Alternaria brown spot (ABS) disease via enhancement of the immune system of Fortune mandarin. The application of 1mM Hx in irrigation water to 2-year-old Fortune plants clearly reduced the incidence of the disease and led to smaller lesions. We observed that several of the most important mechanisms involved in induced resistance were affected by Hx application. Our results demonstrate enhanced callose deposition in infected plants treated with Hx, which suggests an Hx priming mechanism. Plants treated with the callose inhibitor 2-DDG were more susceptible to the fungus. Moreover, polygalacturonase-inhibiting protein (PGIP) gene expression was rapidly and significantly upregulated in treated plants. However, treatment with Hx decreased the levels of reactive oxygen species (ROS) in infected plants. Hormonal and gene analyses revealed that the jasmonic acid (JA) pathway was activated due to a greater accumulation of 12-oxo-phytodienoic acid (OPDA) and JA along with a rapid accumulation of JA-isoleucine (JA-Ile). Furthermore, we observed a more rapid accumulation of abscisic acid (ABA), which could act as a positive regulator of callose deposition. Thus, our results support the hypothesis that both enhanced physical barriers and the JA signaling pathway are involved in hexanoic acid-induced resistance (Hx-IR) to Alternaria alternata.