1. Investigación

We evaluated the ability of metabolic elicitors extracted from Pseudomonas fluorescens N21.4 to induce systemic resistance (ISR) in Arabidopsis thaliana against the pathogen Pseudomonas syringae DC3000. Metabolic elicitors were obtained from bacteria free culture medium with n-hexane, ethyl acetate and n-butanol in three consecutive extractions. Each extract showed plant protection activity. The n-hexane fraction was the most effective and was used to study the signal transduction pathways involved by evaluating expression of marker genes of the salicylic acid (SA) signalling pathway (NPR1, PR1, ICS and PR2) and the jasmonic acid/ethylene (JA/ET) signalling pathway (PDF1, MYC2, LOX2 and PR3). In addition, the level of oxidative stress was tested by determining the activity of enzymes related to the ascorbate-glutathione cycle. N-hexane extracts stimulated both pathways based on overexpression of ICS, PR1, PR2, PDF1 and LOX2 genes. In addition, activity of the pathogenesis-related proteins glucanase (PR2) and chitinase (PR3), lipoxygenase and polyphenol oxidase was enhanced together with an increased capacity to remove reactive oxygen species (ROS). This was associated with less oxidative stress as indicated by a decrease in malondialdehyde (MDA), suggesting a causative link between defensive metabolism against P. syringae and ROS scavenging.

Beneficial rhizobacterium Pseudomonas fluorescens N 21.4 and its metabolic elicitors inoculated to cultivars of blackberry (Rubus spp. Var. Loch Ness) reinforced the plants’ immune system and improved their fitness by increasing photosynthesis, decreasing oxidative stress, and activating pathogenesis-related proteins. They also triggered the leaves’ flavonoid metabolism, enhancing the accumulation of beneficial phenolic compounds such as kaempferols and quercetin derivatives. The elicitation of leaf secondary metabolism allows one to take advantage of the blackberry leaves (a current crop waste), following the premises of the circular economy, to isolate and obtain high added value compounds. The results of this work suggest the use of N 21.4 and/or its metabolic elicitors as plant inoculants as an effective and economically and environmentally friendly agronomic alternative practice in the exploitation of blackberry crops to obtain plants with a better immune system and to revalorize the leaf pruning as a potential source of polyphenols.

In this work, the metabolic elicitors extracted from the beneficial rhizobacterium Pseudomonas fluorescens N 21.4 were sequentially fragmented by vacuum liquid chromatography to isolate, purify and identify the compounds responsible for the extraordinary capacities of this strain to induce systemic resistance and to elicit secondary defensive metabolism in diverse plant species. To check if the fractions sequentially obtained were able to increase the synthesis of isoflavones and if, therefore, they still maintained the eliciting capacity of the live strain, rapid and controlled experiments were done with soybean seeds. The optimal action concentration of the fractions was established and all of them elicited isoflavone secondary metabolism—the fractions that had been extracted with n-hexane being more effective. The purest fraction was the one with the highest eliciting capacity and was also tested in Arabidopsis thaliana seedlings to induce systemic resistance against the pathogen Pseudomonas syringae pv. tomato DC 3000. This fraction was then analyzed by UHPLC/ESI–QTOF–MS, and an alkaloid, two amino lipids, three arylalkylamines and a terpenoid were tentatively identified. These identified compounds could be part of commercial plant inoculants of biological and sustainable origin to be applied in crops, due to their potential to enhance the plant immune response and since many of them have putative antibiotic and/or antifungal potential.

The use of beneficial rhizobacteria (bioeffectors) and their derived metabolic elicitors are efficient biotechnological alternatives in plant immune system elicitation. This work aimed to check the ability of 25 bacterial strains isolated from the rhizosphere of Nicotiana glauca, and selected for their biochemical traits from a group of 175, to trigger the innate immune system of Arabidopsis thaliana seedlings against the pathogen Pseudomonas syringae pv. tomato DC3000. The five strains more effective in preventing pathogen infection were used to elucidate signal transduction pathways involved in the plant immune response by studying the differential expression of Salicylic acid and Jasmonic acid/Ethylene pathway marker genes. Some strains stimulated both pathways, while others stimulated either one or the other. The metabolic elicitors of two strains, chosen for the differential expression results of the genes studied, were extracted using n-hexane, ethyl acetate, and n-butanol, and their capacity to mimic bacterial effect to trigger the plant immune system was studied. N-hexane and ethyl acetate were the most effective fractions against the pathogen in both strains, achieving similar protection rates although gene expression responses were different from that obtained by the bacteria. These results open an amount of biotechnological possibilities to develop biological products for agriculture.

Background: The beneficial rhizobacterium, Pseudomonas fluorescens N 21.4, and its metabolic elicitors were inoculated in com-mercial cultivars of blackberry plants (Rubus cv. Loch Ness). Phenolic compounds present in red and black fruit and the expres-sion of structural marker genes of the phenylpropanoid pathway during fruit ripening were studied.Results: An inverse relationship between gene expression and accumulation of metabolites was seen, except for the RuDFRgene, which had a direct correlation with cyanidin 3-O-glucoside synthesis, increasing its content 1.3 times when RuDFR wasoverexpressed in the red fruit of plants inoculated with the metabolic elicitors of P. fluorescens N 21.4, compared with red fruitof plants inoculated with N 21.4. The RuCHS gene also had a fundamental role in the accumulation of metabolites. Both rhizo-bacterium and metabolic elicitors triggered the flavonoid metabolism, enhancing the catechin and epicatechin contentbetween 1.1 and 1.6 times in the case of red fruit and between 1.1 and 1.8 times in the case of black fruit. Both treatments alsoboosted the anthocyanin, quercetin, and kaempferol derivative content, highlighting the effects of metabolic elicitors in redfruit and the effects of live rhizobacterium in black fruit.Conclusion: The metabolic elicitors' capacity to modulate gene expression and to increase secondary metabolites content wasdemonstrated. This work therefore suggests that they are effective, affordable, easily manageable, and ecofriendly plant inoc-ulants that complement, or are alternatives to, beneficial rhizobacteria.

La presente tesis doctoral se centra en el estudio del microbioma de la rizosfera y en las relaciones planta-microrganismos beneficiosos y su influencia sobre la fisiología de las plantas y sobre el metabolismo secundario defensivo y el sistema inmunitario vegetal en general. Este estudio parte de la consideración de la rizosfera como una fuente especialmente rica en microorganismos beneficiosos, que tienen un papel fundamental en mejorar la capacidad de adaptación de las plantas a ambientes cambiantes y que están adaptados al sistema planta-microorganismo gracias a la presión selectiva que han ejercido las plantas sobre el microbioma durante el largo proceso de coevolución. Además, el trabajo surge ante la actual necesidad de desarrollar nuevas prácticas agrícolas de base biotecnológica, efectivas y a la vez sostenibles que minimicen las pérdidas económicas y materiales debidas a las infecciones patogénicas que atacan a los cultivos y también causadas por condiciones de estrés abiótico. De esta manera se persigue aumentar el rendimiento de los cultivos, tanto en cantidad como en calidad de alimentos, para poder proveer a una población en continuo crecimiento y a la vez cumplir con el concepto de seguridad alimentaria propuesto por la FAO. Por todo lo anterior, el presente trabajo se centra en el uso de rizobacterias beneficiosas (PGPR) y de moléculas derivadas del metabolismo de las mismas (elicitores metabólicos) como inoculantes de plantas capaces de inducir el metabolismo secundario defensivo, dando lugar a plantas con un sistema inmunitario más fuerte y, por tanto, plantas mejor preparadas para combatir el estrés biótico y abiótico. Así, el objetivo último del trabajo es desarrollar inoculantes de plantas de origen biológico, con una capacidad bioestimulante efectiva y por ello, capaces de sustituir a los fitosanitarios químicos. La gran novedad que aporta este trabajo es el uso de moléculas elicitoras derivadas del metabolismo de determinadas rizobacterias. Es por esto, que la especie de rizobacteria beneficiosa seleccionada para estudiar su efecto y el de sus elicitores metabólicos sobre el metabolismo secundario de las plantas fue la cepa Pseudomonas fluorescens N 21.4, una cepa cuya capacidad de elicitación y de inducción de resistencia sistémica en distintas especies vegetales ya ha sido ampliamente demostrada por el grupo de investigación. Se comenzó probando la cepa viva como control positivo y distintas fracciones elicitoras de la misma en plántulas de Arabidopsis thaliana en condiciones controladas de laboratorio, estudiando su capacidad de inducción de resistencia sistémica, el nivel de estrés oxidativo de las plantas tratadas y las rutas de transducción de señal implicadas en la respuesta. Después se probó la cepa viva y sus elicitores metabólicos en condiciones reales de campo en cultivos comerciales de zarzamora (Rubus cv. Loch Ness), estudiándose el fitness general de las plantas a través de marcadores específicos, así como la acumulación de compuestos de interés en las hojas y en los frutos y los genes específicos implicados en la maduración de los frutos. Los resultados mostraron que en las plantas inoculadas con los tratamientos existía una relación causal entre la inducción de resistencia sistémica y una reducción del estrés oxidativo en A. thaliana y un mejor fitness general, menor estrés oxidativo y mayor acumulación de compuestos fenólicos beneficiosos en las hojas y frutos de zarzamora. Al demostrarse la capacidad de los elicitores metabólicos de la cepa P. fluorescens N 21.4 de inducir el metabolismo secundario defensivo, se procedió a la identificación química de las moléculas responsables de dicha capacidad, encontrándose siete compuestos muy prometedores dado su potencial capacidad antibiótica y antifúngica. Este estudio termina con la búsqueda de nuevas cepas PGPR (bioefectores) y de sus elicitores metabólicos y con el estudio de las vías de transducción de señal involucradas en la respuesta de las plantas elicitadas con las mismas. De un grupo inicial de ciento setenta y cinco rizobacterias, se seleccionaron dos por sus prometedoras capacidades para utilizarse como inoculantes de plantas en experimentos futuros.