Aplicación de microorganismos promotores del crecimiento vegetal con actividad quorum quenching frente a estrés biótico y abiótico

Abstract

Samples were taken from the Saladar de El Margen (Granada), to isolate strains which possess the ability to resist adverse climatic conditions such as salinity or drought, while meet these two characteristics. First, sampling in plants, soil and water from the Saladar, a hypersaline and semi-arid environment resulted in the isolation of 320 strains based on their colonial morphology. These, together with 71 strains previously isolated by the BIO 188 group from saline environments made up the collection of study strains of this doctoral thesis. After an initial in vitro screening of characteristics related with plant growth promotion in 391 strains, 14 were selected for their high PGP potential which also demonstrated a high tolerance to salinity. In vivo growth promotion assays showed the ability of some to act positively on the germination and vigour of tomato seeds, as well as in the growth and development of the plant. Some of them exhibited the ability to interfere with QS systems through high versatility of synthetic AHL enzymatic degradation. Taxonomic analysis allowed us to identify them as members of the genera Bacillus, Cobetia, Paenibacillus, Peribacillus, Pseudoalteromonas, Pseudomonas, Psychrobacillus and Staphylococcus, three of them constituting new species, Psychrobacillus vulpis Z8T (Rodríguez et al., 2019), Paenibacillus lutrae N10T (Rodríguez et al., 2019) and Peribacillus castrilensis N3T (article in preparation). Based on their in vitro PGP potential and QQ activity, 3 strains were selected: Staphylococcus equorum EN21, Peribacillus castrilensis N3 and Pseudomonas segetis P6, all of them isolated from the rhizosphere of the halophilic plant Salicornia hispanica. Plant-microorganism interaction was determined through the study of motility, chemotaxis to ɣaminobutyric acid (GABA) and root exudates, as well as the formation of biofilm and the production of exopolysaccharides, all factors associated with plant root colonization. Swimming and swarming motility was demonstrated in P. castrilensis N3 and swimming, swarming and twitching in the P. segetis P6. All showed attraction to the different compounds and plant root exudates tested, as well as the ability to form biofilm in different growing conditions with or without supplementation of plant exudates, as inducers of the formation of these biofilms. By contrast, only P. segetis P6 was able to produce exopolysaccharide. These results were confirmed by genome mining of genes associated with motility, chemotaxis, and exopolysaccharide production. An in vitro root colonization trial on tomato plants finally confirmed that these strains perform efficient colonization of the plant's root. A first in vivo test in tomato plants demonstrated an increase in the weight of the aerial and root part, as well as a high development of its root architecture. These positive effects are the result of several 26 factors: a clear impact on the nitrogen metabolism of plants related with an increase in amino acids and proteins, an increase in the activity of enzymes related to CO2 fixation and ammonium assimilation, and the notable increase in indoleacetic acid concentration in plants treated with these three strains, all this without causing states of stress in the plant. In relation to the activity against phytopathogens the three strains degraded Dickeya solani, Erwinia amylovora, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum and Pseudomonas syringae pv. tomato AHLs, which translates into the interference of certain phenotypic traits that could affect their virulence, such as motility or enzymatic activity. A lactonase-type enzyme was identified in S. equorum EN21, two acylase-type enzymes in P. segetis P6 and one enzyme of each type in P. castrilensis N3, all of which showed homology with other previously described QQ enzymes. These findings were subsequently confirmed by HPLC-MS and by genomic and structural analysis. In the case of acylases of P. segetis P6, the activity was confirmed by a heterologous expression in E. coli DH5α. Results from the interference of virulence assays in potatoes, pears and carrots indicated the ability of P. segetis P6 to completely inhibit the virulence of the tested phytopathogens. By contrast, S. equorum EN21 and P. castrilensis N3 resulted in a partial control of the infection, except for the last strain which totally inhibited the symptomatology produced by D. solani. Regarding the interference tests on P. syringae pv. tomato pathogenicity in tomato plants, all strains reduced the incidence and severity of infection and increased plant biomass and chlorophyll content. An analysis of primary metabolites and redox compounds, enzymatic activity and phytohormone profiling, justified these properties. Finally, the role of these strains on growth promotion under abiotic stress, in particular drought stress, was analysed and we detected clues of the ability of these strains to promote the growth of A. thaliana plants under drought conditions, what was associated with an increase in aerial and root biomass with respect to the control plants. Summarizing our results, we can affirm that saline and arid environments constitute an unexplored source of microorganisms with interesting properties for the agricultural industry constituting an effective, safe and eco-friendly alternative to the use of chemical fertilizers and pesticides in crop fields.