Indigenous Pseudomonas spp. Strains from the Olive (Olea europaea L.) Rhizosphere as Effective Biocontrol Agents against Verticillium dahliae: From the Host Roots to the Bacterial Genomes

Abstract

The use of biological control agents (BCA), alone or in combination with other management measures, has gained attention over the past decades, driven by the need to seek for sustainable and eco-friendly alternatives to confront plant pathogens. The rhizosphere of olive (Olea europaea L.) plants is a source of bacteria with potential as biocontrol tools against Verticillium wilt of olive (VWO) caused by Verticillium dahliae Kleb. A collection of bacterial isolates from healthy nursery-produced olive (cultivar Picual, susceptible to VWO) plants was generated based on morphological, biochemical and metabolic characteristics, chemical sensitivities, and on their in vitro antagonistic activity against several olive pathogens. Three strains (PIC25, PIC105, and PICF141) showing high in vitro inhibition ability of pathogens' growth, particularly against V. dahliae, were eventually selected. Their effectiveness against VWO caused by the defoliating pathotype of V. dahliae was also demonstrated, strain PICF141 being the rhizobacteria showing the best performance as BCA. Genotypic and phenotypic traits traditionally associated with plant growth promotion and/or biocontrol abilities were evaluated as well (e.g., phytase, xylanase, catalase, cellulase, chitinase, glucanase activities, and siderophore and HCN production). Multi-locus sequence analyses of conserved genes enabled the identification of these strains as Pseudomonas spp. Strain PICF141 was affiliated to the “Pseudomonas mandelii subgroup,” within the “Pseudomonas fluorescens group,” Pseudomonas lini being the closest species. Strains PIC25 and PIC105 were affiliated to the “Pseudomonas aeruginosa group,” Pseudomonas indica being the closest relative. Moreover, we identified P. indica (PIC105) for the first time as a BCA. Genome sequencing and in silico analyses allowed the identification of traits commonly associated with plant-bacteria interactions. Finally, the root colonization ability of these olive rhizobacteria was assessed, providing valuable information for the future development of formulations based on these strains. A set of actions, from rhizosphere isolation to genome analysis, is proposed and discussed for selecting indigenous rhizobacteria as effective BCAs.

Bacillales Members from the Olive Rhizosphere Are Effective Biological Control Agents against the Defoliating Pathotype of Verticillium dahliae

“…Strains PIC28, PIC73 and PIC167 were in vitro tested to assess their growth inhibition ability against several olive pathogens (Table 1), following an experimental setup previously described [16,27]. In addition, and in order to quantify the growth inhibition degree of V. dahliae, antagonism tests against a representative of the D pathotype (strain Lebrija 1, [16]) were carried out. Individual drops (10 µL) containing V. dahliae biomass (i.e., mycelium plus conidia, 1 × 10 6 conidia/mL) were placed in the middle of PDA plates, and four equidistant 10-µL drops of each bacterial strain (1 × 10 8 ufc/mL) were inoculated.…”

“…In these bioassays strains P. fluorescens PICF7 [31] and Pseudomonas sp. PICF141 [16] were included for comparative purposes because of their well-known biocontrol activity. Fifty-four (Experiment I; ExpI) ninety and (Experiment II; ExpII) 'Picual' plants (5-month-old) were grown in pots (11 × 11 × 11 cm; one plant per pot), each containing 300 g of potting substrate used in the nursery, and distributed in three random blocks (nine plants in ExpI and fifteen plants in ExpII per treatment) within a greenhouse under natural lighting and day/night temperature of 27/21 • C. For each bacterial treatment, olive plants were inoculated as described by Gómez-Lama Cabanás and co-workers [16].…”

“…Regarding biocontrol of VWO, advances are still limited, although some examples have shown promising results. For instance, olive root-associated fluorescent pseudomonads have been demonstrated to control VWO caused by the highly virulent, defoliating (D) pathotype of V. dahliae in young, nursery-produced plants [13][14][15][16]. Paenibacillus alvei K-165 has also been shown to suppress VWO under both greenhouse and field conditions [17].…”

“…Disease suppressive soils, organic amendments, and healthy plants enduring in pathogen-infested fields are interesting reservoirs of potential BCAs [5,11,16,20]. Microbial communities associated with plant roots are complex and diverse, and they strongly influence the health and development of the host [21].…”

“…In a previous study, we designed a holistic approach to identify, characterize and evaluate new and effective BCAs originating from the roots of healthy nursery-propagated olive plants [16]. This strategy was based on the isolation from the target niche, in vitro antagonism assays against relevant olive pathogens (V. dahliae, Phytophthora cinnamomi Rands., Pseudomonas savastanoi (Janse) Gardan et al), phenotypic and metabolic characterization in order to identify traits associated with biological control or plant growth promotion (PGP), in planta experiments under non-gnotobiotic conditions to assess their effectiveness against the D pathotype of V. dahliae, genome sequencing and in silico analysis, taxonomic identification by multi-locus sequence analyses (MLSA), and evaluation of root colonization abilities [16]. Eventually, we were able to identify and in-depth characterized three novel Gram-negative resident olive rhizobacteria belonging to the Pseudomonas genus (namely, strains PIC25, PIC105 and PICF141) showing good biocontrol ability against VWO.…”