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.
Introduction
Cultivated olive (Olea europaea L. subsp. europaea var. europaea) is one of the most important oil crops in the world. It constitutes an agro-ecosystem of major relevance for the Mediterranean Basin since 90% of the global olive oil and table olive production is concentrated in this area (FAOSTAT, 2016) 1. Severe losses, and even tree death, are caused by a range of olive pathogens. Among them, the soilborne fungus Verticillium dahliae Kleb., causing Verticillium wilt of olive (VWO), represents a major threat in many regions where this tree is cultivated. Currently, however, no individual measure has proven effective to control VWO, and an integrated disease management strategy is therefore highly recommended (López-Escudero and Mercado-Blanco, 2011). Within this holistic framework, the development and implementation of sustainable and eco-friendly disease control measures is essential.
Plants have co-evolved with specific communities of microorganisms (i.e., the plant microbiome) that play crucial roles for the host’s development and health (Berg et al., 2017). Moreover, many components of the plant-associated microbiome, particularly at the rhizosphere level, may constitute the first line of defense against soilborne pathogens (Weller et al., 2007). Hence, the plant rhizosphere constitutes an important, yet insufficiently explored, reservoir of microorganisms with antagonist ability against pathogens. In this sense, the isolation, identification, and characterization of microorganisms with biocontrol potential and able of colonize, endure, and be adapted to a complex niche such as the rhizosphere constitute an interesting disease management strategy. The utilization of biological control agents (BCAs) to suppress pathogens has been studied in several pathosystems involving woody plants (e.g., Pliego and Cazorla, 2012; Kalai-Grami et al., 2014), although it has been implemented to a lesser extent compared to herbaceous species, annual crops, and seedlings (Cazorla and Mercado-Blanco, 2016). Moreover, available information on the diversity and structure of microbial communities associated with woody plants is scant (e.g., Aranda et al., 2011; Zarraonaindia et al., 2015), particularly at the nursery propagation stage (Sun et al., 2014). Likewise, our understanding on woody plant-BCA-pathogen interactions are still limited, i.e., mechanisms underlying biological control, influence of environmental factors, effectiveness of BCAs, interaction between a BCA and the plant microbiome once the former is released, BCA colonization ability, or plant responses to the BCA are issues that still need to be studied in more detail (Cazorla and Mercado-Blanco, 2016). In this sense, omics technologies are contributing to enhance our understanding of these tripartite interactions (Massart et al., 2015). However, their implementation in woody plants is considerably lower than in herbaceous species (e.g., Mgbeahuruike et al., 2013; Gómez-Lama Cabanás et al., 2014; Martínez-García et al., 2015b).
Several studies have shown the potential, or even the true efficacy, of diverse beneficial microorganisms to suppress Verticillium wilts in different hosts (revised by Deketelaere et al., 2017), including olive (e.g., Aranda et al., 2011; Carrero-Carrón et al., 2016; Markakis et al., 2016). Among them, some Pseudomonas spp. strains are highly competent in colonizing the rhizosphere (Lugtenberg et al., 2001) and able to suppress the deleterious effects caused by different pathogens (Haas and Défago, 2005; Mercado-Blanco, 2015). Some strains of P. fluorescens and P. putida have thus been demonstrated as effective BCA against VWO (Mercado-Blanco et al., 2004; Prieto et al., 2009; Maldonado-González et al., 2015b) under different experimental conditions. Nevertheless, most of the studies related with biological control of Verticillium wilts have been conducted under controlled or gnotobiotic conditions. Indeed, BCA performance under field conditions is a scenario not frequently explored in biocontrol research, particularly with woody plants (Markakis et al., 2016). Related to this, it is crucial to understand the complex trophic interactions taking place between a newly-introduced BCA and the indigenous microbial community present in a target site, as well as the influence that diverse biotic and abiotic factors can exert to them, thereby conditioning the performance and effectiveness of the BCA. It seems therefore reasonably to isolate, identify, and characterize beneficial microorganisms from the niche where they will be eventually deployed, since they are theoretically adapted to the environmental conditions they will confront upon release.
Performing in planta assays under non-gnotobiotic conditions is therefore crucial to assess the effectiveness of a potential BCA since parameters such as competition for nutrients and space, colonization ability, time of inoculation, mode and site of application, etc., can be evaluated in a near-natural scenario. This is particularly relevant in plants with large root systems as olive. For instance, P. fluorescens PICF7 (Martínez-García et al., 2015b), a natural root endophyte of olive roots (Prieto and Mercado-Blanco, 2008), has been demonstrated to be an efficient BCA against V. dahliae (Maldonado-González et al., 2015b). However, strain PICF7 seems to require a direct contact with the pathogen to display effective biocontrol, as it was recently demonstrated using a split-root study system (Gómez-Lama Cabanás et al., 2017).
Selection of novel effective BCAs is mostly dependent on the pathosystem under study. The combination of diverse screening methods and the inclusion of the host plant into the screening assay is essential to select BCAs acting through diverse modes of action that are not mutually exclusive (i.e., induced resistance, antibiosis, competition, mycoparasitism, etcetera). In addition, it is necessary to have a comprehensive knowledge of potential traits involved in colonization efficiency of the target site, biocontrol performance, and plant growth promotion efficacy. This key information is currently aided by implementing genomics approaches which, in addition, also assist to discard the presence of potential undesirable traits (i.e., pathogenicity and/or virulence factors) for plants, animals, and/or humans, phenotypes that must be ruled out when aiming to novel BCA-based formulations.
Considering this framework, the main objective of this study was to implement a strategy to isolate, identify, and in-depth characterize novel BCAs from the olive root/rhizosphere. This approach is based on the following major steps: (i) assessment of both in vitro and in planta effectiveness against diverse pathogens, (ii) genotypic and phenotypic characterization including features associated with biological control and plant growth promotion, (iii) metabolic profiling to obtain useful information related to microhabitat adaptation as well as to assist in the future development of bioformulations, (iv) genome sequencing and in silico analyses to allow taxonomic identification and to acquire essential knowledge on both the presence of beneficial traits and the absence of undesirable and/or potentially-harmful genotypes, and (v) evaluation of their root adhesion and colonization abilities. We test the hypothesis that the root/rhizosphere from young olive plants produced at nurseries is already an important source of beneficial bacteria which are also adapted to the target niche where they display biocontrol activity against VWO.
