Verticillium wilt resistant and susceptible olive cultivars express a very different basal set of genes in roots.

BACKGROUND: Olive orchards are threatened by a wide range of pathogens. Of these, Verticillium dahliae has been in the spotlight for its high incidence, the difficulty to control it and the few cultivars that has increased tolerance to the pathogen. Disease resistance not only depends on detection of pathogen invasion and induction of responses by the plant, but also on barriers to avoid the invasion and active resistance mechanisms constitutively expressed in the absence of the pathogen. In a previous work we found that two healthy non-infected plants from cultivars that differ in V. dahliae resistance such as 'Frantoio' (resistant) and 'Picual' (susceptible) had a different root morphology and gene expression pattern. In this work, we have addressed the issue of basal differences in the roots between Resistant and Susceptible cultivars. RESULTS: The gene expression pattern of roots from 29 olive cultivars with different degree of resistance/susceptibility to V. dahliae was analyzed by RNA-Seq. However, only the Highly Resistant and Extremely Susceptible cultivars showed significant differences in gene expression among various groups of cultivars. A set of 421 genes showing an inverse differential expression level between the Highly Resistant to Extremely Susceptible cultivars was found and analyzed. The main differences involved higher expression of a series of transcription factors and genes involved in processes of molecules importation to nucleus, plant defense genes and lower expression of root growth and development genes in Highly Resistant cultivars, while a reverse pattern in Moderately Susceptible and more pronounced in Extremely Susceptible cultivars were observed. CONCLUSION: According to the different gene expression patterns, it seems that the roots of the Extremely Susceptible cultivars focus more on growth and development, while some other functions, such as defense against pathogens, have a higher expression level in roots of Highly Resistant cultivars. Therefore, it seems that there are constitutive differences in the roots between Resistant and Susceptible cultivars, and that susceptible roots seem to provide a more suitable environment for the pathogen than the resistant ones.

Epigenetic Regulation of Verticillium dahliae Virulence: Does DNA Methylation Level Play A Role?

Verticillium dahliae is the etiological agent of Verticillium wilt of olive. The virulence of Defoliating V. dahliae isolates usually displays differences and high plasticity. This work studied whether an epigenetic mechanism was involved in this plasticity. An inverse correlation between virulence and DNA methylation of protein-coding genes was found. A set of 831 genes was selected for their highly consistent inverse methylation profile and virulence in the five studied isolates. Of these genes, ATP-synthesis was highly represented, which indicates that the more virulent D isolates are, the more energy requirements they may have. Furthermore, there were numerous genes in the protein biosynthesis process: genes coding for the chromatin structure, which suggests that epigenetic changes may also affect chromatin condensation; many transmembrane transporter genes, which is consistent with denser compounds, traffic through membranes in more virulent isolates; a fucose-specific lectin that may play a role in the attachment to plant cell walls during the host infection process; and pathogenic cutinases that facilitate plant invasion and sporulation genes for rapid spreading alongside plants. Our findings support the notion that differences in the virulence of the Defoliating V. dahliae isolates may be controlled, at least to some extent, by an epigenetic mechanism.

Efficient colonization of the endophytes Herbaspirillum huttiense RCA24 and Enterobacter cloacae RCA25 influences the physiological parameters of Oryza sativa L. cv. Baldo rice.

Several important bacterial characteristics, such as biological nitrogen fixation, phosphate solubilization, 1-aminocyclopropane-1-carboxylate deaminase activity and production of siderophores and phytohormones, can be assessed as plant growth promotion traits. Our aim was to evaluate the effects of nitrogen fixing and indole-3-acetic acid (IAA) producing endophytes in two Oryza sativa cultivars (Baldo and Vialone Nano). Three bacteria, Herbaspirillum huttiense RCA24, Enterobacter asburiae RCA23 and Staphylococcus sp. 377, producing different IAA levels, were tested for their ability to enhance nifH gene expression and nitrogenase activity in Enterobacter cloacae RCA25. Results showed that H. huttiense RCA24 performed best. Improvement in nitrogen fixation and changes in physiological parameters such as chlorophyll, nitrogen content and shoot dry weight were observed for plants co-inoculated with strains RCA25 and RCA24 in a 10:1 ratio. Based on confocal laser scanning microscopy analysis, strain RCA24 was the best colonizer of the root interior and the only IAA producer located in the same root niche occupied by RCA25 cells. This work shows that the choice of a bio-inoculum having the right composition is one of the key aspects to be considered for the inoculation of a specific host plant cultivar with microbial consortia.

Tolerance of olive (Olea europaea) cv Frantoio to Verticillium dahliae relies on both basal and pathogen-induced differential transcriptomic responses.

Verticillium wilt of olive (VWO) is one of the most serious biotic constraints for this tree crop. Our knowledge of the genetics of the tolerance/resistance to this disease is very limited. Here we show that tolerance of the cv Frantoio relies on both basal and early pathogen-induced differential transcriptomic responses. A comparative transcriptomic analysis (RNA-seq) was conducted in root tissues of cvs Frantoio (VWO-tolerant) and Picual (VWO-susceptible). RNA samples originated from roots of inoculated olive plants during the early infection stages by Verticillium dahliae (highly virulent, defoliating pathotype). A huge number of differentially expressed genes (DEGs) were found between 'Frantoio' and 'Picual' (27 312 unigenes) in the absence of the pathogen. Upon infection with V. dahliae, 'Picual' and 'Frantoio' plants responded differently too. In the early infection stages, four clusters of DEGs could be identified according to their time-course expression patterns. Among others, a pathogenesis-related protein of the Bet v I family and a dirigent-like protein involved in lignification, and several BAK1, NHL1, reactive oxygen species stress response and BAM unigenes showed noticeable differences between cultivars. Tolerance of 'Frantoio' plants to VWO is a consequence of a complex and multifaceted process which involves many plant traits.

Major cereal crops benefit from biological nitrogen fixation when inoculated with the nitrogen-fixing bacterium Pseudomonas protegens Pf-5 X940.

A main goal of biological nitrogen fixation research has been to expand the nitrogen-fixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15 N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops.

Endophytic colonization and biocontrol performance of Pseudomonas fluorescens†PICF7 in olive (Olea europaea L.) are determined neither by pyoverdine production nor swimming motility.

Pseudomonas fluorescens PICF7 is an indigenous inhabitant of olive (Olea europaea L.) rhizosphere, able to display endophytic lifestyle in roots, to induce a wide range of defence responses upon colonization of this organ and to exert effective biological control against Verticillium wilt of olive (VWO) (Verticillium dahliae). We aimed to evaluate the involvement of specific PICF7 phenotypes in olive root colonization and VWO biocontrol effectiveness by generating mutants impaired in swimming motility (fliI) or siderophore pyoverdine production (pvdI). Besides, the performance of mutants with diminished in vitro growth in potato dextrose agar medium (gltA) and cysteine (Cys) auxotrophy was also assessed. Results showed that olive root colonization and VWO biocontrol ability of the fliI, pvdI and gltA mutants did not significantly differ from that displayed by the parental strain PICF7. Consequently, altered in vitro growth, swimming motility and pyoverdine production contribute neither to PICF7 VWO suppressive effect nor to its colonization ability. In contrast, the Cys auxotroph mutant showed reduced olive root colonization capacity and lost full biocontrol efficacy. Moreover, confocal laser scanning microscopy revealed that all mutants tested were able to endophytically colonize root tissue to the same extent as wild-type PICF7, discarding these traits as relevant for its endophytic lifestyle.

Mediterranean pine forest decline: A matter of root-associated microbiota and climate change.

Forest ecosystems worldwide currently face worrying episodes of forest decline, which have boosted weakening and mortality of the trees. In the Mediterranean region, especially in the southeast Iberian Peninsula, Pinus sylvestris forests are severely affected by this phenomenon, and it has been commonly attributed to drought events. Remarkably, the role of root microbiota on pine decline has been overlooked and remains unclear. We therefore used metabarcoding to identify the belowground microbial communities of decline-affected and unaffected pine trees. Taxonomic composition of bacterial and fungal rhizosphere communities, and fungal populations dwelling in root endosphere showed different profiles depending on the health status of the trees. The root endosphere of asymptomatic trees was as strongly dominated by 'Candidatus Phytoplasma pini' as the root of decline-affected pines, accounting for >99 % of the total bacterial sequences in some samples. Notwithstanding, the titer of this phytopathogen was four-fold higher in symptomatic trees than in symptomless ones. Furthermore, the microbiota inhabiting the root endosphere of decline-affected trees assembled into a less complex and more modularized network. Thus, the observed changes in the microbial communities could be a cause or a consequence of forest decline phenomenon. Moreover, 'Ca. Phytoplasma pini' is positively correlated to Pinus sylvestris decline events, either as the primary cause of pine decline or as an opportunistic pathogen exacerbating the process once the tree has been weaken by other factors.

Pitting the olive seed microbiome.

BACKGROUND: The complex and co-evolved interplay between plants and their microbiota is crucial for the health and fitness of the plant holobiont. However, the microbiota of the seeds is still relatively unexplored and no studies have been conducted with olive trees so far. In this study, we aimed to characterize the bacterial, fungal and archaeal communities present in seeds of ten olive genotypes growing in the same orchard through amplicon sequencing to test whether the olive genotype is a major driver in shaping the seed microbial community, and to identify the origin of the latter. Therefore, we have developed a methodology for obtaining samples from the olive seed's endosphere under sterile conditions. RESULTS: A diverse microbiota was uncovered in olive seeds, the plant genotype being an important factor influencing the structure and composition of the microbial communities. The most abundant bacterial phylum was Actinobacteria, accounting for an average relative abundance of 41%. At genus level, Streptomyces stood out because of its potential influence on community structure. Within the fungal community, Basidiomycota and Ascomycota were the most abundant phyla, including the genera Malassezia, Cladosporium, and Mycosphaerella. The shared microbiome was composed of four bacterial (Stenotrophomonas, Streptomyces, Promicromonospora and Acidipropionibacterium) and three fungal (Malassezia, Cladosporium and Mycosphaerella) genera. Furthermore, a comparison between findings obtained here and earlier results from the root endosphere of the same trees indicated that genera such as Streptomyces and Malassezia were present in both olive compartments. CONCLUSIONS: This study provides the first insights into the composition of the olive seed microbiota. The highly abundant fungal genus Malassezia and the bacterial genus Streptomyces reflect a unique signature of the olive seed microbiota. The genotype clearly shaped the composition of the seed's microbial community, although a shared microbiome was found. We identified genera that may translocate from the roots to the seeds, as they were present in both organs of the same trees. These findings set the stage for future research into potential vertical transmission of olive endophytes and the role of specific microbial taxa in seed germination, development, and seedling survival.

Confronting stresses affecting olive cultivation from the holobiont perspective.

The holobiont concept has revolutionized our understanding of plant-associated microbiomes and their significance for the development, fitness, growth and resilience of their host plants. The olive tree holds an iconic status within the Mediterranean Basin. Innovative changes introduced in olive cropping systems, driven by the increasing demand of its derived products, are not only modifying the traditional landscape of this relevant commodity but may also imply that either traditional or emerging stresses can affect it in ways yet to be thoroughly investigated. Incomplete information is currently available about the impact of abiotic and biotic pressures on the olive holobiont, what includes the specific features of its associated microbiome in relation to the host's structural, chemical, genetic and physiological traits. This comprehensive review consolidates the existing knowledge about stress factors affecting olive cultivation and compiles the information available of the microbiota associated with different olive tissues and organs. We aim to offer, based on the existing evidence, an insightful perspective of diverse stressing factors that may disturb the structure, composition and network interactions of the olive-associated microbial communities, underscoring the importance to adopt a more holistic methodology. The identification of knowledge gaps emphasizes the need for multilevel research approaches and to consider the holobiont conceptual framework in future investigations. By doing so, more powerful tools to promote olive's health, productivity and resilience can be envisaged. These tools may assist in the designing of more sustainable agronomic practices and novel breeding strategies to effectively face evolving environmental challenges and the growing demand of high quality food products.

Co-occurrence network analysis unveils the actual differential impact on the olive root microbiota by two Verticillium wilt biocontrol rhizobacteria.

BACKGROUND: Verticillium wilt of olive (VWO), caused by Verticillium dahliae Kleb, is one of the most threatening diseases affecting olive cultivation. An integrated disease management strategy is recommended for the effective control of VWO. Within this framework, the use of biological control agents (BCAs) is a sustainable and environmentally friendly approach. No studies are available on the impact that the introduction of BCAs has on the resident microbiota of olive roots. Pseudomonas simiae PICF7 and Paenibacillus polymyxa PIC73 are two BCAs effective against VWO. We examined the effects of the introduction of these BCAs on the structure, composition and co-occurrence networks of the olive (cv. Picual) root-associated microbial communities. The consequences of the subsequent inoculation with V. dahliae on BCA-treated plants were also assessed. RESULTS: Inoculation with any of the BCAs did not produce significant changes in the structure or the taxonomic composition of the 'Picual' root-associated microbiota. However, significant and distinctive alterations were observed in the topologies of the co-occurrence networks. The introduction of PIC73 provoked a diminution of positive interactions within the 'Picual' microbial community; instead, PICF7 inoculation increased the microbiota's compartmentalization. Upon pathogen inoculation, the network of PIC73-treated plants decreased the number of interactions and showed a switch of keystone species, including taxa belonging to minor abundant phyla (Chloroflexi and Planctomycetes). Conversely, the inoculation of V. dahliae in PICF7-treated plants significantly increased the complexity of the network and the number of links among their modules, suggestive of a more stable network. No changes in their keystone taxa were detected. CONCLUSION: The absence of significant modifications on the structure and composition of the 'Picual' belowground microbiota due to the introduction of the tested BCAs underlines the low/null environmental impact of these rhizobacteria. These findings may have important practical consequences regarding future field applications of these BCAs. Furthermore, each BCA altered the interactions among the components of the olive belowground microbiota in idiosyncratic ways (i.e. PIC73 strongly modified the number of positive relations in the 'Picual' microbiota whereas PICF7 mostly affected the network stability). These modifications may provide clues on the biocontrol strategies used by these BCAs.

Thriving beneath olive trees: The influence of organic farming on microbial communities.

Soil health and root-associated microbiome are interconnected factors involved in plant health. The use of manure amendment on agricultural fields exerts a direct benefit on soil nutrient content and water retention, among others. However, little is known about the impact of manure amendment on the root-associated microbiome, particularly in woody species. In this study, we aimed to evaluate the effects of ovine manure on the microbial communities of the olive rhizosphere and root endosphere. Two adjacent orchards subjected to conventional (CM) and organic (OM) management were selected. We used metabarcoding sequencing to assess the bacterial and fungal communities. Our results point out a clear effect of manure amendment on the microbial community. Fungal richness and diversity were increased in the rhizosphere. The fungal biomass in the rhizosphere was more than doubled, ranging from 1.72 × 106 ± 1.62 × 105 (CM) to 4.54 × 106 ± 8.07 × 105 (OM) copies of the 18 S rRNA gene g-1 soil. Soil nutrient content was also enhanced in the OM orchard. Specifically, oxidable organic matter, total nitrogen, nitrate, phosphorous, potassium and sulfate concentrations were significantly increased in the OM orchard. Moreover, we predicted a higher abundance of bacteria in OM with metabolic functions involved in pollutant degradation and defence against pathogens. Lastly, microbial co-occurrence network showed more positive interactions, complexity and shorter geodesic distance in the OM orchard. According to our results, manure amendment on olive orchards represents a promising tool for positively modulating the microbial community in direct contact with the plant.

Physiological and Structural Responses of Olive Leaves Related to Tolerance/Susceptibility to Verticillium dahliae.

Verticillium wilt of olive (VWO), caused by the soil borne fungus Verticillium dahliae, is one of the most relevant diseases affecting this crop worldwide. One of the best VWO management strategies is the use of tolerant cultivars. Scarce information is available about physiological and structural responses in the leaves of olive cultivars displaying different levels of tolerance to VWO. To identify links between this phenotype and variations in functional characteristics of the leaves, this study examined the structural and physiological traits and the correlations among them in different olive varieties. This evaluation was conducted in the presence/absence of V. dahliae. On the one hand, no leaf trait but the area was related to VWO tolerance in the absence of the pathogen. On the other hand, after inoculation, susceptible cultivars showed lower leaf area and higher leaf mass per area and dry matter content. Furthermore, at the physiological level, these plants showed severe symptoms resembling water stress. Analyzing the relationships among physiological and structural traits revealed differences between tolerant and susceptible cultivars both in the absence and in the presence of V. dahliae. These results showed that olive leaves of VWO-tolerant and VWO-susceptible cultivars adopt different strategies to cope with the pathogen.

Unveiling Differences in Root Defense Mechanisms Between Tolerant and Susceptible Olive Cultivars to Verticillium dahliae.

Verticillium wilt of olive (VWO), caused by the soil-borne vascular pathogen Verticillium dahliae, is one of the most devastating diseases affecting olive, the woody crop. One of the best VWO management measures is the use of tolerant cultivars. Yet, our knowledge about defense mechanisms that operate at the root level to explain tolerance to this disease is incomplete. Moreover, most of the approaches so far followed focus only on a specific mechanistic level (e.g., genetic, physiological, or biochemical) rather than on a holistic/multilevel perspective. In this study, eighteen root functional traits, the time-course expression of nine defense-related genes, the root lignin content, and the root membrane permeability were evaluated in six olive varieties differing in their level of tolerance/susceptibility to VWO. The aim was to find links between the level of tolerance to VWO and specific root defense mechanisms at the structural, genetic, biochemical, and physiological levels. Tolerant and susceptible cultivars showed substantial differences in the root system architecture and root lignin content. VWO-susceptible cultivars presented roots with higher specific length and area, but lower diameter and larger number of forks and tips compared to tolerant varieties that also showed less branched roots, higher root diameter, and larger basal content of lignin. Interestingly, VWO-tolerant varieties significantly increased their root lignin content and root membrane permeability after inoculation with V. dahliae. These results were seldom (or not at all) observed in the susceptible plants. At the genetic level, genes related to defense mechanisms, such as cell wall lignin biosynthesis (C4H and CO-MT), production of hydrolytic enzymes able to degrade the fungal cell wall (ß-1.3-glucanase), and activation of innate immunity (BAK1 and WRKY5) increased their expression in tolerant cultivars from early moments after inoculation, in contrast to the susceptible ones. These results showed that differences in the root system architecture and lignin content may greatly determine the performance of olive against colonization and invasion by V. dahliae. Moreover, the increase in root membrane permeability in the presence of the pathogen was a typical response of tolerant cultivars. Finally, VWO-tolerant cultivars were able to mount a more intense and rapid defense-related genetic response to respond to the attack by V. dahliae.

Identification of Volatile Organic Compounds Emitted by Two Beneficial Endophytic Pseudomonas Strains from Olive Roots.

The production of volatile organic compounds (VOCs) represents a promising strategy of plant-beneficial bacteria to control soil-borne phytopathogens. Pseudomonas sp. PICF6 and Pseudomonas simiae PICF7 are two indigenous inhabitants of olive roots displaying effective biological control against Verticillium dahliae. Additionally, strain PICF7 is able to promote the growth of barley and Arabidopsis thaliana, VOCs being involved in the growth of the latter species. In this study, the antagonistic capacity of these endophytic bacteria against relevant phytopathogens (Verticillium spp., Rhizoctonia solani, Sclerotinia sclerotiorum and Fusarium oxysporum f.sp. lycopersici) was assessed. Under in vitro conditions, PICF6 and PICF7 were only able to antagonize representative isolates of V. dahliae and V. longisporum. Remarkably, both strains produced an impressive portfolio of up to twenty VOCs, that included compounds with reported antifungal (e.g., 1-undecene, (methyldisulfanyl) methane and 1-decene) or plant growth promoting (e.g., tridecane, 1-decene) activities. Moreover, their volatilomes differed strongly in the absence and presence of V. dahliae. For example, when co incubated with the defoliating pathotype of V. dahliae, the antifungal compound 4-methyl-2,6-bis(2-methyl-2-propanyl)phenol was produced. Results suggest that volatiles emitted by these endophytes may differ in their modes of action, and that potential benefits for the host needs further investigation in planta.

Designing a synthetic microbial community devoted to biological control: The case study of Fusarium wilt of banana.

Fusarium oxysporum f. sp. cubense (Foc) tropical race 4 (TR4) is threatening banana production because of its increasing spread. Biological control approaches have been widely studied and constitute interesting complementary measures to integrated disease management strategies. They have been based mainly on the use of single biological control agents (BCAs). In this study, we moved a step forward by designing a synthetic microbial community (SynCom) for the control of Fusarium wilt of banana (FWB). Ninety-six isolates of Pseudomonas spp., Bacillus spp., Streptomyces spp., and Trichoderma spp. were obtained from the banana rhizosphere and selected in vitro for the antagonism against Foc TR4. In pot experiments, a large community such as SynCom 1.0 (44 isolates with moderate to high antagonistic activity) or a small one such as SynCom 1.1 (seven highly effective isolates) provided similar disease control (35% symptom severity reduction). An in vitro study of the interactions among SynCom 1.1 isolates and between them and Foc revealed that beneficial microorganisms not only antagonized the pathogen but also some of the SynCom constituents. Furthermore, Foc defended itself by antagonizing the beneficial microbes. We also demonstrated that fusaric acid, known as one of the secondary metabolites of Fusarium species, might be involved in such an interaction. With this knowledge, SynCom 1.2 was then designed with three isolates: Pseudomonas chlororaphis subsp. piscium PS5, Bacillus velezensis BN8.2, and Trichoderma virens T2C1.4. A non-simultaneous soil application of these isolates (to diminish cross-inhibition) delayed FWB progress over time, with significant reductions in incidence and severity. SynCom 1.2 also performed better than two commercial BCAs, BioPak® and T-Gro. Eventually, SynCom 1.2 isolates were characterized for several biocontrol traits and their genome was sequenced. Our data showed that assembling a SynCom for biocontrol is not an easy task. The mere mixtures of antagonists (e.g., SynCom 1.0 and 1.1) might provide effective biocontrol, but an accurate investigation of the interactions among beneficial microorganisms is needed to improve the results (e.g., SynCom 1.2). SynCom 1.2 is a valuable tool to be further developed for the biological control of FWB.

Impacts of the Biocontrol Strain Pseudomonas simiae PICF7 on the Banana Holobiont: Alteration of Root Microbial Co-occurrence Networks and Effect on Host Defense Responses.

The impact of the versatile biocontrol and plant-growth-promoting rhizobacteria Pseudomonas simiae PICF7 on the banana holobiont under controlled conditions was investigated. We examine the fate of this biological control agent (BCA) upon introduction in the soil, the effect on the banana root microbiota, and the influence on specific host genetic defense responses. While the presence of strain PICF7 significantly altered neither the composition nor the structure of the root microbiota, a significant shift in microbial community interactions through co-occurrence network analysis was observed. Despite the fact that PICF7 did not constitute a keystone, the topology of this network was significantly modified-the BCA being identified as a constituent of one of the main network modules in bacterized plants. Gene expression analysis showed the early suppression of several systemic acquired resistance and induced systemic resistance (ISR) markers. This outcome occurred at the time in which the highest relative abundance of PICF7 was detected. The absence of major and permanent changes on the banana holobiont upon PICF7 introduction poses advantages regarding the use of this beneficial rhizobacteria under field conditions. Indeed a BCA able to control the target pathogen while altering as little as possible the natural host-associated microbiome should be a requisite when developing effective bio-inoculants.

Corrigendum to "Coupling the endophytic microbiome with the host transcriptome in olive roots" [Comput. Struct. Biotechnol. J. 19 (2021) 4777-4789].

[This corrects the article DOI: 10.1016/j.csbj.2021.08.035.].

Root hairs play a key role in the endophytic colonization of olive roots by Pseudomonas spp. with biocontrol activity.

The use of indigenous bacterial root endophytes with biocontrol activity against soil-borne phytopathogens is an environmentally-friendly and ecologically-efficient action within an integrated disease management framework. The earliest steps of olive root colonization by Pseudomonas fluorescens PICF7 and Pseudomonas putida PICP2, effective biocontrol agents (BCAs) against Verticillium wilt of olive (Olea europaea L.) caused by the fungus Verticillium dahliae Kleb., are here described. A gnotobiotic study system using in vitro propagated olive plants, differential fluorescent-protein tagging of bacteria, and confocal laser scanning microscopy analysis have been successfully used to examine olive roots-Pseudomonas spp. interactions at the single-cell level. In vivo simultaneous visualization of PICF7 and PICP2 cells on/in root tissues enabled to discard competition between the two bacterial strains during root colonization. Results demonstrated that both BCAs are able to endophytically colonized olive root tissues. Moreover, results suggest a pivotal role of root hairs in root colonization by both biocontrol Pseudomonas spp. However, colonization of root hairs appeared to be a highly specific event, and only a very low number of root hairs were effectively colonized by introduced bacteria. Strains PICF7 and PICP2 can simultaneously colonize the same root hair, demonstrating that early colonization of a given root hair by one strain did not hinder subsequent attachment and penetration by the other. Since many environmental factors can affect the number, anatomy, development, and physiology of root hairs, colonization competence and biocontrol effectiveness of BCAs may be greatly influenced by root hair's fitness. Finally, the in vitro study system here reported has shown to be a suitable tool to investigate colonization processes of woody plant roots by microorganisms with biocontrol potential.

Functional Traits of Olive Varieties and Their Relationship with the Tolerance Level towards Verticillium Wilt.

Verticillium wilt of olive (VWO), caused by the soil-borne pathogen Verticillium dahliae, is considered one of the most important diseases affecting this tree crop. One of the best VWO management measures is the use of tolerant cultivars. Remarkably, no information is available about olive functional traits and their potential relationship with tolerance to V. dahliae. Twenty-five selected functional traits (for leaf, stem, root and whole plant) were evaluated in six olive varieties differing in their VWO tolerance level to identify possible links between this phenotype and functional traits' variation. High intervarietal diversity was found among cultivars and several functional traits were related with VWO tolerance. Tolerant varieties showed higher leaf area, dry matter content (leaf, stem and plant) and mass fraction for stems, but lower for leaves. Significant differences were also detected for root functional traits, tolerant cultivars displaying larger fine root diameter and lignin content but smaller specific length and area of thick and fine roots. Correlations were found among functional traits both within varieties and between levels of tolerance/susceptibility to VWO. Associations were observed between biomass allocation, dry matter content and VWO tolerance. The most relevant difference between tolerant and susceptible cultivars was related to root system architecture.

The Banana Root Endophytome: Differences between Mother Plants and Suckers and Evaluation of Selected Bacteria to Control Fusarium oxysporum f.sp. cubense.

This study aimed to disentangle the structure, composition, and co-occurrence relationships of the banana (cv. Dwarf Cavendish) root endophytome comparing two phenological plant stages: mother plants and suckers. Moreover, a collection of culturable root endophytes (>1000) was also generated from Canary Islands. In vitro antagonism assays against Fusarium oxysporum f.sp. cubense (Foc) races STR4 and TR4 enabled the identification and characterization of potential biocontrol agents (BCA). Eventually, three of them were selected and evaluated against Fusarium wilt of banana (FWB) together with the well-known BCA Pseudomonas simiae PICF7 under controlled conditions. Culturable and non-culturable (high-throughput sequencing) approaches provided concordant information and showed low microbial diversity within the banana root endosphere. Pseudomonas appeared as the dominant genus and seemed to play an important role in the banana root endophytic microbiome according to co-occurrence networks. Fungal communities were dominated by the genera Ophioceras, Cyphellophora, Plecosphaerella, and Fusarium. Overall, significant differences were found between mother plants and suckers, suggesting that the phenological stage determines the recruitment and organization of the endophytic microbiome. While selected native banana endophytes showed clear antagonism against Foc strains, their biocontrol performance against FWB did not improve the outcome observed for a non-indigenous reference BCA (strain PICF7).