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.

Knowledge on the genetic basis underlying interactions between beneficial bacteria and woody plants is still very limited, and totally absent in the case of olive. We aimed to elucidate genetic responses taking place during the colonization of olive roots by the native endophyte Pseudomonas fluorescens PICF7, an effective biocontrol agent against Verticillium wilt of olive. Roots of olive plants grown under non-gnotobiotic conditions were collected at different time points after PICF7 inoculation. A Suppression Subtractive Hybridization cDNA library enriched in induced genes was generated. Quantitative real time PCR (qRT-PCR) analysis validated the induction of selected olive genes. Computational analysis of 445 olive ESTs showed that plant defence and response to different stresses represented nearly 45% of genes induced in PICF7-colonized olive roots. Moreover, quantitative real-time PCR (qRT-PCR) analysis confirmed induction of lipoxygenase, phenylpropanoid, terpenoids and plant hormones biosynthesis transcripts. Different classes of transcription factors (i.e., bHLH, WRKYs, GRAS1) were also induced. This work highlights for the first time the ability of an endophytic Pseudomonas spp. strain to mount a wide array of defence responses in an economically-relevant woody crop such as olive, helping to explain its biocontrol activity.

Important losses in strawberry production are caused by species of the fungus Colletotrichum, the causal agent of anthracnose. However, very limited studies at molecular level exist of the mechanisms related to strawberry susceptibility against this pathogen. We have analysed a moderately resistant cultivar (cv. Andana) together with a very susceptible one (cv. Camarosa) during the process of infection with Colletotrichum acutatum at a molecular level. To gain insight into this interaction we have identified a large number of strawberry genes involved in signalling, transcriptional control, defence and many genes with unknown function with altered expression in response to C. acutatum infection. Spatial and temporal gene expression profiles after infection showed that the response was dependant on the tissue and cultivar analysed and also quicker and/or stronger in the moderately resistant cultivar (cv. Andana) than in the susceptible one (cv. Camarosa). Interestingly, we found that genes described as being induced during pathogen infection such as g-thionins, peroxidases, chitinases and b-1-3-glucanases were downregulated in fruit and/ or crown tissues of the very susceptible cultivar. Our results yielded a first insight on some of the genes responding to this plant-pathogen interaction at molecular level and suggest that pathogen progression can be dependent upon a reduction of the active defences of strawberry and this is genotype and tissue dependent.