Pseudomonas fluorescens PICF7 traits involved in the biocontrol of Verticillium wilt of olive

Ponencia presentada en el 11 th International Verticillium Symposium, celebrado en Göttingen (Alemania) del 5 al 8 de mayo de 2013.
Verticillium wilt of olive (VWO), caused by Verticillium dahliae Kleb., is one of the most important biotic constrains affecting olive. The incidence and severity of this disease has increased during the last decades and its effective control requires implementation of an integrated disease management strategy, with emphasis on preventive measures. The use of biological control agents (BCA) appears as an excellent before-planting measure since they can be applied during the nursery production stage. Pseudomonas fluorescens PICF7 is a natural inhabitant of olive roots and an effective BCA against V. dahliae. Strain PICF7 displays some traits in vitro which may explain its biocontrol activity. For instance, growth inhibition of V. dahliae and production of salicylic acid and the siderophore pyoverdine have been demonstrated, as well as swimming motility which can be important for rhizosphere colonization. Moreover, PICF7 is able to endophytically colonize olive roots and induces a broad range of defense responses. However, mechanism(s) underlying its biocontrol activity remain mostly unknown. To unravel traits involved in the suppressive effect against VWO, a mutant bank of P. fluorescens PICF7 was generated (>9.000 insertions) by random transposon Tn5 mutagenesis. Phenotypes affected in swimming motility, siderophore production and growth inhibition of V. dahliae were screened. A collection of selected mutants altered in one of these phenotypes were analyzed by nested-PCR to localize the Tn5 insertion site. Adjacent DNA regions to the insertion point were sequenced and compared against available databases. Four mutants were finally chosen to conduct in planta bioassays: ME424, affected in motility (insertion in fliI homologue); ME589, a siderophore mutant with insertion located in a gene coding for a putative pyoverdine nonribosomal peptide synthetase; ME419, showing enhanced growth inhibition of V. dahliae (undetermined Tn5 insertion); and ME1508, a mutant displaying diminished growth inhibition of the pathogen (insertion in a gene encoding a putative sulfite reductase). Biocontrol bioassays using nursery-produced olive plants (cv. Picual) have consistently shown that mutant ME1508 has significantly lost the ability to control V. dahliae (defoliating [D] pathotype). However, the remaining mutants displayed variable biocontrol performance among bioassays. Arabidopsis thaliana has been also used to evaluate these mutants. This model plant was first checked for: i) Verticillium wilt symptoms development upon inoculation with an olive V. dahliae D isolate; ii) successful rhizosphere colonization by strain PICF7; and iii) effective control of the disease by the BCA. Results were similar to those observed in olive plants: mutant ME1508 had significantly lost the biocontrol ability compared to PICF7. Thus, the gene disrupted in ME1508 seems to play an important role in biocontrol of PICF7 against V. dahliae. However, phenotypes such as motility and siderophore production either do not participate in the suppressive effect or are greatly influenced by the experimental conditions used.
Research supported by grants AGL2009-07275 from Spanish MICINN/MINECO and P07-CVI-02624 from J. Andalucía (Spain), both co-financed by ERDF of the EU.