Identification, characterization and involvement of specific Pseudomonas simiae PICF7 genes in belowground colonization and biocontrol of Verticillium wilt of olive

Abstract

Olive is the most iconic tree crop in the Mediterranean Basin. Verticillium wilt of olive (VWO), caused by Verticillium dahliae, is present in most of the olive growing areas. The disease is very difficult to control, posing a major threat for the olive oil industry. The use of beneficial microorganisms represents an excellent option to be used in combination with other control tools within an integrated disease management strategy. Pseudomonas simiae(fluorescens) PICF7 is an indigenous inhabitant of the olive rhizosphere/root endosphere and an effective biocontrol agent against VWO. Two objectives were pursued: the identification and characterization of genes involved in phenotypes such as rhizosphere/soil persistence (copper resistance), efficient root colonization (biofilm formation), plant growth promotion (phytase activity) and VWO biocontrol. An available Tn5 random insertion mutant bank was screened (> 5,500 tetracycline-resistant colonies) to select mutants affected in one of the traits mentioned above. The identification of the disrupted genes was performed by nested-PCR and DNA sequencing. Mutants that showed disruption of putative genes coding for: (i) the transcriptional regulator CusR or the chemotaxis protein CheW, impairing PICF7’s growth in medium supplemented with Cu2+, (ii) a membrane protein or a flagellar regulatory protein, generating biofilm formation defective phenotypes, and (iii) histidinol-phosphate aminotransferase or hemolysin D that abolished phytase activity, were eventually selected. Biofilm-defective and copper-sensitive mutants displayed the same antagonistic effect against V. dahliaethan the parental strain in different culturing media (Potato dextrose agar,[PDA], nutrient agar and Waksman's agar). In contrast, phytase-defective mutants lost this ability in PDA. In planta bioassays were conducted using the olive cultivar Picual. Root colonization ability of PICF7 mutants was assessed by confocal laser scanning microscope and fluorescently- labelled derivatives of each mutant. Colonization pattern of PICF7 and that of phytase and copper mutants were similar. Biofilm-defective mutants showed good olive rhizosphere/rhizoplane colonization, although no evidence of endophytic behaviour was detected in this case. Results from greenhouse biological control assays showed that all tested mutants displayed similar VWO control performance than strain PICF7. This indicates that the genes here studied are not involved in biocontrol of VWO.