Use of Arabidopsis thaliana to study mechanisms of control of Verticillium wilt by Pseudomonas fluorescens PICF7.

Verticillium wilt (VW), caused by Verticillium dahliae Kleb., is an important disease in many crops and its effective management has proven difficult. Among the various disease control measures to be implemented, the use of microbial antagonists (biological control agents, BCAs) constitutes an environmentally-friendly approach fitting criteria of modern sustainable agriculture. Pseudomonas fluorescens PICF7 was isolated from root tissues of nursery--propagated olive plants. Selection of this strain was based on in vitro growth inhibition of V. dahliae, colonizing ability of olive roots, endophytic lifestyle, and control of the highly-virulent defoliating (D) pathotype of V. dahliae in olive planting stocks. The mode of action by which PICF7 controls VW in olive is as yet unknown; moreover, to uncover potential biocontrol mechanisms poses additional difficulties in this pathosystem because the target is a tree. Therefore we used the model plant Arabidopsis thaliana to study: i) if PICF7 colonizes the rhizosphere of A. thaliana; ii) disease symptoms caused by V. dahliae in A. thaliana; iii) control of VW by PICF7 in different accessions and mutants of A. thaliana; and iv) if motility, antibiosis and/or siderophores are involved in control of V. dahliae by PICF7. Diverse bioassays were conducted and in all of them both the BCA and the pathogen were introduced in the rhizosphere of A. thaliana. Both D and non-defoliating isolates of V. dahliae caused disease symptoms in A. thaliana. PICF7 colonized and persisted in the rhizosphere of different Arabidopsis accessions and could control the D pathotype in some of them. PICF7 mutants affected in antibiosis significantly lost their ability to control VW in A. thaliana. We conclude that the model plant A. thaliana is useful to unravel interactions between this BCA and V. dahliae.

No role for bacterially produced salicylic Acid in rhizobacterial induction of systemic resistance in Arabidopsis.

ABSTRACT The role of bacterially produced salicylic acid (SA) in the induction of systemic resistance in plants by rhizobacteria is far from clear. The strong SA producer Pseudomonas fluorescens WCS374r induces resistance in radish but not in Arabidopsis thaliana, whereas application of SA leads to induction of resistance in both plant species. In this study, we compared P. fluorescens WCS374r with three other SA-producing fluorescent Pseudomonas strains, P. fluorescens WCS417r and CHA0r, and P. aeruginosa 7NSK2 for their abilities to produce SA under different growth conditions and to induce systemic resistance in A. thaliana against bacterial speck, caused by P. syringae pv. tomato. All strains produced SA in vitro, varying from 5 fg cell(-1) for WCS417r to >25 fg cell(-1) for WCS374r. Addition of 200 muM FeCl(3) to standard succinate medium abolished SA production in all strains. Whereas the incubation temperature did not affect SA production by WCS417r and 7NSK2, strains WCS374r and CHA0r produced more SA when grown at 33 instead of 28 degrees C. WCS417r, CHA0r, and 7NSK2 induced systemic resistance apparently associated with their ability to produce SA, but WCS374r did not. Conversely, a mutant of 7NSK2 unable to produce SA still triggered induced systemic resistance (ISR). The possible involvement of SA in the induction of resistance was evaluated using SA-nonaccumulating transgenic NahG plants. Strains WCS417r, CHA0r, and 7NSK2 induced resistance in NahG Arabidopsis. Also, WCS374r, when grown at 33 or 36 degrees C, triggered ISR in these plants, but not in ethylene-insensitive ein2 or in non-plant pathogenesis- related protein-expressing npr1 mutant plants, irrespective of the growth temperature of the bacteria. These results demonstrate that, whereas WCS374r can be manipulated to trigger ISR in Arabidopsis, SA is not the primary determinant for the induction of systemic resistance against bacterial speck disease by this bacterium. Also, for the other SAproducing strains used in this study, bacterial determinants other than SA must be responsible for inducing resistance.

Repeated introduction of genetically modified Pseudomonas putida WCS358r without intensified effects on the indigenous microflora of field-grown wheat.

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 10(7) CFU per g of roots soon after sowing to less than 10(2) CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.