3.5.13

INFLUENCE OF THE STATIONARY-PHASE SIGMA FACTOR ON FITNESS OF PSEUDOMONAS FLUORESCENS ON PLANT SURFACES


VO STOCKWELL1, MD HAGEN1, C WHISTLER2 and JE LOPER1

1US Department of Agriculture, Agricultural Research Service, Horticultural Crops Research Laboratory, 3420 N.W. Orchard Ave., Corvailis, Oregon 97330; 2Molecular and Cellular Biology Program, Oregon State University, Corvallis, Oregon 97331, USA

Background and objectives
Fluorescent pseudomonads are ubiquitous inhabitants of plant surfaces, including roots, leaves, and floral tissues; and certain strains are promising agents for the biological control of plant diseases caused by phytopathogenic fungi and bacteria. Antibiotic production and the capacity to survive exposure to environmental and physiological stresses encountered on plant surfaces are characteristics contributing to the biological control activities of fluorescent pseudomonads, and these characteristics are expressed preferentially by stationary-phase cells in culture. Bacterial cells undergo complex physiological and morphological changes upon entry into stationary phase, which are controlled by the stationary-phase sigma factor RpoS. The objective of this research was to evaluate the role of RpoS in the stress response and environmental fitness of two strains of P. fluorescens that suppress plant diseases.

Materials and methods
P. fluorescens Pf-5 is a rhizosphere bacterium that produces several antifungal metabolises and suppresses a number of soilborne diseases. In a previous study [1], the rpos gene of Pf -5 was cloned and sequenced, and an RpoS - mutant of Pf-5 was obtained by transposon mutagenesis. P. fluorescens A506, an epiphytic bacterium isolated from pear, is registered in the United States as a biological control agent for suppression of fire blight and frost injury of pear [2].

Results and conclusions
In culture, stationary-phase cells of an RpoS - mutant of Pf -5 were more sensitive than the wildtype to desiccation, oxidative stress, and freezing. In replicated field trials, Pf-5 established greater population sizes than the RpoS - mutant in the rhizosphere of bean grown in dry soils, whereas the strains established similar rhizosphere population sizes in moist soils. These data provide indirect evidence that, during at least a portion of its life in the rhizosphere, P. fluorescens is in a physiological state where the stationary-phase sigma factor RpoS is operative. Furthermore, RpoS contributes significantly to the fitness of Pf-5 in the rhizosphere of of bean grown in dry soils, which may be due to its role in desiccation tolerance.
Stock cultures of P.fluorescens A506 had anrpoS gene with a frameshift mutation that resulted in a truncated RpoS, as determined by western analysis. We repaired the rpos mutation in the genome of A506 through a combination of PCR and marker exchange techniques, and compared the repaired strain to the original A506 for stress response in culture and survival on apple leaves in the field. In field experiments, near-isogenic strains with truncated and full-length RpoS proteins established similar population sizes on apple leaf surfaces. Therefore, RpoS played a less significant role in the survival of A506 than of Pf-5 in the field environments evaluated in this study.

References
1. Sarniguet A, Kraus J, Henkels MD., Muehichen AM., and Loper JE. 1995. Proceedings of the National Academy of Sciences (USA) 92, 12255-12259.
2. Stockwell VO, Johnson KB, and Loper JE. 1996. Phytopathology 86, 834-840.