Biological Control

Anderson, L. M., Stockwell, V. O., and Loper, J. E. 2004. An extracellular protease of Pseudomonas fluorescens inactivates antibiotics of Pantoea agglomerans. Phytopathology 94:1228-1234. Pseudomonas fluorescens A506 and Pantoea agglomerans strains Eh252 and C9-1 are biological control agents that suppress fire blight, an important disease of pear and apple caused by the bacterium Erwinia amylovora. Pseudomonas fluorescens strain A506 suppresses disease largely through competitive exclusion of E. amylovora on surfaces of blossoms, the primary infection court, whereas Pantoea agglomerans strains Eh252 and C9-1 produce antibiotics that are toxic to E. amylovora. In this study, an extracellular protease produced by A506 is characterized and evaluated for its capacity to inactivate the antibiotics produced by the strains of Pantoea agglomerans. Activity of the extracellular protease was optimal at pH 9 and inhibited by zincor calcium-chelators, indicating that the protease is an alkaline metalloprotease. In an agar plate bioassay, partially purified extracellular protease inactivated the antibiotics mccEh252 and herbicolin O, which are produced by Pantoea agglomerans strains Eh252 and C9-1, respectively. Derivatives of A506 deficient in extracellular protease production were obtained by transposon mutagenesis, and the aprX gene encoding the protease was cloned and sequenced. Strain A506 inactivated mccEh252 and herbicolin O in agar plate bioassays, whereas the aprX mutant did not inactivate the antibiotics. Both A506 and the aprX mutant were insensitive to antibiosis by C9-1 and Eh252; thus, the protease was not required to protect A506 from antibiosis. These data highlight a previously unknown role of the extracellular protease produced by Pseudomonas fluorescens A506 in interactions among plant-associated microbes. Additional keyword: Erwinia herbicola. Biological control is a promising approach for management of plant diseases but its use in commercial agriculture remains limited. Among the factors that limit the adoption of biological control into mainstream agriculture is the variation in efficacy that characterizes many biological control systems. One approach toward enhancing the reliability of biological control has been to combine biological control agents into mixtures, which can provide more effective and consistent disease suppression than is attained by application of individual agents (7,11,13,23,24). The compatibility of the biological control agents is recognized as an important factor in designing such mixtures, and monitoring the population sizes of all microbial components in the mixture is typically advised to assure that the biological control agents do not antagonize one another in the formulation or, after application, on plant surfaces. Aside from that compatibility, however, factors influencing the success of biological control mixtures are poorly understood. In this study, we explored a second factor for consideration in designing mixtures of biological agents: the capacity of one biological control agent to interfere with the biological control activity of another. Specifically, we describe a mechanism by which the biological control agent Pseudomonas fluorescens A506 interferes with the inhibition of Erwinia amylovora, the fire blight pathogen, by a second biological control agent, Pantoea agglomerans. Fire blight is a major disease of pear and apple that was once controlled by application of commercial antibiotics. The emergence of streptomycin-resistant strains of E. amylovora (22) made it necessary to develop alternate methods for managing the disease. One such approach is biological control achieved by application of nonpathogenic bacterial antagonists of E. amylovora to blossoms, the primary site of infection by the fire blight pathogen (33). Pseudomonas fluorescens strain A506, a commercially available biological control agent for fire blight, suppresses the growth of E. amylovora on stigmatic surfaces of blossoms through preemptive exclusion (38). Preemptive exclusion is a process whereby the biological control agent suppresses growth of the pathogen by prior colonization of physical sites and utilization of limited resources required by the pathogen (38). The epiphytic bacteria Pantoea agglomerans (syn. Erwinia herbicola) strains Eh252 and C9-1 also are used experimentally to suppress populations of E. amylovora on blossom surfaces and limit severity of fire blight disease in pear and apple orchards (14–16,34– 37,40). Each biocontrol agent suppresses fire blight by an average of 50%, but the level of disease control varies among field trials (15,20,21,34,35). In an effort to improve the biological control of fire blight, combinations of A506 and strains of Pantoea agglomerans have been examined for suppression of disease when applied to blossoms as a mixture. Although strain A506 and Pantoea agglomerans successfully co-colonize blossoms, mixtures of these bacteria generally have not been more effective than single strains in controlling fire blight (14,15,33,35,36). Many strains of Pantoea agglomerans produce one or more compounds inhibitory to E. amylovora (12,16,37,39,41). Strain Eh252 produces a single antibiotic, mccEh252 (35,37), and strain C9-1 produces two distinct antibiotics, herbicolin O and herbiCorresponding author: V. O. Stockwell E-mail address: [email protected] Publication no. P-2004-0916-02R This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological