Competition Between Ice Nucleation-Active Wild Type and Ice Nucleation-Deficient Deletion Mutant Strains ofPseudomonas syringaeandP. fluorescensBiovar I and Biological Control of Frost Injury on Strawberry Blossoms

Lindemann, J., and Suslow, T. V. 1987. Competition between ice nucleation-active wild type and ice nucleation-deficient deletion mutant strains of Pseudomonas syringae and P. fluorescens biovar I and biological control of frost injury on strawberry blossoms. Phytopathology 77:882-886. Ice nucleation-deficient (INA-) mutants of Pseudomonas strains derived inhibited INAderivatives when inoculum doses were reversed. Inhibition by site-directed mutagenesis were tested for their efficacy as biological was incomplete unless doses differed by about 10-fold. No inhibition control agents of frost injury on blossoms of greenhouse-grown strawberry occurred when two strains were inoculated simultaneously at equal doses plants. Inhibition of one bacterial strain by its near-isogenic counterpart (either 102 or 107 cfu per blossom). The INAP. syringae strain protected was dose dependent rather than strain dependent. The INAdeletion blossoms against freezing by other P. syringae strains but did not inhibit or mutants of P. syringae and P.fluorescens biovar 1, inoculated at 107 cfu per protect against INA+ P. fluorescens. The INAP. fluorescens strain was blossom, inhibited growth of their ice nucleation-active (INA') parental much more effective as an inhibitor of P. syringae strains than of other P. strains inoculated at 102 cfu per blossom. The INA+ parental strains fluorescens strains. Procedures for screening the efficacy of potential biological MATERIALS AND METHODS control agents frequently involve assays for inhibition in vitro. However, in some cases, protection from infection by Bacterial cultures. Ice nucleation-active (INA+) strains S203 and phytopathogenic bacteria has been achieved with microbes that MS 1650 were originally isolated from healthy strawberry plants in demonstrated no inhibitory properties in vitro (2,10,16,19). commercial fruit production fields in California. Based on the Lindow isolated mutants of antagonistic bacteria after chemical combined results from 19 diagnostic tests, S203 was identified as P. mutagenesis that were no longer able to inhibit Erwinia amylovora syringae and MS1650 was identified as P. fluorescens biovar I and Pseudomonas syringae van Hall in plate assays (10). In most (Trevisan) Migula (5,18). Strain S203 closely resembled the group cases, these mutant organisms were still effective biological control lb pathovars of P. syringae, based on the LOPAT tests (5), but agents in greenhouse assays. could not be assigned to a specific pathovar since it demonstrated Since detectable antibiosis is apparently not necessary for no pathogenic potential on the 26 plant species tested (7; efficacy in vivo, bacterial pests containing deletion mutations unpublished data). (which attenuate their harmful property) may be effective Ice nucleation-deficient (INA-) deletion mutants of these two biological control agents. Lindow and co-workers have reported strains, derived by site-directed mutagenesis as described elsewhere that chemical mutants deficient in ice nucleation activity (INA-) (21), are designated RGP36 (P. syringae) and GJP17B (P. gave protection from frost injury under field conditions (9,15). fluorescens). The deletion mutant strains had no detectable ice Lindow also found that genetically engineered INAdeletion nucleation activity (less than one ice nucleus per 109 cells) at10 C mutants were effective in greenhouse tests (11). It has been when assayed by a droplet freezing assay (13,20). Wild type and proposed that genetically engineered pathogenicity-minus mutants deletion mutant strains were subjected to a battery of 19 standard of phytopathogenic bacteria may be effective disease control diagnostic tests to verify their identity as described previously (7). agents (6,17). These strains were also screened for their ability to utilize 31 The most rigorous method of evaluating the effects of single compounds as sole carbon sources in a minimal medium (1), their genes or single phenotypic characteristics on competition and sensitivity to 19 antibiotics, and their growth rates in vitro and in biological control is construction of isogenic lines of an organism, vivo. The INAdeletion mutant strains were identical to their each with a specific mutation in a uniform genetic background. In INA+ wild type parental strains in all tests except ice nucleation. the present study, near-isogenic lines of Pseudomonas strains Variants expressing spontaneous resistance to rifampicin and (differing only in ice nucleation activity and antibiotic resistance) nalidixate were selected by plating about 108 cells from log phase were used to study the phenomena of reciprocal competition, cultures onto plates containing the appropriate antibiotic at 50 competitive exclusion, and the effect of the INA phenotype on /Ag/ml and isolating individual colonies after incubation. The epiphytic colonization ability. To our knowledge, this is the first antibiotic-resistant mutants that were chosen for this study were study showing that, under appropriate circumstances, a plant pest those that demonstrated a colonization competence on strawberry (in this case, an INA+ bacterial strain) can inhibit epiphytic blossoms equivalent to that of the parental strain. Parental and multiplication of a biological control agent (the INAbacterial derivative strains used in greenhouse studies are referred to as near strain). This phenomenon, therefore, places limits on biological isogenic, rather than isogenic, because they differed in both control strategies that rely on competition rather than antibiosis antibiotic resistance and ice nucleation activity. Additional strains and also has important implications for the fate of recombinant of P. syringae and P. fluorescens were isolated from strawberry bacteria that may be released into the environment, plants. Two strains (from different locations) of each species were included in competition studies. Bacterial stock cultures were maintained in a freezer at -80 C in 1:10 (v/v) DMSO:Luria broth. Fresh inoculum for colonization studies was always prepared from the frozen stock cultures. @1987 The American Phytopathological Society Bacteria were streaked onto King's medium B (KB) (4)