Temporary loss of antibiotic resistance by marked bacteria in the rhizosphere of spruce seedlings

Abstract Survival and persistence of two plant growth-promoting rhizobacteria strains in the rhizosphere of young spruce seedlings were evaluated in field plots and soil microcosms. Bacillus polymyxa strain Pw-2R and Pseudomonas fluorescens strain Sw5-RN are spontaneous antibiotic-resistant derivatives of the naturally occurring parental strains B. polymyxa Pw-2 and P. fluorescens Sw5, respectively. Resistance to 200 μg ml−1 rifamycin in strain Pw-2R, and to 100 μg ml−1 each of rifamycin and nalidixic acid in Sw5-RN, facilitated monitoring of these bacteria in rhizosphere samples. Strains Pw-2R and Sw5-RN were each inoculated into the rhizosphere of spruce seedlings in field plots as well as in intact soil core microcosms that were incubated under controlled environmental conditions. Bacterial survival data based on the abilities of Pw-2R and Sw5-RN to be re-isolated from rhizosphere samples, and by growing on agar containing antibiotics, were collected over a 2-year period as part of a larger study. The population sizes of both bacterial strains appeared to drop below detection limits by the beginning of the second year as they failed to grow on primary isolation media containing antibiotics. However, strains Pw-2R and Sw5-RN grew on isolation medium with antibiotics if they were first isolated on agar without antibiotics and then replica-plated onto agar media containing antibiotics. A similar temporary loss of antibiotic resistance has been observed with endophytic bacteria, but our results suggest such masking may be of much wider significance than previously thought. If it commonly occurs in rhizosphere colonizing bacteria as well as endophytes, significant underestimates of bacterial population sizes in similar environmental samples may result.