No abstract

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.

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 microg ml(-1) rifamycin in strain Pw-2R, and to 100 microg 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.

Pseudomonas chloroaphis 3732 RN-L11 is a genetically modified bacterial strain that contains the lacZY marker genes in its chromosome. This strain is known to be a vigorous colonizer of plant roots and rhizosphere soil, and has been used as a model to evaluate survival and persistence of field-released genetically engineered microorganisms (GEMs). However, the possibility that strain 3732 RN-L11 may also colonize internal plant tissues has not previously been investigated. Using spring wheat as a model system, we studied the ability of strain 3732 RN-L11 to colonize external and internal root tissues after seed inoculation. Strain 3732 RN-L11 was recovered from rhizosphere soil of 28-, 42-, and 56-day-old seedlings with mean population sizes of 3.3 × 10 5 , 7.5 × 10 4 , and 2.2 × 10 5 CFU·g -1 fresh root tissue, respectively. In addition, this strain was consistently recovered from surface-sterilized root tissues of 28-to 56-day-old seedlings with mean population sizes of 1.0 × 10 2 to 6.2 × 10 3 CFU·g -1 fresh root tissue. Our results indicate that evaluation of plant-associated GEM populations after field release should include all possible colonization niches, including internal plant tissues.