In recent years, the interest in the use of bacteria for biological control of plant-pathogenic fungi has increased. We studied the possible side effects of coating barley seeds with the antagonistic strain Pseudomonas fluorescens DR54 or a commercial fungicide, imazalil. This was done by monitoring the number of indigenous Pseudomonas organisms and actinomycetes on barley roots during growth in soil, harvest after 50 days, and subsequent decomposition. Bacteria were enumerated by traditional plate spreading on Gould's S1 agar (Pseudomonas) and as filamentous colonies on Winogradsky agar (actinomycetes) and by two quantitative competitive PCR assays. For this we developed an assay targeting Streptomyces and closely related genera. DR54 constituted more than 75% of the Pseudomonas population at the root base during the first 21 days but decreased to less than 10% at day 50. DR54 was not successful in colonizing root tips. Initially, DR54 affected the number of indigenous Pseudomonas organisms negatively, whereas imazalil affected Pseudomonas numbers positively, but the effects were transient. Although plate counts were considerably lower than the number of DNA copies, the two methods correlated well for Pseudomonas during plant growth, but after plant harvest Pseudomonas-specific DNA copy numbers decreased while plate counts were in the same magnitude as before. Hence, Pseudomonas was 10-fold more culturable in a decomposition environment than in the rhizosphere. The abundance of actinomycetes was unaffected by DR54 or imazalil amendments, and CFU and quantitative PCR results correlated throughout the experiment. The abundance of actinomycetes increased gradually, mostly in numbers of DNA copies, confirming their role in colonizing old roots.

We used a quantitative PCR method targeting 16S ribosomal DNA using competitive PCR for specific detection of indigenousPseudomonas DNA in soil hot spots. The amount ofPseudomonas DNA corresponded to the number of culturablePseudomonas bacteria on Gould’s S1 agar. This represents the first use of PCR for quantification of indigenous bacteria in more than one sample of soil.

The protocol used in the present study was a long-term mesocosm experiment where the microbial succession around live barley roots and subsequent decomposing roots was assessed after seed coating with either the antagonistic strain Pseudomonas fluorescens DR54-BN14 or the fungicide imazalil. Four diversity measures were used: community level physiological profiles (CLPP), Bacteria-specific polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), actinomycete-specific PCR-DGGE and phospholipid fatty acid (PLFA), as well as total cell counts, colony-forming units (CFU) and culturable spore formers, and spore counts of the Bacillus cereus group. Analysis of non-treated plants provided a baseline description of the natural microbial succession from which effects of the treatments could be evaluated. A microbial succession occurred both in the rhizosphere and around decomposing roots, shown with all three diversity measures. A clear response to root death was found, and a clear distinction between root tip and root base samples. Using the recommended concentration of imazalil and a realistic number of DR54-BN14 for seed coating, transient, initial effects of both treatments on the microbial communities were observed at the root base with the PLFA analysis only. The lack of lasting significant side effects of DR54-BN14 is in agreement with an initial fast reduction in culturable DR54-BN14.