External and internal root colonization of lodgepole pine seedlings by two growth-promotingBacillusstrains originated from different root microsites

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Abstract
Root colonization and in vitro carbon substrate utilization by two seedling growth-promoting Bacillus strains that originated from different root microsites were studied in greenhouse and growth chamber experiments. Strain L6, identified as Bacillus polymyxa, was previously isolated from rhizosphere soil containing roots of pasture plants, and Pw-2, tentatively identified also as B. polymyxa, was isolated from within surface-sterilized lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) roots. Rifamycin-resistant strains derived spontaneously from wild-type strains L6 and Pw-2, designated strain L6-16R and Pw-2R, respectively, were used to monitor lodgepole pine root colonization in a closed tube assay system. Three-week-old pine seedlings were inoculated with 105 colony-forming units (cfu) of strain Pw-2R or 106 cfu of strain L6-16R, and external and internal root colonization was assessed 2 and 4 weeks later. Strains L6-16R and Pw-2R were both recovered from pine rhizosphere samples with > 5 × 107 cfu/g fresh root tissue 2 weeks after inoculation, but neither strain was detected in the root interior. When root colonization was assessed 4 weeks after inoculation, the rhizosphere populations of both strains had declined slightly to between 5 × 106 and 5 × 107 cfu/g fresh root tissue, but strain Pw-2R was also detected within root tissues with 105 cfu/g fresh root tissue. Lateral root formation was abundant 4 weeks after inoculation and may have facilitated colonization of internal root tissues by strain Pw-2R. Both strains possessed pectolytic activity, although differences between the strains were detected in in vitro substrate utilization capabilities using BIOLOG assays. These differences may be related to their abilities to colonize internal root tissues. On the basis of our results, we hypothesize that internal root colonization by Bacillus strains is not a random event and that root-endophytic Bacillus strains possess specific physiological and (or) biochemical characteristics that facilitate colonization of internal root tissues.Key words: Bacillus, PGPR, rhizosphere, endophytes, colonization.