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In a previous study, we demonstrated that transgenic Lotus plants producing opines (which are small amino acid and sugar conjugates) specifically favor growth of opine-degrading rhizobacteria. The opine-induced bias was repeated and demonstrated with another soil type and another plant species (Solanum nigrum). This phenomenon is therefore independent of both soil type and plant species.
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The results shown in Table , obtained 10 weeks after transfer of the plants to the greenhouse, indicate that the densities of the total cultivable bacteria isolated from the roots of both WT plants and opine-producing plants cultured in La-Côte-Saint-André soil did not differ significantly. A similar conclusion was drawn for the fluorescent Pseudomonaceae isolated from the roots of both WT plants and opine-producing plants. However, the densities of mannopine, nopaline, and octopine utilizers were 300 to 1,000 times higher in the rhizospheres of the plants producing the opines, including the previously untested compound octopine, than in the rhizospheres of the WT plants. In addition, octopine utilizers were also significantly more abundant in the rhizospheres of Lotus plants producing nopaline than in the rhizospheres of WT plants and plants producing mannopine (Table ). Although not investigated, this cross-selection could be attributed to the very similar structures of the opines nopaline and octopine (Fig. ) (for a review, see reference ), which could therefore be degraded by the same single catabolic system in bacteria. In agreement with this hypothesis, related proteins encoded by related genes in Agrobacterium are involved in nopaline and octopine degradation (, ). In addition, proteins involved in nopaline catabolism can also use octopine as a substrate ().
The bacterial populations colonizing the root systems of WT Lotus plants and of transformed Lotus plants harboring the oncogenes but devoid of genes encoding opine biosynthesis (ONC plants) were examined. The densities of total cultivable bacteria were identical whatever the plant of origin (WT and ONC plants) (data not shown). Similar results were obtained upon comparison of the densities of fluorescent Pseudomonas and the densities of opine utilizers. Consequently, the growth stimulation of opine-degrading bacteria observed around the root systems of opine-producing plants is related to expression of the opine biosynthesis genes and not to the transformed status of the plants or the presence of the pRi transferred DNA (T-DNA) oncogenes. Overall, our results indicate that the opine-dependent bias induced by transgenic, opine-producing plants also occurred with octopine-producing plants and was not specific for the soil from La Mérantaise that we used in earlier studies (, ). This opine-induced bias is therefore not restricted to one soil type.
The bacterial populations of the rhizospheres were recovered and analyzed as indicated by Oger et al. (, ). Total cultivable bacterial populations and the fluorescent Pseudomonas populations were counted on modified Luria-Bertani medium (which contained 5 g of NaCl per liter instead of 10 g/liter) at 28°C and on King's B medium () at 25°C, respectively. Fluorescence of colonies was assessed under UV light at 365 nm. Both media were supplemented with cycloheximide (250 mg/liter). The densities of cultivable organisms that utilized opines (mannopine, nopaline, and octopine) were evaluated by inoculating 50 μl of AT minimal medium () supplemented with the appropriate opine(s) (5 mM each) and cycloheximide (250 mg/liter) with serial dilutions of the bacterial suspensions obtained from the plant rhizospheres as indicated above and were deduced from the value of the last active dilution that induced disappearance of opines in each case. This was assessed after 7 days of incubation at 28°C by high-voltage paper electrophoresis at pH 1.9 (). The values presented below resulted from three independent experiments (see above), with all enumerations performed in triplicate. An analysis of variance and a Student t test were performed on all data collected. Values were considered significantly different at a P of 0.05.
In the second part of this study, we investigated whether the marked opine bias induced by the Lotus plants was specific for this plant species. We repeated the above experiments using nightshade (S. nigrum) plants, which are taxonomically unrelated to the genus Lotus, engineered to produce opines (see above). These plants were grown and transferred to the greenhouse in the La-Côte-Saint-André soil, as indicated above, and microbes associated with their root systems were analyzed as described above for the Lotus plants.
The results (Table ) indicate that the densities of total cultivable bacteria isolated from the rhizospheres of the S. nigrum plants producing opines were identical to the densities of total cultivable bacteria isolated from the rhizospheres of the WT plants. A similar observation was made for the fluorescent Pseudomonaceae component of the microflora. However, as observed with the Lotus plants, the concentrations of mannopine-, nopaline-, and octopine-utilizing bacteria were 30 to ca. 1,000 times higher in the rhizospheres of opine-producing Solanum plants than in the rhizospheres of WT plants. In addition, octopine utilizers were also significantly more abundant in the rhizospheres of S. nigrum plants producing nopaline than in the rhizospheres of WT plants (Table ). A comparison of the values obtained for ONC and WT plants (data not shown) suggested that the elevated densities of opine-degrading bacteria in the rhizospheres of opine-producing S. nigrum plants resulted from expression of opine biosynthesis genes and not from the transformed status of the plants. Additional measurements were obtained at 10, 14, and 18 weeks. The results of this series of experiments clearly indicated that the population density of total cultivable bacteria and the population density of the fluorescent Pseudomonaceae component of the rhizosphere were stable over the observation time (Fig. ), from 6 to 18 weeks following installation of the plants in microcosms. Furthermore, the opine-induced bias appeared to be consistently detected over time under our experimental conditions (Fig. ). Similar results have been obtained using transgenic Lotus plants producing opines, albeit only after 6, 10, and 14 weeks as the experiment was discontinued after 14 weeks (data not shown). This result is of interest because it has been shown previously that the microbial community selected by a plant varies according to the developmental stage of the plant (, , ), a feature that also relates to legume species (). The apparent stability of the opine-induced bias suggests that the compositions of the root exudates of the Lotus and S. nigrum plants used in this study remained steady while the experiment lasted.
The two soil types used in our studies had different physical and chemical characteristics and originated from different geographical regions. Therefore, the microflora inhabiting these two soils were most likely different (, ). As a consequence, our results obtained with the loamy soil from La-Côte-Saint-André demonstrated that the opine-induced bias generated by the opine-producing plants was not specific for a single microflora inhabiting the clay-rich soil from La Mérantaise, which was used previously (, ). This conclusion is supported by our results obtained with transgenic, opine-producing nightshade (S. nigrum) plants. Indeed, it is reasonable to assume that the microbial community inhabiting the root systems of the Lotus plants differed from that inhabiting the root systems of S. nigrum plants because the microbial communities colonizing plant roots are determined by the plant genus, species, or cultivar (, , , , , , ).