Additional file 6 of A minimized symbiotic gene set from the 1.68 Mb pSymB chromid of Sinorhizobium meliloti reveals auxiliary symbiotic loci

Additional file 6: Fig. S4. Symbiotic phenotype of key strains on the alternate S. meliloti host Melilotus officinalis (yellow sweet clover) and the consistent nodule morphology of exclusively Fix− SmB1.0 nodules. (A) The shoot dry weight of M. officinalis inoculated with SmB1.0 was consistently Fix− and indistinguishable from un-inoculated controls (red). This appears to be due to the simultaneous removal of the B301 and B307 regions as seen when M. officinalis is inoculated with SmB1.0+ pSymBΔB302ΔB307. The addition of pSymBΔB302 to SmB1.0 was able to restore the symbiosis wild-type levels as was seen with alfalfa (light blue). SmB2.0 was Fix+ with M. officinalis at a level below that seen with alfalfa (orange). Mean shoot height differences were assessed using one-way ANOVA followed by a Tukey multiple comparison test. (B-D) Wild-type-inoculated nodules of M. officinalis 5 weeks post-inoculation displayed typical development with highly elongated bacteroids. (E-J) SmB1.0-inoculated nodules of M. officinalis 5 weeks post-inoculation exhibited consistent early arrest of nodule development (E-F, further magnified in G) with poorly elongated bacteria (H). Proximal part of the nodule was often devoid of infected cells and possessed numerous infection threads (I-J). Nodules were fixed and stained with the DNA-binding dye SYTO13. Images are representative of a minimum of five independent nodules examined. Scale bars: (B, E-G) 100 μm; (C-D, H-J) 25 μm. Arrowheads point to examples of bacteria in infection threads.