Figure 1.

Polymerase chain reaction (PCR) based on repetitive sequences (rep-PCR) analysis of rhizobial strains. (A) BOXA1R-PCR fingerprinting of rhizobial strains used as bioinocula along with related species from the genus Rhizobium. M) Marker, 1Kb Plus DNA Ladder (Invitrogen, Life Technologies, Grand Island, NY). 1) 102H2, 2) 102A13, 3) 102B11, 4) WSM540, 5) W118, 6) M2, 7) WSM1115, 8) Sinorhizobium medicae USDA 1037, 9) RR1128, 10) M18, 11) M49, 12) 102F85, 13) Sinorhizobium meliloti USDA 1002, 14) Bradyrhizobium japonicum USDA 6, 15) Bradyrhizobium japonicum USDA 123, 16) Mesorhizobium ciceri USDA 3383, 17) Mesorhizobium huakuii USDA4779, and 18) Rhizobium legumenosarum bv. viciae USDA 2370. (B) Cluster analysis using simple matching (SM) coefficient and unweighted pair-group method using arithmetic average (UPGMA) clustering method of BOXA1R-PCR fingerprinting showing diversity of rhizobial strains. The scale bar represents similarity coefficient.

 


Figure 2.

Neighbor joining phylogenetic tree based on 16S and the intergenic spacer (IGS) region between the 16S and 23s ribosomal DNA (rDNA) genes. (A) Phylogenetic tree based on 16S gene sequences. (B) Phylogenetic tree based on IGS sequences showing the position of different strains compared with those of related species of genus Rhizobium. Bioinoculum isolates are in bold. Bootstrap values calculated for 1000 replications are indicated on branch nodes. The bar represents one nucleotide substitution per 100 nucleotides. Bradyrhizobium japonicum, Azorhizobium caulinodans, Agrobacterium tumefaciens, Rhizobium galegae, Mesorhizobium loti, Mesorhizobium ciceri, Mesorhizobium haukuii, Mesorhizobium tianshanens, Mesorhizobium mediterraneum, Agrobacterium rhizogenes, Rhizobium tropici, Rhizobium etli, Rhizobium leguminosarum bv. viciae, Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium terangae, Sinorhizobium melilotii, Sinorhizobium medicae, Sinorhizobium kostiense, and Sinorhizobium xinjiangense.

 


Figure 3.

Effectiveness study comparing single specific Rhizobial strains with the NFmix3 blend (WSM1115, 102F85, and M49) on the annual medics Medicago minima, Medicago orbicularis, Medicago rigidula, and Medicago rigiduloides. (A) Rhizobia effective index as affected by medic species × rhizobium interaction (p < 0.0001) at 45 d after emergence. SE = 31.6 of the interaction mean. (B) Comparison of total plant dry biomass (g per plant) as affected by medic species × rhizobium interaction (p < 0.0001) at 45 d after emergence. SE = 0.13 of the interaction mean. Means followed by the same letter above the bar do not differ by the least squares means test (p > 0.05) whereby lowercase letters are within each rhizobial treatment and uppercase letters are within each plant species. UTC, untreated control.