Tn5 mutagenesis of Chinese Rhizobium fredii for siderophore overproduction

In the midwestern United States, members of USDA Bradyrhizobium japonicum serogroup 135 dominate in alkaline soils for nodulation of soybean [Glycine max (L.) Merr.], whereas members of USDA serogroup 123 dominate in non-alkaline soils. A possible explanation for the dominance of 135 in alkaline soils is that native 135 strains can produce siderophores that assist in acquiring iron. Our objectives were to test six reference strains (three slow-growing B. japonicum and three fast-growing Rhizobium fredii) for siderophore production and to evaluate mutants of R. fredii (developed by Tn5-insertion for overproduction of siderophores) for their competitive abilities in an alkaline soil. B. japonicum reference strains USDA 110, 123 and 135 were tested for siderophore production in liquid culture by using Neilands' CAS Assay Solution. Mutants were developed for siderophore overproduction by introducing transposon Tn5 (Km[r]) into a siderophore-producing Chinese R. fredii strain. Two siderophore overproducing mutants that produced mature and pink nodules on soybean in growth pouches were examined for the presence of Tn5 by Southern hybridization with a Tn5 probe. The Tn5-carrying mutants were further tested in the greenhouse for their competitiveness against native strains present in an alkaline soil of Iowa and for their symbiotic effectivity. Among the three slow-growing bradyrhizobia tested for siderophore production, USDA 135 was the only strain producing siderophore in a liquid medium low in iron. Nodule occupancies in the greenhouse by the two siderophore over-producing mutants of R. fredii were 3 and 4%, compared with 19% for the wild-type strain. Thus, either the wild-type was acquiring all the iron needed for maximum growth and nodulation, or the insertion of Tn5 and the overproduction of siderophore resulted in less competitive strains.