Isolation and Characterization of Alfalfa-Nodulating Rhizobia Present in Acidic Soils of Central Argentina and Uruguay

Abstract

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809–1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean andLeucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N2-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.

Attachment to plant roots and nod gene expression are not affected by pH or calcium in the acid-tolerant alfalfa-nodulating bacteria Rhizobium sp. LPU83

“…Rhizobium sp. strain LPU83, recently isolated from acid soils of Argentina, is one example of this type of bacteria [2,18]. Data presented in this manuscript show that the addition of Ca has no marked effect on the nodulation kinetics of this bacterium and that Ca treatment increases nodulation of alfalfa by more effective S. meliloti strains, supporting an old field observation [33].…”

“…To the best of our knowledge, this is the first time that an alfalfa-nodulating Rhizobium with such characteristics has been described. Like S. meliloti, LPU83 displays an impaired nodulation phenotype on alfalfa plants in mildly acidic conditions (pH 5.6) [2,18]. Although this result could tempt us to speculate that alfalfa may play a role in restricting nodulation under acidic conditions, it is also possible that the limitation of nodulation observed for this rhizobial strain is correlated to a specific bacterial problem.…”

“…Progressive soil acidification is a worldwide problem with important economical consequences for countries which rely on alfalfa (Medicago sativa L.) production for cattle nutrition [1,2]. The poor performance of alfalfa plants in moderately acid soils (pH 5.5-6.5) is mainly due to the effects of low pH on the establishment of the symbiosis between this legume and the nitrogen-fixing bacteria Sinorhizobium meliloti [3][4][5][6].…”

“…Recently, another factor has been suggested to affect the S. meliloti-alfalfa symbiosis in some acidic soils which has drastic effects on alfalfa yield. Acid-tolerant but ineffective alfalfa-nodulating rhizobia have been identified in the acidic soils of Argentina and Uruguay [2,18]. These bacteria, which show surprising genetic uniformity [19], are related to Rhizobium sp.…”

Laccase activity in melanin-producing strains of Sinorhizobium meliloti

“…Twenty-four sinorhizobia strains, part of a large collection of S. meliloti isolates from Uruguayan soils [17], were analysed to determine the production of melanin. Castro et al [18] found that 62% of them produced melanin (Mel+).…”