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Appl Environ Microbiol. 1991 July; 57(7): 2005-2011
Copyright © 1991, American Society for Microbiology. All Rights Reserved.

Construction of an Acid-Tolerant Rhizobium leguminosarum Biovar Trifolii Strain with Enhanced Capacity for Nitrogen Fixation

Hancai Chen^*, Alan E. Richardson, Elena Gartner, Michael A. Djordjevic, Rodney J. Roughley and Barry G. Rolfe

¹ Plant Microbe Interaction Group, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra City, Australian Capital Territory 2601, and NSW Agriculture and Fisheries, Gosford, New South Wales 2250, ² Australia

ABSTRACT

Strain ANU1173 is an acid-tolerant Rhizobium leguminosarum biovar trifolii strain that is able to nodulate subterranean clover plants growing in agar culture at pH 4.4 At pH 6.5, its symbiotic effectiveness in association with Trifolium subterraneum cv. Mt. Barker was 80% relative to that of strain ANU794, a Sm^r derivative of the commercial inoculant R. leguminosarum bv. trifolii TA1. Strain ANU1173 contained four indigenous megaplasmids, the smallest of these being the symbiotic (Sym) plasmid. The critical pH requirement for growth of strain ANU1173 in laboratory media was shown not to be associated with this plasmid. When the Sym plasmid of strain ANU1173(pSym-1173) was mobilized into a Nod^- strain of R. leguminosarum bv. viciae, the plasmid conferred to the transconjugant a level of symbiotic effectiveness in association with T. subterraneum that was similar to that observed with ANU1173. The symbiotic effectiveness of strain ANU1173 was improved by first curing pSym-1173 (generating strain ANU1184) and replacing it with another R. leguminosarum bv. trifolii Sym plasmid, pBR1AN. Subterranean clover plants inoculated with strain ANU1184 (pBR1AN) exhibited a 35 or 53% increase in acetylene reduction activity and a 20 or 17% increase in dry weight when grown at pH 6.5 and pH 4.4, respectively, compared with plants inoculated with strain ANU1173 and grown under the same pH conditions. It was further shown that pBR1AN was stably maintained in strain ANU1184 under free-living and symbiotic conditions. These results indicate that it is possible to construct an acid-tolerant strain of R. leguminosarum bv. trifolii with an enhanced capacity for nitrogen fixation.

^* Corresponding author.

Present address: Commonwealth Scientific and Industrial Research Organisation, Division of Plant Industry, Canberra City, ACT 2601, Australia.

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