Summary• The ability of Burkholderia phymatum STM815 to effectively nodulate Mimosa spp., and to fix nitrogen ex planta , was compared with that of the known Mimosa symbiont Cupriavidus taiwanensis LMG19424.• Both strains were equally effective symbionts of M. pudica , but nodules formed by STM815 had greater nitrogenase activity. STM815 was shown to have a broader host range across the genus Mimosa than LMG19424, nodulating 30 out of 31 species, 21 of these effectively. LMG19424 effectively nodulated only nine species. GFP-marked variants were used to visualise symbiont presence within nodules.• STM815 gave significant acetylene reduction assay (ARA) activity in semisolid JMV medium ex planta , but no ARA activity was detected with LMG19424. 16S rDNA sequences of two isolates originally from Mimosa nodules in Papua New Guinea (NGR114 and NGR195A) identified them as Burkholderia phymatum also, with nodA , nodC and nifH genes of NGR195A identical to those of STM815.• B. phymatum is therefore an effective Mimosa symbiont with a broad host range, and is the first reported beta-rhizobial strain to fix nitrogen in free-living culture.

Haemoglobin has previously been recorded in plants only in the nitrogen-fixing nodules formed by symbiotic association between Rhizobium or Frankia and legume or non-legume hosts. Structural similarities amongst these and animal haemoglobins at the protein and gene level suggested a common evolutionary origin. This suggests that haemoglobin genes, inherited from an ancestor common to plants and animals, might be present in all plants. We report here the isolation of a haemoglobin gene from Trema tomentosa, a non-nodulating relative of Parasponia (Ulmaceae). The gene has three introns located at positions identical to those in the haemoglobin genes of nodulating plant species, strengthening the case for a common origin of all plant haemoglobin genes. The data argue strongly against horizontal haemoglobin gene transfer from animals to plants. The Trema gene has a tissue-specific pattern of transcription and translation, producing monomeric haemoglobin in Trema roots. We have also found that the Parasponia haemoglobin gene is transcribed in roots of non-nodulated plants. These results suggest that haemoglobin has a role in the respiratory metabolism of root cells of all plant species. We propose that its special role in nitrogen-fixing nodules has required adaptation of the haemoglobin-gene regulation pathway, to give high expression in the specialized environment of the nodule.

M . J. TRINICK This paper reports on the infective ability, morphology, colony characteristics, growth in litmus milk, precipitation in calcium glycerophosphate medium, carbohydrate utilization and serology of fast-growing rhizobia from the tropical legumes M . inoisa, M . pudicu, A . farnesiuna, S. grundijora, Lublub purpureus and Leucuenu Ieucocephula. The relationship of this group to other established host groups is examined. Materials and Methods PIunt hostsLeucuenu leucocephala (Lam.) de Wit., Mimosa incisa Mart., M . pudica L., Acacia furnesiana (L.) Willd. and Seshaniu grand$ora Poir. are nodulated only with fastgrowing root-nodule bacteria. Only one fast-growing nodule symbiont was isolated from Lublub purpureus (L.) Sweet which is usually nodulated, like other tropical legumes, with slow-growing types. These hosts will be referred to by their generic name throughout the text and M . incisu is the test species of Mimosa when not otherwise specified. Other tropical legumes are listed in Tables 1, 2 and 3 and were nodulated in the field only by slow-growing rhizobia. Source of Rhizobium struinsThe NGR prefix indicates a source in Papua New Guinea. The fast-growing rootnodule bacteria from Leucuenu and Mimosa spp. were isolated from nodulated plants growing in slightly acid (pH 5 5 ) , neutral and alkaline soils (pH 8.3) in Papua New Guinea. Only one isolate from Acacia and two from Seshania growing in alkaline soils were used in this study. The isolate, NGR 234, from Lahlab was the only fast-growing isolate obtained from 30 nodules collected from plants growing in a soil with pH 8.5. The other 29 isolates were slow-growing. The fast-growing isolates were shown to be free from associated slow-growing rhizobia by their repeated streaking for isolated colonies on yeast-extract mannitol medium (YMA) containing brom thyniol blue after shaking in water for 10 min and after growth in broth culture: also only fast-growers were re-isolated from nodulated plants inoculated with low rhizobial populations (1 to 10 cells). The slow-growing strains of Rhizobium were isolated from tropical legumes and the non-legume Parusponia andersonii (Trinick 1980) grown in similar locations in Papua New Guinea. All strains of Rhizobium were isolated on YMA and were preserved by lyophilization. Characterization of the, fust-growing rhizobirr Staining and morphology old cultures grown on YMA at 27°C. Cultures were examined for motility, cell morphology and Gram reaction on2 and 3 d Colony characteristics colonies of R. meliloti and cowpea-type strains. Characteristics of 7 and 10 d old isolated colonies on YMA were compared with

A dimeric hemoglobin was purified from nitrogen-fixing root nodules formed by association of Rhizobium with a nonleguminous plant, Parasponia. The oxygen dissociation rate constant is probably sufficiently high to allow Parasponia hemoglobin to function in a fashion similar to that of leghemoglobin, by oxygen buffering and transport during symbiotic nitrogen fixation. The identification of hemoglobin in a nonlegume raises important questions about the evolution of plant hemoglobin genes.