Alan H. Gibson

Differentiation of Rhizobium strains using the polymerase chain reaction with random and directed primers

Total genomic DNA isolated from Rhizobium spp was analysed by the polymerase chain reaction (PCR) in conjunction with either arbitrary oligonucleotide primers or a 20-base oligonucleotide primer corresponding to a conserved nif gene promoter region. The amplified fragment length polymorphisms (amplification profiles) generated by the 2 arbitrary primers (RPO4 and RPO5), effectively differentiated a diverse collection of Rhizobium meliloti, R. leguminosarum bv. trifolii and R. l. bv. viciae strains. The nif-directed primer (RPO1) was also highly discriminatory on Rhizobium DNA and generated unique amplification profiles for each strain. For selected strains, reproducible amplification profiles were obtained using a variety of DNA sources, and these were achievable over a 125-fold range in template concentration. Amplification profiles generated by the primers RPO5 and RPO1 were shown to be temperature dependant, with the RPO1 primer capable of generating amplification profiles at annealing temperatures up to 65°C. Reproducible amplification profiles were generated from either purified total genomic DNA or from template DNA present in freeze-thaw bacterial cell lysates. Moreover, the RPO1 primer was used to positively identify specific R. l. bv. trifolii strains directly in crude extracts prepared from squashed clover-root nodules. The significance of these results is discussed in relation to the development of strategies for using PCR-based methodologies in Rhizobium ecological studies.

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (μmol C2H4g−1 nodule fr. wt. h−1), were both affected by the added nitrate.

Studies of field populations of rhizobium: Effectiveness of strains of rhizobium trifolii associated with trifolium subterraneum L. pastures in South-Eastern Australia

Abstract Field populations of Rhizobiuin trifollii from eight regions in south-eastern Australia were sampled over a period of 5 years from 1966 to 1970. The R. trifolii isolates were tested under bacteriologically-and environmentally-controlled conditions for effectiveness of nitrogen fixation in combination with Trifalium subterraneum L. cv. Bacchus Marsh; the effective strain TA1 was used as a standard of comparison. Mean effectiveness of the R. trifolii populations for any region at any sampling varied between 62 and 93 percent of the effectiveness of the standard strain. The principal feature was the large variability within sampling sites, between sites within paddocks, and between paddocks within regions. In addition there was some variability with time in the range of effectiveness values of isolates within a site and in the absolute values for both sites and paddocks. Effectiveness values were not related to soil pH, size of population of R. trifolii, inoculation procedure at sowing, age of pasture, annual rate of fertilizer application, or mean annual rainfall.

Nitrogenase activity and respiration of cultures of Rhizobium spp. with special reference to concentration of dissolved oxygen

Studies of nitrogenase in cultures of the cowpea rhizobia (Rhizobium spp.) strains 32H1 and CB756 are reported. Preliminary experiments established that, even when agar cultures were grown in air, suspensions of bacteria prepared anaerobically from them were most active at low concentrations of free dissolved O2. Consequently, assays for activity used low concentrations of O2, stabilized by adding the nodule pigment leghaemoglobin. In continuous, glutamine-limited cultures of 32H1, nitrogenase activity appeared only when the concentration of dissolved O2 in the cultures approached 1 muM. Lowering the glutamine concentration in the medium supplied to the culture from 2 to 1 mM halved the cell yield and nitrogenase activity was also diminished. Omitting succinate from the medium caused the concentration of dissolved O2 to rise and nitrogenase activity was lost. Upon restoration of the succinate supply, the O2 concentration immediately fell and nitrogenase was restored. The activity doubled in about 8 h, whereas the doubling time of this culture was 14 h. Sonic extracts of 32H1 cells from continuous cultures with active nitrogenase contained components reacting with antiserum against nitrogenase Mo-Fe protein from soybean bacteroids. Continuous cultures grown at higher O2 concentration, with only a trace of active nitrogenase, contained less of these antigens and they were not detected in highly aerobic cultures. Nitrogenase activity of a continuous culture was repressed by NH+4; the apparent half-life was about 90 min. Cells of 32H1 from a continuous culture growing at between 30 and 100 muM dissolved O2 possessed a protective mechanism which permitted respiration to increase following exposure to a rapid increase in O2 concentration from low levels (O2 shock). This effect disappeared as the O2 concentration for growth was reduced towards 1 muM.

Competitiveness and persistence of strains of rhizobia for faba bean in acid and alkaline soils

Abstract Two ecological aspects of the rhizobial inoculation of faba beans were examined, namely their persistence in acid soils and their competitiveness in soils with high naturally-occurring populations of rhizobia. Of 8 Rhizobium leguminosarum bv. viciae strains introduced into 3 acid soils (pH range 4.8–5.7), only 3 consistently maintained soil populations in excess of 100 g −1 2 years after planting. This poor persistence was reflected in nodulation scores and plant growth of uninoculated faba bean sown 2 years after the initial introduction of inocula. Only plants in plots originally inoculated with WSM1014 had yields that were comparable to that of plots sown with freshly-inoculated (commercial strain SU303) seed. In the same area in southwest Victoria, soils of 11 commercial faba bean crops that had been inoculated with SU303, were sampled 1 year later and the total number of rhizobia in the soils were compared with those in soil from adjacent paddocks with no grain legume cropping history. Below pH 6, numbers in the sown soils were low, with little or no increase over numbers in unsown soil. Above pH 6, the numbers in the unsown soil varied from “not detectable” to 10 4 g −1 , and the sown soils showed a variable increase. In only 3 inoculated and 2 uninoculated soils (pH 6.08–8.25) did rhizobia equal or exceed 10 3 g −1 . Serological examination of isolates from MPN plants indicated poor establishment of the inoculant strain. In conclusion, the persistence of R. leguminosarum bv. viciae strains in acid soils was poor, however strains with improved persistence may be selected. Five potential inoculant strains were introduced as inoculants on faba bean seed into 7 neutral-to-alkaline soils in the Northern Wimmera, which possessed high populations of competitive native strains. At 12 wk, all plants, including the uninoculated controls, were well-nodulated. At 16 wk, serological examinations indicated only a low level of infection by the inoculant strains. At harvest, there were no yield increases due to inoculation. At one site, isolates from nodules in uninoculated plots were serological identical to one of the inoculant strains, but the application of molecular biology techniques showed they were not the inoculant strain. Another strain showed a loss of infectivity in the peat inoculant. These results demonstrated the difficulty of demonstrating a response to inoculation in the presence of a substantial native population.

Diversity and genetic structure of a natural population of Rhizobium leguminosarum bv. trifolii isolated from Trifolium subterraneum L.

A collection of 121 isolates of Rhizobium leguminosarum biovar (bv.) trifolii was obtained from root nodules of Trifolium subterraneum L. (subclover) plants growing in an established pasture. The collection consisted of a single isolate from each of 18 plants sampled from seven microplots. The following year, a further 28 and 27 isolates were collected from the first and seventh sampling points, respectively. Analysis of restriction fragment length polymorphisms (RFLPs) of both chromosomal and Sym (symbiotic) plasmid DNA and multilocus enzyme electrophoresis (MLEE) were used to assess the diversity, genetic relationships and structure of this population. Symbiotic effectiveness tests were used to examine the symbiotic phenotype of each isolate collected in the first year. Analysis of RFLPs of the first year isolates revealed 13 chromosomal types and 25 Sym plasmid types. Similar Sym plasmid types were grouped into 14 families containing 1–6 members. No new chromosomal types and six new Sym plasmid types were detected in the second year. The symbiotic effectiveness of the first year isolates of the same Sym plasmid type was similar. Significant differences in symbiotic effectiveness were detected between different Sym plasmid types in the same plasmid family. Representative isolates of each chromosomal type Sym plasmid type identified in the first year were analysed using multilocus enzyme electrophoresis. Mean genetic diversity per locus was high (0.559). Enzyme electrophoresis revealed 17 electrophoretic types (ETs). Ouster analysis of the enzyme data revealed large genetic diversity amongst the ETs. Strong linkage disequilibrium was observed for the population as a whole, i.e. clonal population structure, but significantly less disequilibrium was observed among a cluster of ETs suggesting that recombination occurred between ETs within the cluster. Our results revealed that a population of naturally occurring isolates of Rhizobium leguminosarum bv. trifolii can be genetically diverse and support the possibility that recombination plays a role in generating new genotypes.

The induction of nitrogenase activity in Rhizobium by non-legume plant cells

SummaryNitrogen fixation was induced in a strain of “cowpea” rhizobia, 32Hl, when it was grown in association with cell cultures of the non-legume, tobacco (Nicotiana tabacum). Rhizobia grown alone on the various media examined did not show nitrogenase activity, indicating the involvement of particular plant metabolites in nitrogenase induction. Nitrogenase activity, as measured by C2H2 reduction, was maximized at an O2 concentration of 20% and at an assay temperature of 30°C, the conditions under which the plant cell-rhizobia associations developed. Glutamine, as a nitrogen source, could be replaced by other organic nitrogen sources, but NH4+ and NO3- repressed nitrogenase activity. Nitrogenase activity induced in rhizobia when cultured adjacent to, but not in contact with, the plant cells could be stimulated by providing succinate in the medium. At least 12 other strains of rhizobia also reduced C2H2 in association with tobacco cells; the highest levels of activity were found among cowpea strains.

Nitrogenase activity in cultured Rhizobium sp. strain 32H1

Nutritional and physical conditions affecting nitrogenase activity in the strain of “cowpea” rhizobia, 32H1, were examined using cultures grown on agar medium. Arabinose in the basic medium (CS7) could be replaced by ribose, xylose, or glycerol, but mannitol, glucose, sucrose, or galactose only supported low nitrogenase (C2H2 reduction) activity. Succinate could be replaced by pyruvate, fumarate, malate, or 2-oxoglutarate, but without any carboxylic acid, nitrogenase activity was low or undetectable unless a high level of arabinose was provided. Inositol was not essential. Several nitrogen sources could replace glutamine including glutamate, urea, (NH4)2SO4 and asparagine.The maximum nitrogenase activity of cultures grown in air at 30°C was observed under assay conditions of pO2=0.20–0.25 atm and 30°C incubation. Greatest activity occurred after a period of rapid bacterial growth, when viable cell count was relatively constant.Compared with results obtained on the CS7 medium, nitrogenase activity could be substantially increased and/or sustained for longer periods of time by using 12.5 mM succinate and 100 mM arabinose, by increasing phosphate concentration from 2 to 30–50 mM, or by culturing the bacteria at 25°C.

A comparison of competitiveness and persistence amongst five strains of Rhizobium tripolii

Abstract Five strains of Rhizohium trifolii were used to inoculate Trifolium subterraneum cv. Woogenellup sown into two soils with naturally-occurring populations of R. trifolii . In the 1st year all inoculant strains used singly were present at high frequency in the sampled nodule populations from the inoculated plots. Where an inoculant containing a mixture of equal parts of the 5 strains was used. one strain (WU95) predominated at both sites. The persistence of the strains was followed for a further 3 years at one site. Three of the strains WU95, CC2480 a and WU290, were maintained at a high frequency (>75% of nodules sampled) for the entire period, but the other two strains showed poor persistence in this environment. Highly effective strains of rhizobia, not identifiable as inoculant strains, nor present in the nodule population at the first sampling, appeared during the course of the study. One strain. WU290, showed a high degree of variation in symbiotic effectiveness between single colony isolates from the stock culture and also between field isolates that were serologically identical with this strain.

Nitrogen fixation in cultured cowpea rhizobia: Inhibition and regulation of nitrogenase activity

Abstract Nitrogenase activity in agar cultures of cowpea rhizobia, strain 32H1, was rapidly inhibited by NH 4 + but this was relieved by increased O2 tension. Inhibition was more rapid than that caused by inhibitors of protein synthesis and was not relieved by methionine sulfoximine or methionine sulfone. Under conditions were nitrogenase activity was inhibited by NH 4 + , glutamine synthetase and glutamate synthase were substantially unaffected. Glutamate dehydrogenase was undetected in either nitrogenase active or NH 4 + inhibited cultures. These results indicate that NH 4 + inhibition of nitrogenase activity in strain 32H1 is not effected through glutamine synthetase regulation of nitrogenase synthesis.