Clover Rhizobia Research Articles

Survival of white clover rhizobium isolate S11N9 in different delivery systems: assessment of potential for commercialisation

Survival of white clover rhizobium isolate S11N9 in different delivery systems: assessment of potential for commercialisation

Ecological consequences of urbanization on a legume–rhizobia mutualism

Mutualisms are key determinants of community assembly and composition, but urbanization can alter the dynamics of these interactions and associated effects on ecosystem functions. Legume–rhizobia mutualisms are a model interaction to evaluate the ecological and ecosystem‐level effects of urbanization, particularly urban‐driven eutrophication and nitrogen (N) deposition. Here, we evaluated how urbanization affected the ecology of the mutualism between white cloverTrifolium repensand its rhizobial symbiontRhizobium leguminosarumsymbiovartrifoliialong an urbanization gradient. We found that the abundance of rhizobium nodules on white clover decreased with urbanization. White clover acquired N from mixed sources of N fixation and uptake from the soil for the majority of the urbanization gradient, but white clover primarily acquired N from the soil rather than N fixation by rhizobia at the urban and rural limits of the gradient. Importantly, we identified soil N as a critical nexus for urban‐driven changes in the white clover–rhizobium mutualism. Taken together, our results demonstrate that urbanization alters the ecological consequences of a legume–rhizobium mutualism, with direct and indirect effects of the urban landscape on an ecologically‐important mutualistic interaction.

A Small Amount of Nitrogen Transfer from White Clover to Citrus Seedling via Common Arbuscular Mycorrhizal Networks

Few studies have examined if perennial leguminous cover crops are able to transfer nitrogen (N) via common mycorrhizal networks (CMNs) to neighboring fruit trees; the gradient of such N transfer could affect the N nutrition of both plants. Using separated three-column chambers to grow plants in a greenhouse, 99 atom% 15N as (15NH4)2SO4 was applied to leaves of white clover (Trifolium repens L.) and 15N was then traced in neighboring citrus (Citrus sinensis (L.) Osbeck) seedlings interconnected by an arbuscular mycorrhizal fungus (AMF, Rhizophagus intraradices). A range of 66.85–68.74% mycorrhizal colonization in white clover (mycorrhizal and/or Rhizobium trifolii inoculated) and 19.29–23.41% in citrus (non-mycorrhizal inoculated) was observed after 12 months of AMF inoculation in the white clover, indicating a successful CMN linkage was established between these two plant species. This CMN establishment resulted in significant increases in biomass, N accumulation, and 15N content of citrus when accompanied with nodulated and mycorrhizal fungus colonized white clover. N transfer from white clover to citrus was significantly greater under nodulation plus mycorrhization (46.23 mg N per pot, 1.71% of N transferred) than under non-inoculated control (4.36 mg N per pot, 0.21% of N transferred), and higher than sole mycorrhization (36.34 mg N per pot, 1.42% of N transferred). The percentage of N in citrus derived from white clover under nodulated/mycorrhization was 1.83–1.93%, and was highest in leaves (3.31%), moderate in stems (2.47%), and lowest in roots (0.41%) of citrus. In summary, results from this experiment demonstrated that nearly 2.0% of N transferred from white clover to citrus via CMN. Further studies are needed to quantify N transfer between white clover and citrus by other routes, including soil or root exudation pathways.

Enhancing glutaric acid production in Escherichia coli by uptake of malonic acid

Glutaric acid is an important organic acid applied widely in different fields. Most previous researches have focused on the production of glutaric acid in various strains using the 5-aminovaleric acid (AMV) or pentenoic acid synthesis pathways. We previously utilized a five-step reversed adipic acid degradation pathway (RADP) in Escherichia coli BL21 (DE3) to construct strain Bgl146. Herein, we found that malonyl-CoA was strictly limited in this strain, and increasing its abundance could improve glutaric acid production. We, therefore, constructed a malonic acid uptake pathway in E. coli using matB (malonic acid synthetase) and matC (malonic acid carrier protein) from Clover rhizobia. The titer of glutaric acid was improved by 2.1-fold and 1.45-fold, respectively, reaching 0.56g/L and 4.35g/L in shake flask and batch fermentation following addition of malonic acid. Finally, the highest titer of glutaric acid was 6.3g/L in fed-batch fermentation at optimized fermentation conditions.

A model bioassay for influence of heavy metal salts toxicity on the viability of nodule-forming bacteria <i>Rhizobium meliloti</i>

The study consists of a bioassay of a medium experimentally contaminated with heavy metals using Rhizobium meliloti microorganisms as a test object. In vitro, the viability reaction of clover and alfalfa nodule bacteria to lead, cadmium, copper and zinc was established. The effect of the studied metals on the number of colonies depends on the concentration of the chemical element, physiological action of the microelement and biological characteristics of the strains used. The studies performed have determined a direct relationship of reducing the growth in the rhizobia colonies number of clover and alfalfa with an increase in the concentration of salts of heavy metals. Inhibition of the colonies number had a high regularity when the environment was contaminated with lead, copper and cadmium at a concentration of 0,3%; with a subsequent increase, complete death of microorganisms was observed. With the introduction of zinc into the nutrient medium in small concentrations, a positive resistance tendency of the clover and alfalfa bacteria was observed: thus, at 0,010,1% of the zinc salt content, the number of grown colonies was above the control level and amounted to 714987 pcs. at the control value of 578 pcs. In an environment with a concentration of zinc 0,5%, a significant decrease in the growth of clover rhizobia was noted up to 65 pieces of colonies. At the same time, in the alfalfa rhizobia colonies in these concentrations a lower degree of survival was observed and the environment of zinc 0,3% became critical.

Population structure of the clover rhizobia Rhizobium leguminosarum bv. trifolii upon transition from soil into the nodular niche

High-throughput sequencing of the amplicon gene library revealed variations in the population structure of clover rhizobia (Rhizobium leguminosarum bv. trifolii) upon transition from soil into the root nodules of the host plant (Trifolium-hybridum). Analysis of rhizobial-diversity using the nodA gene revealed 3258 and 1449 nucleotide sequences (allelic-variants) for the soil and root nodule population, respectively. They were combined into 29 operational taxonomic units (OTU) according to the 97% identity level; 24 OTU were. foundin the soil population, 12 were present in the root nodulepopulation, and 7 were common. The predominant OTE13 (77.4 and 91.5% of the soil and root nodule populations, respectively) contained 155 and -200 variants of the soil and root nodule populations, respectively, with the nucleotide diversity increasing significantly upon the "soil-->root" transition. The "moving window" approach was used to reveal the sites of the nodA gene in which polymorphism, including that associated with increased frequency of non-synonymous substitution frequency, increased sharply upon transition from soil into root nodiles. PCR analysis of the IGS genotypes of individual strains revealed insignificant changes in rhizobial diversity upon transition from soil into root nodules. These results indicate that acceleration of rhizobial evolutioin in the course of symbiosis may be associated with development of highly polymorphic virulent:subpopulations subjected to directional selection in the "plant-soil" system.

Genetic structure of the introduced and local populations of Rhizobioum leguminosarum in plant-soil systems

Comparative study of Rhizobium leguminosarum populations formed under the conditions of the Srednii Island (White Sea) demonstrated the introduced clover rhizobia (R. l. bv. trifolii) to be more variable than the aboriginal vetch/vetchling rhizobia (R. l. bv. viceae) in the chromosomal IGS locus, while being less variable in the plasmid-located symbiotic genes nodD and nifH. The analysis of these genes revealed the most pronounced differences between the clover and vetch/vetchling rhizobia populations. These differences, together with the results of ERIC-fingerprinting, indicated that the evolution of the clover rhizobia was mainly linked with the adaptation to local soil environment, and the evolution of the vetch/vetchling rhizobia, to the adaptation to various species of the host plants. High panmixia of R. leguminosarum population suggests its evolution to be based on the combinatory variability associated with the transfer of Sym-plasmids between R. l. bv. trifolii and R. l. bv. viceae, as well as with genomic rearrangements in the resulting recombinants.

White clover seed inoculation and coating in New Zealand

This paper reviews the requirement for inoculation of white clover (Trifolium repens) seed with rhizobia bacteria in New Zealand. The pastoral industry relies on the nitrogen fixed by clover's rhizobia bacteria. These rhizobia were not present in soils prior to European settlement, but were introduced as contaminants, and naturally spread with pastoral development. The advent of large scale land development in the 1950s identified areas where clovers failed to nodulate and establish due to the absence of rhizobia, which led to the development of inoculated lime coated seed. Rhizobia have spread widely throughout New Zealand, and in the great majority of situations where pasture is being sown, soils now contain high levels of resident rhizobia capable of nodulating white, red and alsike clover, and inoculation of clover seed is not required. However, it is suggested the use of inoculated clover seed should be considered in the following three situations as an insurance against nodulation failure: undeveloped grasslands with no evidence of resident clover; virgin pastoral land cleared directly from scrub; and paddocks cropped with maize continually for over 10 years. It has been hypothesised that inoculating clover seed may improve clover growth through introducing more effective rhizobia with better nitrogen fixation ability, but the research in this area suggests there is little likelihood of any significant response from doing this. Keywords: rhizobia, inoculation, coated seed, white clover, red clover, alsike clover

Symbiotic competence of rose clover (Trifolium hirtum All.)

Rose clover (Trifolium hirtum All.) is a forage plant that is well adapted to acidic and mildly alkaline soils of low natural fertility in southern Australia and to climates with a winter-dominant annual rainfall of 300 mm and above. Reports of low concentrations of nitrogen in rose clover foliage have been attributed to poor N2 fixation and may have discouraged its use in Australia. This investigation, conducted in tube culture, examined the ability of four lines of rose clover to nodulate and fix N2 with effective strains of clover rhizobia (Rhizobium leguminosarum bv. trifolii) and with soils (as a source of naturalised rhizobia) collected from field sites in New South Wales and South Australia. Comparisons with other Trifolium spp. were also made. It was confirmed that there was a low concentration of N in the shoots of the rose clover cvv. SARDI Rose and Hykon. This occurred even where rose clover nodulated and fixed N2 effectively with well known inoculant strains of clover rhizobia and with soil samples collected in the field (provided that the populations of resident clover rhizobia in the soil were at least 150/g). Individual plants were uniform in response to inoculation. Rose clover cv. SARDI Rose was closely related to six of the nine other lines of clover with which it was compared. It was concluded that the registered cultivars of rose clover, cvv. SARDI Rose and Hykon, are symbiotically competent plants. It appears that low N in rose clover foliage is an intrinsic characteristic of the species unconnected with its symbiotic characteristics.

Initial results from long‐term field studies at three sites on the effects of heavy metal‐amended liquid sludges on soil microbial activity

AbstractIn a long‐term study of the effects on soil fertility and microbial activity of heavy metals contained in sewage sludges, metal‐amended liquid sludges each with elevated Zn, Cu or Cd concentrations were applied over a 3‐year period (1995–1997) to three sites in England. The experiments were sited adjacent to experimental plots receiving metal‐rich sludge cakes enabling comparisons to be made between the effects of heavy metal additions in metal‐amended liquid sludges and sludge cakes. The liquid sludge additions were regarded as ‘worst case’ treatments in terms of likely metal availability, akin to a long‐term situation following sewage sludge additions where organic matter levels had declined and stabilised. The aim was to establish individual Zn (50–425 mg kg−1), Cu (15–195 mg kg−1) and Cd (0.3–4.0 mg kg−1) metal dose–response treatments at each site, but with significantly smaller levels of organic matter addition than the corresponding sludge cake experiments. There were no differences (P > 0.05) in soil respiration rates, biomass carbon concentrations or most probable numbers of clover Rhizobium between the treatments at any of the sites at the end of the liquid sludge application programme. Soil heavy metal extractability differed between the metal‐amended liquid sludge and metal‐rich sludge cake treatments; Zn and Cd extractabilities were higher from the liquid sludge additions, whereas Cu extractability was higher from the sludge cake application. These differences in metal extractability in the treated soil samples reflected the contrasting NH4NO3 extractable metal contents of the metal‐amended liquid sludges and sludge cakes that were originally applied.

Initial results from a long‐term, multi‐site field study of the effects on soil fertility and microbial activity of sludge cakes containing heavy metals

AbstractIn a long‐term study of the effects on soil fertility and microbial activity of heavy metals contained in sewage sludges, metal‐rich sludge cakes each with high Zn, Cu or Cd concentrations were applied annually for 4 years (1994–1997) to nine sites throughout Britain. These sites were selected to represent agricultural soils with a range of physical and chemical properties, typical of those likely to be amended with sewage sludge. The aim was to establish individual total Zn (approx. 60–450 mg kg−1), total Cu (approx. 15–200 mg kg−1) and total Cd (approx. 0.2–4 mg kg−1) metal dose–response treatments at each site. Sludges with low metal concentrations were added to all treatments to achieve as constant an addition of organic matter as possible. Across the nine sites, soil pH was the single most important factor controlling Zn (P < 0.001; r2 = 92%) and Cd extracted with 1 m NH4NO3 (P < 0.001; r2 = 72%), and total iron content the most important factor controlling Cu extracted with 1 m NH4NO3 (P < 0.001; r2 = 64%). There were also positive relationships (P < 0.001) between soil organic carbon (C) concentrations and soil biomass C and respiration rates across the nine sites. Oxidation of sludge C following land application resulted in approximately 45% of the digested sludge cake C and approximately 64% of the ‘raw’ sludge cake C being lost by the end of the 4‐year application period. The sludge cake applications generally increased soil microbial biomass C and soil respiration rates, whilst most probable numbers of clover Rhizobium were generally unchanged. Overall, there was no evidence that the metal applications were damaging soil microbial activity in the short term after the cessation of sludge cake addition.

Genetic diversity of a natural population of Rhizobium leguminosarum biovar trifolii analysed from field nodules and by a plant infection technique

We compared trapping of Rhizobium leguminosarum biovar trifolii isolates under field soil and non-soil environmental conditions. Isolates were obtained from white clover ( Trifolium repens L.) and red clover ( T. pratense L.) grown directly in the field and from plants inoculated with soil from the same site using a plant infection technique. Isolates were identified by genomic polymerase chain reaction (PCR) fingerprinting using primers derived from the enterobacterial repetitive intergenic consensus (ERIC) sequences. The isolates trapped from soil dilutions in the laboratory included a number of major ERIC types that were not found in the field trapped nodules, suggesting that sampling of clover Rhizobium strains from soil dilutions may not be representative of the field nodulating population.

Characterization and evaluation of North American Trifolium ambiguum -nodulating rhizobia

Kura clover ( Trifolium ambiguum M. Bieb.) is a promising persistent forage legume, whose use is currently limited by establishment difficulties in part attributable to nodulation problems. In this study, we phenotypically characterized 18 Kura clover rhizobia including 12 newly isolated North American strains using 111 growth tests. The symbiotic performances of these 12 strains when used to inoculate hexaploid Kura clover plants were then evaluated in a growth chamber and compared to five strains commonly used in commercial Kura clover inoculants. Non-inoculated plants with or without N fertilization were used as controls. Field evaluations were also conducted in 2001 and 2002 in Montreal, Quebec and Becker, Minnesota. Hexaploid Kura clover was seeded with one of three North American strains or three commercial strains and compared to non-inoculated controls with or without N fertilizer. Phenotypic diversity observed among the 12 North American strains was limited. The North American strains were often more efficient than strains commonly used in commercial rhizobial inoculants in both growth chamber and field trials. In the growth chamber study, 60 days after seeding, shoot dry matter accumulation was overall 205% greater with North American strains than with commercial strains and 45% greater than with the best commercial strain tested. Some of the strains currently used in commercial inoculants failed to nodulate Kura clover plants effectively. North American strains tested in field trials (i.e., CT1-1, CT1-2, and WI4-4) overall increased total and shoot dry matter accumulation, 100 days after seeding, in three of four environments by 27% and 31% respectively, when compared to commercial inoculant strains. While these strains appear to have potential they still produced less dry matter than non-inoculated N-fertilized controls.

Diversity and symbiotic effectiveness of Rhizobium leguminosarum bv. trifolii isolates from pasture soils in south-western Australia

The abundance of the Australian inoculant strain of Rhizobium leguminosarum bv. trifolii for subterraneum clover (WU95) and the diversity of naturalised rhizobia were assessed in 3 subterranean clover pastures in the Albany region of south-western Western Australia. Most probable number, enzyme linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) techniques were used. A putative strain similar to inoculant strain WU96 was uncommon at one site (South Stirling) and not isolated at 2 other sites. Randomly amplified polymorphic DNA (RAPD) PCR fingerprinting using the RPO1 primer identified 45 different profiles amongst the 208 isolates examined. RAPD-PCR fingerprinting using the primers RPO4 and RPO5 confirmed most groupings based on RPO1 fingerprint patterns and revealed further genetic diversity within some groups. Overall, 54 putative strains were defined by RAPD-PCR fingerprint profiles across the 3 sites. Subterranean clover rhizobia at the Manypeaks and Mount Shadforth sites were dominated by isolates with 1 or 2 RPO1 RAPD profiles at 2 sampling times, while the population at South Stirling was much more diverse. The symbiotic effectiveness of 11 rhizobial isolates, representing the major RPO1 RAPD profile groups within naturalised rhizobial populations, were compared in pot culture with those of the 2 commercial inoculant strains for subterranean clover, WU95 and TA1, on 3 cultivars. Differences in effectiveness among 3 of the 11 isolates were observed in comparison to both the commercial strains and other naturalised isolates. The nitrogen fixing effectiveness of 8 isolates representing different subgroups from one RP01 group was not the same. The use of all 3 primers increased the precision in defining putative strains of Rhizobium leguminosarum bv. trifolii, and although naturalised rhizobia from these pastures are saprophytically competent, their dominance in nodules does not appear to be linked to symbiotic effectiveness.

Erosion of root epidermal cell walls by Rhizobium polysaccharide-degrading enzymes as related to primary host infection in the Rhizobium-legume symbiosis.

A central event of the infection process in the Rhizobium-legume symbiosis is the modification of the host cell wall barrier to form a portal of entry large enough for bacterial penetration. Transmission electron microscopy (TEM) indicates that rhizobia enter the legume root hair through a completely eroded hole that is slightly larger than the bacterial cell and is presumably created by localized enzymatic hydrolysis of the host cell wall. In this study, we have used microscopy and enzymology to further clarify how rhizobia modify root epidermal cell walls to shed new light on the mechanism of primary host infection in the Rhizobium-legume symbiosis. Quantitative scanning electron microscopy indicated that the incidence of highly localized, partially eroded pits on legume root epidermal walls that follow the contour of the rhizobial cell was higher in host than in nonhost legume combinations, was inhibited by high nitrate supply, and was not induced by immobilized wild-type chitolipooligosaccharide Nod factors reversibly adsorbed to latex beads. TEM examination of these partially eroded, epidermal pits indicated that the amorphous, noncrystalline portions of the wall were disrupted, whereas the crystalline portions remained ultrastructurally intact. Further studies using phase-contrast and polarized light microscopy indicated that (i) the structural integrity of clover root hair walls is dependent on wall polymers that are valid substrates for cell-bound polysaccharide-degrading enzymes from rhizobia, (ii) the major site where these rhizobial enzymes can completely erode the root hair wall is highly localized at the isotropic, noncrystalline apex of the root hair tip, and (iii) the degradability of clover root hair walls by rhizobial polysaccharide-degrading enzymes is enhanced by modifications induced during growth in the presence of chitolipooligosaccharide Nod factors from wild-type clover rhizobia. The results suggest a complementary role of rhizobial cell-bound glycanases and chitolipooligosaccharides in creating the localized portals of entry for successful primary host infection.

Distribution, abundance and symbiotic effectiveness of Rhizobium leguminosarum bv. trifolii from alkaline pasture soils in South Australia

The current dissatisfaction with low productivity of annual medic (Medicago spp.) pastures has highlighted the need to seek alternative legumes to provide efficient N2 fixation in low rainfall, alkaline soil environments of southern Australia. Clover species adapted to these environments will have limited N2 fixation if effective rhizobia are not present in sufficient quantities. A survey of 61 sites was conducted across South Australia to determine the size, distribution and effectiveness of Rhizobium leguminosarum bv. trifolii (clover rhizobia) populations resident in these low rainfall, alkaline soil environments. Clover rhizobia were detected at 56 of the sites, with a median density of 230–920 rhizobia/g soil. Most rhizobial populations were poor in their capacity to fix nitrogen. Rhizobial populations from fields provided 11–89% and 10–85% of the shoot biomass of commercial reference strains when inoculated onto host legumes T. purpureum (purple clover) and T. resupinatum (persian clover), respectively. Rhizobial population size was correlated negatively to pH and the percentage of CaCO3 in the soil, and was significantly increased in the rhizospheres of naturalised clover, found at 17 sites. Management options for rhizobial populations to improve legume diversity and productivity are discussed in terms of rhizobial population dynamics and likely soil constraints to successful rhizobial colonisation.

Differentiation of Clover Rhizobium Isolated from Biosolids‐Amended Soils with Varying pH

AbstractMetal contamination may alter the diversity of microbes residing in soil. The genetic structure and phenotypic characteristics of clover Rhizobium isolated from contaminated and control soils were compared. Plant infection and symbiotic competence tests were used for phenotypic characterization. Variation across isolates in fingerprint patterns determined with primers for repetitive extragenic palindromic (REP) sequences and the polymerase chain reaction (PCR) were used for genetic characterization. Two phenotypic groups of effective and ineffective isolates were identified using the symbiotic effectiveness test. Soil pH was the primary factor influencing this phenotypic characteristic. Effective isolates were associated with higher soil pH and ineffective isolates were associated with lower soil pH regardless of soil metal content. The isolates were genetically diverse. The variation of isolates from the different soils overlapped, indicating that neither the heavy metals nor the low soil pH resulted in the selection of a single genotype. Isolates from the most heavily contaminated soils were more variable than isolates from control soils. Soil pH, and not heavy metal content, was important in the selection of rhizobia that formed ineffective N2‐fixing symbioses.

Effects of previous aluminium exposure on motility and nodulation by Rhizobium and Bradyrhizobium

Abstract Substrains of rhizobia and bradyrhizobia which had been previously exposed to Al were tested for motility, infectiveness and effectiveness. The rate of motility of some substrains of soyabean Bradyrhizobium strain USDA 143 were enhanced after exposure to Al, and nodule number and nitrogen fixation were reduced. Infectiveness of clover Rhizobium strain RDG 2002 was also reduced. No significant change between Al-treated and untreated substrains of lotus rhizobia strain NZP 2037 was observed. These results suggest that ecologically important traits in Rhizobium and Bradyrhizobium may be permanently affected by prior exposure of strains to Al, and support the hypothesis that Al is potentially mutagenic.

Numbers of clover rhizobia needed for crown nodulation and early growth of clover in soil

Low soil populations of Rhizobium leguminosarum biovar trifolii indicate a need for inoculating clovers (Trifolium sp.) at planting. The number of rhizobia in soil varies considerably from field to field and the number needed for nodulation on the upper taproot and for vigorous seedling development is not known. Two experiments were undertaken using arrowleaf clover (T. vesiculosum Savi) and crimson clover (T. incarnatum L.) grown in pots filled with soil. Two soils were used; one contained 10 indigenous rhizobia g-1 and the other contained fewer than three. The treatments consisted of amending each soil with two strains of inoculant rhizobia to contain from 10 to approximately 1×106 rhizobia g-1 followed by planting to clover. The number of nodules near the top of the root increased as the number of rhizobia in the soil increased to the highest inoculum level. A low number (approximately 1×103 to 1×104) of rhizobia was sufficient for maximal N content of seedlings. It seems that soil containing 100 or fewer rhizobia g-1 may respond to inoculation with increased crown nodulation and seedling vigor.

Population densities of clover rhizobia in Texas pastures and response to liming

Clovers are widely used forage legumes on acidic soils in Texas and need inoculation with appropriate rhizobia when first introduced. Acidic soils are not conducive to survival of clover rhizobia. A survey of pastures was undertaken to determine the number of rhizobia present. The effect of liming acidic soils on the survival of clover rhizobia was also evaluated in the laboratory. The number of clover rhizobia was more than 100 cells g-1 soil in 70% of the pastures surveyed but populations within pastures varied by more than two orders of magnitude. The number of years of clover production beyond 1 year did not affect the rhizobial population density. The soil pH of twelve samples was below 5.0 and six samples had populations of rhizobial lower than 100 g-1 soil. Eleven out of sixteen samples from fields that had grown clover and had pH values above 6.0 had populations exceeding 1000 g-1 soil and only three samples had populations lower than 100 g-1 soil. Incubating indigenous or inoculated rhizobia in well-mixed soils having pH values of 5.1 or below resulted in populations declining to below 10 g-1 soil in 6 weeks. Mixing of soils with pH values of up to 5.4 induced reduction of rhizobial numbers, possibly by destroying microsites. Liming of soils to increase pH values above 5.5 improved survival of native or inoculated rhizobia in most cases.