Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.     

Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.     

Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in Sinorhizobium meliloti increases its ability to nodulate alfalfa

1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants. The ACC deaminase of Rhizobium leguminosarum bv. viciae 128C53K was previously reported to be able to enhance nodulation of peas. The ACC deaminase structural gene (acdS) and its upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene (lrpL), from R. leguminosarum bv. viciae 128C53K were introduced into Sinorhizobium meliloti, which does not produce this enzyme, in two different ways: through a plasmid vector and by in situ transposon replacement. The resulting ACC deaminase-producing S. meliloti strains showed 35 to 40% greater efficiency in nodulating Medicago sativa (alfalfa), likely by reducing ethylene production in the host plants. Furthermore, the ACC deaminase-producing S. meliloti strain was more competitive in nodulation than the wild-type strain. We postulate that the increased competitiveness might be related to utilization of ACC as a nutrient within the infection threads.     

1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Genes in Rhizobia from Southern Saskatchewan

A collection of 233 rhizobia strains from 30 different sites across Saskatchewan, Canada was assayed for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, with 27 of the strains displaying activity. When all 27 strains were characterized based on 16S rRNA gene sequences, it was noted that 26 strains are close to Rhizobium leguminosarum and one strain is close to Rhizobium gallicum. Polymerase chain reaction (PCR) was used to rapidly isolate ACC deaminase structural genes from the above-mentioned 27 strains; 17 of them have 99% identities with the previously characterized ACC deaminase structural gene (acdS) from R. leguminosarum bv. viciae 128C53K, whereas the other ten strains are 84% identical (864~866/1,020 bp) compared to the acdS from strain 128C53K. Southern hybridization showed that each strain has only one ACC deaminase gene. Using inverse PCR, the region upstream of the ACC deaminase structural genes was characterized for all 27 strains, and 17 of these strains were shown to encode a leucine-responsive regulatory protein. The results are discussed in the context of a previously proposed model for the regulation of bacterial ACC deaminase in R. leguminosarum 128C53K. An erratum to this article can be found at     

Disparate role of rhizobial ACC deaminase in root-nodule symbioses

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase converts ACC, a precursor of the plant hormone ethylene, into ammonia and ??-ketobutyrate. ACC deaminase is widespread among the rhizobia in which it might play a crucial role in protecting rhizobia against inhibitory effects of ethylene synthesized by the host plant in response to the nodulation process. The beneficial action of this enzyme was demonstrated in several rhizobia such as Mesorhizobium loti and Rhizobium leguminosarum where knock-out mutants of the ACC deaminase gene showed nodulation defects. The genome of the slow-growing rhizobial species Bradyrhizobium japonicum also carries an annotated gene for a putative ACC deaminase (blr0241). Here, we tested the possible importance of this enzyme in B. japonicum by constructing an insertion mutant of blr0241 and studying its phenotype. First, the activity of ACC deaminase itself was measured. Unlike the B. japonicum wild type, the blr0241 mutant did not show any enzymatic activity. By contrast, the mutant was not impaired in its ability to nodulate soybean, cowpea, siratro, and mungbean. Likewise, symbiotic nitrogen fixation activity remained unaffected. Furthermore, a co-inoculation assay with the B. japonicum wild type and the blr0241 mutant for soybean and siratro nodulation revealed that the mutant was not affected in its competitiveness for nodulation and nodule occupation. The results show that the role previously ascribed to ACC deaminase in the rhizobia cannot be generalized, and species-specific differences may exist.     

Prevalence of 1-aminocyclopropane-1-carboxylate deaminase in Rhizobium spp

This is the first report documenting the presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Rhizobium. This enzyme, previously found in free-living bacteria, yeast and fungi, degrades ACC, the immediate precursor of ethylene in higher plants. Thirteen different rhizobial strains were examined by Southern hybridization, Western blots and ACC deaminase enzyme assay. Five of them tested positive for ACC deaminase. Induction of the expression of ACC deaminase was examined in one of the positively tested strains, Rhizobium leguminosarum bv. viciae 128C53K. This rhizobial ACC deaminase had a trace basal level of expression without ACC, but could be induced by a concentration of ACC as low as 1 μM. The more ACC added to this Rhizobium the higher the expression level of the ACC deaminase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Quorum Sensing in Nitrogen-Fixing Rhizobia

Members of the rhizobia are distinguished for their ability to establish a nitrogen-fixing symbiosis with leguminous plants. While many details of this relationship remain a mystery, much effort has gone into elucidating the mechanisms governing bacterium-host recognition and the events leading to symbiosis. Several signal molecules, including plant-produced flavonoids and bacterially produced nodulation factors and exopolysaccharides, are known to function in the molecular conversation between the host and the symbiont. Work by several laboratories has shown that an additional mode of regulation, quorum sensing, intercedes in the signal exchange process and perhaps plays a major role in preparing and coordinating the nitrogen-fixing rhizobia during the establishment of the symbiosis. Rhizobium leguminosarum, for example, carries a multitiered quorum-sensing system that represents one of the most complex regulatory networks identified for this form of gene regulation. This review focuses on the recent stream of information regarding quorum sensing in the nitrogen-fixing rhizobia. Seminal work on the quorum-sensing systems of R. leguminosarum bv. viciae, R. etli, Rhizobium sp. strain NGR234, Sinorhizobium meliloti, and Bradyrhizobium japonicum is presented and discussed. The latest work shows that quorum sensing can be linked to various symbiotic phenomena including nodulation efficiency, symbiosome development, exopolysaccharide production, and nitrogen fixation, all of which are important for the establishment of a successful symbiosis. Many questions remain to be answered, but the knowledge obtained so far provides a firm foundation for future studies on the role of quorum-sensing mediated gene regulation in host-bacterium interactions.     

Microscopic analysis of the effect ofRhizobium leguminosarum biovartrifolii host specific nodulation genes in the infection of white clovers

Summary A microscopic assessment is presented of the comparative infection capacity of wild-type and hybrid strains ofRhizobium leguminosarum bv.viciae withR. l. bv.trifolii strain ANU 843 on white clover seedlings. TheR. l. bv.viciae hybrid strains contained defined DNA segments coding for different combinations ofR. l. bv.trifolii host-specific nodulation genes. White clover plants were examined over a 72 h period to assessRhizobium infectivity, the morphological changes in root hair growth; colonisation ability of rhizobia; infection thread initiation and the ability to induce cortical cell division.R. l. bv.viciae strain 300 induced root hair curling more slowly than strain ANU 843 or any of the hybrid strain 300 bacteria, and when curling had taken place, there was poorer colonization by strain 300 within the folded hair cell, no evidence of infection thread formation and only limited cortical cell division 72 h after inoculation. The addition of the host-specific nodulation genes ofR. l. bv.trifolii to strain 300 was necessary to induce infection threads and establish a normal pattern of nodulation of the roots of white clovers.     

The Influence of Lipopolysaccharides and Glucans from Two Rhizobium leguminosarum bv. viciae Strains on the Formation and Efficiency of Their Symbioses with Pea Plants

The influence of lipopolysaccharides (LPS), glucans, and their unseparated complexes on nodulation activity of rhizobia and efficiency of their symbioses with pea plants was studied in vegetation tests. Two Rhizobium leguminosarum bv. viciae strains which differed in their symbiotic properties were used: strain 31 (fix⁺, efficient, moderately virulent, and moderately competitive) and strain 248b (fix^–, inefficient, highly virulent, and highly competitive). Preparations of LPS–glucan complex and the respective LPS from the highly virulent strain 248b increased the nodulation activity of both strains by 10–26%. Analogous preparations from a less virulent strain 31 did not have this ability. Unseparated LPS–glucan complexes from these strains increased the productivity of plants infected with the efficient strain by 18–23% but did not change it in plants inoculated with the other, inefficient strain. No significant influence of LPS preparations on the symbiosis productivity was observed. Glucans from both strains enhanced the nodulation ability of the highly virulent strain by 36–56%. In addition, treatment of pea plants with glucan from strain 248b increased nitrogen fixation by root nodules by 27% in plants inoculated with strain 31. In general, the formation and efficiency of the symbiosis of R. leguminosarum bv. viciae with pea plants was more influenced by preparations from strain 248b, highly virulent but deficient in nitrogen fixation, than by preparations from the nitrogen fixation–proficient but less virulent strain 31.     

Identification of a nodD-like gene in Frankia by direct complementation of a Rhizobium nodD-mutant.

Summary Clones from aFrankia At4 gene bank were pooled into groups and mass conjugated into anodD mutant ofRhizobium leguminosarum bv.viciae by triparental matings. When peas were inoculated with the pooled transconjugants, nodulation was observed. A plasmid, pAt2GX containingFrankia DNA, was isolated from bacteria recovered from these nodules. This plasmid was shown to complement anodD mutant ofR. leguminosarum bv.viciae. Thus pAt2GX contains aFrankia gene that is functionally equivalent tonodD ofR. leguminosarum bv.viciae.     

Regulation of Ethylene Biosynthesis Under Salt Stress in Red Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) by 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-producing Halotolerant Bacteria

The present study was carried out to understand the mechanism of salt stress amelioration in red pepper plants by inoculation of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing halotolerant bacteria. In general, ethylene production, ACC concentration, ACC synthase (ACS), and ACC oxidase (ACO) enzyme activities increased with increasing levels of salt stress. Treatment with halotolerant bacteria reduced ethylene production by 47–64%, ACC concentration by 47–55% and ACO activity by 18–19% in salt-stressed (150 mmol NaCl) red pepper seedlings compared to uninoculated controls. ACS activity was lower in red pepper seedlings treated with Bacillus aryabhattai RS341 but higher in seedlings treated with Brevibacterium epidermidis RS15 (44%) and Micrococcus yunnanensis RS222 (23%) under salt-stressed conditions as compared to uninoculated controls. A significant increase was recorded in red pepper plant growth under salt stress when treated with ACC deaminase-producing halotolerant bacteria as compared to uninoculated controls. The results of this study collectively suggest that salt stress enhanced ethylene production by increasing enzyme activities of the ethylene biosynthetic pathway. Inoculation with ACC deaminase-producing halotolerant bacteria plays an important role in ethylene metabolism, particularly by reducing the ACC concentration, although a direct effect on reducing ACO activity was also observed. It is suggested that growth promotion in inoculated red pepper plants under inhibitory levels of salt stress is due to ACC deaminase activity present in the halotolerant bacteria.     

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Trifolitoxin Production and Nodulation Are Necessary for the Expression of Superior Nodulation Competitiveness by Rhizobium leguminosarum bv. trifolii Strain T24 on Clover

Rhizobium leguminosarum bv. trifolii T24 is ineffective in symbiotic nitrogen fixation, produces a potent antibiotic (referred to here as trifolitoxin) that is bacteriostatic to certain Rhizobium strains, and is very competitive for clover root nodulation (EA Schwinghamer, RP Belkengren 1968 Arch Mikrobiol 64: 130-145). The primary objective of this work was to demonstrate the roles of nodulation and trifolitoxin production in the expression of nodulation competitiveness by T24. Unlike wildtype T24, transposon mutants of T24 lacking trifolitoxin production were unable to decrease clover nodulation by an effective, trifolitoxin-sensitive strain of R. leguminosarum bv. trifolii. A non-nodulating transposon mutant of T24 prevented clover nodulation by a trifolitoxin-sensitive R. leguminosarum bv. trifolii when co-inoculated with a T24 mutant lacking trifolitoxin production. Neither mutant alone prevented nodulation by the trifolitoxin-sensitive strain. These results demonstrate that trifolitoxin production and nodulation are required for the expression of nodulation competitiveness by strain T24. A trifolitoxin-sensitive strain of R. meliloti did not nodulate alfalfa when co-inoculated with T24 and a trifolitoxin-resistant strain of R. meliloti. Thus, a trifolitoxin-producing strain was useful in regulating nodule occupancy on a legume host other than clover. Trifolitoxin production was constitutive in both minimal and enriched media. Trifolitoxin was found to inhibit the growth of 95% of all strains of R. leguminosarum bvs. trifolii, viceae, and phaseoli tested. Strains of all 13 biotypes of R. leguminosarum bv. trifolii were inhibited by trifolitoxin. Three strains of R. fredii were also inhibited. Strain T24 ineffectively nodulated 46 clover species, did not nodulate Trifolium ambiguum, and induced partially effective nodules on Trifolium micranthum. Since T24 produced partially effective nodules on T. micranthum and since a trifolitoxin-minus mutant of T24 induced ineffective nodules, trifolitoxin production is not the cause of the symbiotic ineffectiveness of T24.     

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29–20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.     

ACC Deaminase-Containing Bacillus subtilis Reduces Stress Ethylene-Induced Damage and Improves Mycorrhizal Colonization and Rhizobial Nodulation in Trigonella foenum-graecum Under Drought Stress

This study was aimed at protecting Trigonella plants by reducing stress ethylene levels through ACC (1-aminocyclopropane-1-carboxylic acid) deaminase-containing Bacillus subtilis (LDR2) and promoting plant growth through improved colonization of beneficial microbes like Ensifer meliloti (Em) and Rhizophagus irregularis (Ri) under drought stress. A plant growth-promoting rhizobacterium strain possessing high levels of ACC deaminase characterized as B. subtilis was selected. Application of this strain considerably protected Trigonella plants under severe drought stress conditions; this protection was correlated with reduced levels of ACC (responsible for generation of stress ethylene). The experiment consisted of eight inoculation treatments with different combinations of ACC deaminase-containing rhizobacteria LDR2, Ri, and Em under three water regimes. The tripartite combination of LDR2 + Ri + Em acted synergistically to induce protective mechanisms against decreased soil water availability in Trigonella plants and improved plant weight by 56 % with lower ACC concentration (39 % less than stressed noninoculated plants) under severe drought conditions. Drought-induced changes in biochemical markers like reduced chlorophyll concentration, increased proline content, and higher lipid peroxidation were monitored and clearly indicated the protective effects of LDR2 under drought stress. Under drought conditions, apart from alleviating ethylene-induced damage, LDR2 enhanced nodulation and arbuscular mycorrhizal fungi colonization in the plants resulting in improved nutrient uptake and plant growth.     

Analysis of N-acyl homoserine-lactone quorum-sensing molecules made by different strains and biovars of Rhizobium leguminosarum containing different symbiotic plasmids

Strains of Rhizobium leguminosarum use a cell density-dependent gene regulatory system to assess their population density. This is achieved by the accumulation of N-acyl-homoserine lactones (AHLs) in the environment during growth of the bacteria and these AHLs stimulate the induction of various bacterial genes that are up-regulated in the late-exponential and stationary phases of growth. A genetically well-characterised strain of R. leguminosarum biovar viciae was found to have four genes, whose products synthesise different AHLs. We have analysed AHL production by four genetically distinct isolates of R. leguminosarum, three of bv. viciae and one of bv. phaseoli. Distinct differences were seen in the pattern of AHLs produced by the bv. viciae strains compared with bv. phaseoli and the increased levels and diversity of AHLs found in bv. viciae strains can be attributed to the rhiI gene, which is located on the symbiotic (Sym) plasmid and is up-regulated when the bacteria are grown in the rhizosphere. Additional complexity to the profile of AHLs is found to be associated with highly transmissible plasmid pRL1JI of R. leguminosarum bv. viciae, but this is not observed with some other strains, including those carrying different transmissible plasmids. In addition to AHLs produced by the products of genes on the symbiotic plasmid, there is clear evidence for the presence of other AHL production loci. Expression levels and patterns of AHLs can change markedly in different growth media. These results indicate that there is a network of quorum-sensing loci in different strains of R. leguminosarum and these loci may play a role in adapting to rhizosphere growth and plasmid transfer.     

A quantitative analysis of root distortion from contrasting wheat cropping systems

Aims

Bacterial ACC deaminase is one of the key tools to ameliorate plant stress by lowering ethylene level in plants. The effects of ACC deaminase-producing bacteria on the volatile profiles in plants have not been examined to date. To address this, we performed metabolic profiling of volatiles in carrots following inoculation of the bacteria producing ACC deaminase.

Methods

We isolated ACC deaminase-producing bacteria from the inner part of the fruits and vegetables grown on organic farms by culturing on ACC-containing media, and screened them with PCR for the acdS gene, mungbean growth assay, and in vitro ACC deaminase activity. The isolated endophytes were evaluated for their ability to alter volatile profiles in carrots.

Results

Eleven bacterial strains possessing the activity to cleave ACC were selected among the 60 isolates grown on the medium containing ACC as a sole N source. Three of them that belonged to Pseudomonas could reduce the levels of (E)-2-hexenal and the other green leaf volatiles (GLVs) and terpenoids in the carrot leaves following inoculation of the seeds.

Conclusions

The isolated endophytes with ACC deaminase activity could alter the composition of volatiles in plants, probably through lowering ethylene level in the plant.

     

Effect of transferring 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative CHA96 on their growth-promoting and disease-suppressive capacities

Pseudomonas fluorescens strain CHA0, a root colonizing bacterium, has a broad spectrum of biocontrol activity against plant diseases. However, strain CHA0 is unable to utilize 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of plant ethylene, as a sole source of nitrogen. This suggests that CHA0 does not contain the enzyme ACC deaminase, which cleaves ACC to ammonia and alpha-ketobutyrate, and was previously shown to promote root elongation of plant seedlings treated with bacteria containing this enzyme. An ACC deaminase gene, together with its regulatory region, was transferred into P. fluorescens strains CHA0 and CHA96, a global regulatory gacA mutant of CHA0. ACC deaminase activity was expressed in both CHA0 and CHA96. Transformed strains with ACC deaminase activity increased root length of canola plants under gnotobiotic conditions, whereas strains without this activity had no effect. Introduction of ACC deaminase genes into strain CHA0 improved its ability to protect cucumber against Pythium damping-off, and potato tubers against Erwinia soft rot in small hermetically sealed containers. In contrast, ACC deaminase activity had no significant effect on the ability of CHA0 to protect tomato against Fusarium crown and root rot, and potato tubers against soft rot in large hermetically sealed containers. These results suggest that (i) ACC deaminase activity may have lowered the level of plant ethylene thereby increasing root length; (ii) the role of stress-generated plant ethylene in susceptibility or resistance depends on the host-pathogen system, and on the experimental conditions used; and (iii) the constructed strains could be developed as biosensors for the role of ethylene in plant diseases.     

Drought tolerance ofRhizobium leguminosarum andR. meliloti

Cell suspension ofRhizobium leguminosarum bv.viciae D-253,R. leguminosarum bv.viciae D-560 andR. meliloti D-557 were incorporated into sterile diatomaceous earth (DE) and dried at room temperature. Initial numbers of colony-forming units (CFU), expressed as log₁₀, were 8.27, 8.36 and 8.51, respectively. After 5 months of storage the CFU numbers were 0.00, 5.99 and 7.43, respectively.R. meliloti D-557 showed only minor lowering of the CFU number even after 16 months of storage (log₁₀=7.07). After 7 months of storage in DE some single-colony isolates of D-253 produced 10–100 times higher CFU numbers than the original strain. The isolates of D-560 were much more drought-tolerant. The cells of the original strain died after 7 months of storage, log₁₀ of CFU was 6–7 in the isolates. In both strains some of their drought-tolerant isolates had the same specific acetylene-reducing activity of nodule tissue as the original strains. Diatomaceous earth seems to be a prospective carrier for the formulation of bacterization preparations.     

Genetic Diversity among Rhizobium leguminosarum bv. Trifolii Strains Revealed by Allozyme and Restriction Fragment Length Polymorphism Analyses

Allozyme electrophoresis and restriction fragment length polymorphism (RFLP) analyses were used to examine the genetic diversity of a collection of 18 Rhizobium leguminosarum bv. trifolii, 1 R. leguminosarum bv. viciae, and 2 R. meliloti strains. Allozyme analysis at 28 loci revealed 16 electrophoretic types. The mean genetic distance between electrophoretic types of R. leguminosarum and R. meliloti was 0.83. Within R. leguminosarum, the single strain of bv. viciae differed at an average of 0.65 from strains of bv. trifolii, while electrophoretic types of bv. trifolii differed at a range of 0.23 to 0.62. Analysis of RFLPs around two chromosomal DNA probes also delineated 16 unique RFLP patterns and yielded genetic diversity similar to that revealed by the allozyme data. Analysis of RFLPs around three Sym (symbiotic) plasmid-derived probes demonstrated that the Sym plasmids reflect genetic divergence similar to that of their bacterial hosts. The large genetic distances between many strains precluded reliable estimates of their genetic relationships.