Some antigenic properties of cultured cell and bacteroid forms of fast- and slow-growing strains of Lotus rhizobia.

Immunodiffusion cross-reactions of 62 fast- and 76 slow-growing of Lotus rhizobia with antisera to four of the fast-growing and five of the slow-growing strains were studied. No sharing of antigens by both fast- and slow-growing strains was found. Somatic antigens were very strain specific with only eight of the fast-growing and five of the slow-growing strains tested having somatic antigens identical to those of one or more of the strains of the same group used for antisera production. In contrast, internal antigens were shared by all fast-growing strains and with seven exceptions by all slow-growing strains. Antigens of cultured rhizobia, and bacteroids from nodules formed on different legumes by the same strain of Rhizobium, were similar. However, incontrast to cultured cells, bacteroids generally required no pretreatment (heat or ultrasonic disruption) to give a strong somatic antigen reaction in immunodiffusions.     

Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soils

Tropical cowpea rhizobia are often presumed to be generally promiscuous but poor N fixers. This study was conducted to evaluate symbiotic interactions of 59 indigenous rhizobia isolates (49 of them from cowpea (Vigna unguiculata)), with up to 13 other (mostly tropical) legume species. Host ranges averaged 2.4 and 2.3 legume species each for fast- and slow-growing isolates respectively compared to 4.3 for slow-growing reference cowpea strains. An average of 22% and 19% of fast- and slow-growing cowpea isolates respectively were effective on each of 12 legume species tested. We conclude that the indigenous cowpea rhizobia studied have relatively narrow host ranges. The ready nodulation of different legumes in tropical soils appears due to the diversity of indigenous symbiotic genotypes, each consisting of subgroups compatible with a limited number of legume species.     

Metabolism of glucose and gluconate in fast- and slow-growing rhizobia

Enzymatic evidence was sought for the operation of pathways involved in glucose and gluconate catabolisms in fast- and slow-growing Rhizobium species including members of the cowpea group. Enzymes of the Entner-Doudoroff pathway, pentose phosphate pathway and tricarboxylic acid cycle were detected in fast-growing rhizobia but the pentose phosphate pathway was absent in slow-growers, regardless of the carbon source used. When analysed for enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways in glucose-grown cells, the pathways were found to operate simultaneously in rhizobia.     

Relationships Among Rhizobia from Native Australian Legumes

Isolates from 12 legumes at three sites in Victoria showed a wide range of morphological, cultural, symbiotic, and serological properties. Isolates from Acacia longifolia var. sophorae and Kennedia prostrata were fast growing but nodulated ineffectively Macroptilium atropurpureum and all native legumes except Swainsonia lessertiifolia. Isolates from S. lessertiifolia showed anomalous properties intermediate between fast- and slow-growing rhizobia. All isolates from the other two sites were slow-growing “cowpea” rhizobia. Symbiotic effectiveness was usually poor, and there was no relationship between effectiveness and host taxonomy or serological affinities of the isolates. This is the first report of fast-growing rhizobia from temperate Australian woody legumes and the first report of the symbiotic effectiveness of native Australian legumes with indigenous rhizobia.     

Characterization of rhizobia fromLeucaena

Seventy-six rhizobia were isolated from the nodules ofLeucaena plants of various genotypes growing in a wide range of soil types and climatic regions. The isolates were fast-growing and acid-producing. In establishing a serological grouping for the isolates, the intrinsic antibiotic resistance (IAR) patterns to low concentrations of eight antibiotics was helpful for selecting the strains for immunization purposes. Eight distinct somatic serogroups ofLeucaena rhizobia were identified by using strain-specific fluorescent antibodies. The results indicated that use of serological markers is a more specific technique than IAR pattern for strain identification. Strains from some different serogroups had the same IAR patterns. The immunofluorescence cross-reactions ofLeucaena rhizobia serogroups among themselves and with other species of fast- and slow-growing rhizobia were very low. Sero-grouping is ideal for use in further ecological studies in field inoculation trials.     

Indigenous plasmids and cultural characteristics of rhizobia nodulating chickpeas (Cicer arietinum L.)

We examined 27 strains of chickpea rhizobia from different geographic origins for indigenous plasmids, location and organization of nitrogen fixation (nif) genes, and cultural properties currently used to separate fast- and slow-growing groups of rhizobia. By using an in-well lysis and electrophoresis procedure one to three plasmids of molecular weights ranging from 35 to higher than 380 Mdal were demonstrated in each of 19 strains, whereas no plasmids were detected in the eight remaining strains. Nitrogenase structural genes homologous to Rhizobium meliloti nifHD, were not detected in plasmids of 26 out of the 27 strains tested. Hybridization of EcoRI digested total DNA from these 26 strains to the nif probe from R. meliloti indicated that the organization of nifHD genes was highly conserved in chickpea rhizobia. The only exception was strain IC-72 M which harboured a plasmid of 140 Mdal with homology to the R. meliloti nif DNA and exhibited also a unique organization of nifHD genes. The chickpea rhizobia strains showed a wide variation of growth rates (generation times ranged from 4.0 to 14.5 h) in yeast extract-mannitol medium but appear to be relatively homogeneous in terms of acid production in this medium and acid reaction in litmus milk. Although strains with fast and slow growth rates were identified, DNA/DNA hybridization experiments using a nifHD-specific probe, and the cultural properties examined so far do not support the separation of chickpea rhizobia into two distinct groups of the classical fast- and slow-growing types of rhizobia.     

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.     

Growth of Fast- and Slow-Growing Rhizobia on Ethanol

Free-living soybean rhizobia and Bradyrhizobium spp. (lupine) have the ability to catabolize ethanol. Of the 30 strains of rhizobia examined, only the fast- and slow-growing soybean rhizobia and the slow-growing Bradyrhizobium sp. (lupine) were capable of using ethanol as a sole source of carbon and energy for growth. Two strains from each of the other Rhizobium species examined (R. meliloti, R. loti, and R. leguminosarum biovars phaseoli, trifolii, and viceae) failed to grow on ethanol. One Rhizobium fredii (fast-growing) strain, USDA 191, and one (slow-growing) Bradyrhizobium japonicum strain, USDA 110, grew in ethanol up to concentrations of 3.0 and 1.0%, respectively. While three of the R. fredii strains examined (USDA 192, USDA 194, and USDA 205) utilized 0.2% acetate, only USDA 192 utilized 0.1% n-propanol. None of the three strains utilized 0.1% methanol, formate, or n-butanol as the sole carbon source.     

Nodulation of acacia species by fast- and slow-growing tropical strains of Rhizobium

Thirteen Acacia species were classified into three groups according to effective nodulation response patterns with fast- and slow-growing tropical strains of Rhizobium. The first group nodulated effectively with slow-growing, cowpea-type Rhizobium strains; the second, with fast-growing Rhizobium strains; and the third, with both fast- and slow-growing Rhizobium strains. The Rhizobium requirements of the Acacia species of the second group were similar to those of Leucaena leucocephala.     

DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics

Genetic studies of Mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system. To address this issue we have developed a viral system through a detailed characterization of mycobacteriophage L5, a temperate phage that infects both fast- and slow-growing mycobacteria. We describe here the complete DNA sequence of the L5 genome and initial characterization of L5 virion structure and gene expression. In addition to providing a genetic‘toolbox’for the mycobacteria we find that L5 offers a new paradigm for dsDNA phages, being phenotypically temperate but employing genetic strategies for phage growth usually associated with lytic bacteriophages.     

Nodulation Efficiency of Legume Inoculation as Determined by Intrinsic Antibiotic Resistance

Patterns of intrinsic resistance and susceptibility to different levels of antibiotics were determined for strains of both fast- and slow-growing rhizobia. These patterns were stable to plant passage when they were used to identify Rhizobium strains in nodule suspensions or nodule isolates. The method of identification by intrinsic resistance and susceptibility patterns was reliable for identifying strains in field nodules when strains were first isolated from the nodules to provide a standard inoculum size and then typed on antibiotic-containing media. Other patterns of resistance were encountered during identification of field isolates; these patterns may have resulted from acquired resistance to certain antibiotics or from mixed infections of the nodules. The occurrence of resistance patterns identical to those of inoculant strains among native strains was directly related to the size of the soil population. High strain recovery was associated directly with high rates of inoculation.     

Exopolysaccharide depolymerases induced by Rhizobium bacteriophages.

Enzymes induced by two Rhizobium trifolii bacteriophages caused depolymerization of exopolysaccharides from most R. trifolii and R. leguminosarum strains tested, but did not, in general, attack the exopolysaccharides of R. meliloti, the slow-growing rhizobia, or Agrobacterium. Ca2+ and (or) Mg2+ were required for enzyme activity. In all strains tested, depolymerization of exopolysaccharide occurred when there was successful phage infection, but depolymerization also occurred with exopolysaccharides from nonsusceptible strains.     

Chemical composition of 24 wild species differing in relative growth rate

The chemical composition of 24 plant species which showed a three-fold range in potential growth rate was investigated. The carbon content of whole plants was lower for fast-growing species than for slow-growing ones. Fast-growing species accumulated more organic N-compounds, organic acids and minerals, whereas slow-growing species accumulated more (hemi)cellulose, insoluble sugars and lignin. No correlations with relative growth rate were found for soluble phenolics, soluble sugars and lipids. The costs to construct 1 g of plant biomass were rather similar for fast- and slow-growing species, both when expressed as C needed for C-skeletons, as glucose to provide ATP and NAD(P)H, and as total glucose costs. Therefore, we conclude that, despite the differences in chemical composition between fast- and slow-growing species, variation in the costs of synthesis of whole plant biomass cannot explain interspecific variation in relative growth rate of herbaceous species.     

Underexpression of Ap from R-Plasmids in Fast-Growing Rhizobium Species

The presence of the plasmid RP1 in the cells of Rhizobium leguminosarum strains Rld1, 300, and 248, R. phaseoli 1233, R. trifolii strains T1 and 6661, and R. meliloti 4013 was found to appreciably increase bacterial resistance toward kanamycin and tetracycline but not toward ampicillin. The presence of 16 other R-plasmids in R. leguminosarum was also found to either not increase or only marginally increase bacterial resistance toward ampicillin. It appears now that underexpression of the plasmid-specified ampicillin function is common to most fast- and slow-growing rhizobia.     

Recombineering mycobacteria and their phages

Bacteriophages are central components in the development of molecular tools for microbial genetics. Mycobacteriophages have proven to be a rich resource for tuberculosis genetics, and the recent development of a mycobacterial recombineering system based on mycobacteriophage Che9c-encoded proteins offers new approaches to mycobacterial mutagenesis. Expression of the phage exonuclease and recombinase substantially enhances recombination frequencies in both fast- and slow-growing mycobacteria, thereby facilitating construction of both gene knockout and point mutants; it also provides a simple and efficient method for constructing mycobacteriophage mutants. Exploitation of host-specific phages thus provides a general strategy for recombineering and mutagenesis in genetically naive systems.     

Phenotypic and genotypic analysis of rhizobia isolated from pasture legumes native of Sardinia and Asinara Island

Thirty-five rhizobial strains were isolated from nodules of Lotus edulis, L. ornithopodioides, L. cytisoides, Hedysarum coronarium, Ornithopus compressus and Scorpiurus muricatus growing in Sardinia and Asinara Island. Basic characteristics applied to identification of rhizobia such as symbiotic properties, antibiotic- and salt-resistance, temperate-sensitivities, utilization of different sources of carbon and nitrogen were studied. The results from the 74 metabolic tests were used for cluster analysis of the new rhizobial isolates and 28 reference strains, belonging to previously classified and unclassified fast-, intermediate- and slow-growing rhizobia. All strains examined were divided into two large groups at a linkage distance of 0.58. None of the reference strains clustered with the new rhizobial isolates, which formed five subgroups almost respective of their plant origin. RFLP analysis of PCR-amplified 16S-23S rDNA IGS showed that the levels of similarity between rhizobial isolates from Ornithopus, Hedysarum and Scorpiurus, and the type strains of Rhizobium leguminosarum, Mesorhizobium loti, M. ciceri, M. mediterraneum, Sinorhizobium meliloti and Bradyrhizobium japonicum were not more than 30%. Thus, it can be assumed that these groups of new rhizobial isolates are not closely related to the validly described rhizobial species.     

Inoculant Production with Diluted Liquid Cultures of Rhizobium spp. and Autoclaved Peat: Evaluation of Diluents, Rhizobium spp., Peats, Sterility Requirements, Storage, and Plant Effectiveness

Fully grown broth cultures of various fast- and slow-growing rhizobia were deliberately diluted with various diluents before their aseptic incorporation into autoclaved peat in polypropylene bags (aseptic method) or mixed with the peat autoclaved in trays (tray method). In a factorial experiment with the aseptic method, autoclaved and irradiated peat samples from five countries were used to prepare inoculants with water-diluted cultures of three Rhizobium spp. When distilled water was used as the diluent, the multiplication and survival of rhizobia in the peat was similar to that with diluents having a high nutrient status when the aseptic method was used. In the factorial experiment, the mean viable counts per gram of inoculant were log 9.23 (strain TAL 102) > log 8.92 (strain TAL 82) > log 7.89 (strain TAL 182) after 24 weeks of storage at 28°C. The peat from Argentina was the most superior for the three Rhizobium spp., with a mean viable count of log 9.0 per g at the end of the storage period. The quality of inoculants produced with diluted cultures was significantly (P = 0.05) better with irradiated than with autoclaved peat, as shown from the factorial experiment. With the tray method, rhizobia in cultures diluted 1,000-fold or less multiplied and stored satisfactorily in the presence of postinoculation contaminants, as determined by plate counts, membrane filter immunofluorescence, and plant infection procedures. All strains of rhizobia used in both the methods showed various degrees of population decline in the inoculants when stored at 28°C. Fast- and slow-growing rhizobia in matured inoculants produced by the two methods showed significant (P < 0.01) decline in viability when stored at 4°C, whereas the viability of some strains increased significantly (P < 0.01) at the same temperature. The plant effectiveness of inoculants produced with diluted cultures and autoclaved peat did not differ significantly from that of inoculants produced with undiluted cultures and gamma-irradiated peat.     

Predominance of Fast-Growing Rhizobium japonicum in a Soybean Field in the People's Republic of China

Soybean rhizobia were isolated from two soils with different cropping histories from Hubei province in central China. The first, from Honghu county, has been under soybean cultivation for decades. All of the isolates obtained from nodules on soybeans growing in this soil were fast-growing, acid-producing rhizobia. However, slow-growing, alkali-producing isolates were obtained at higher dilutions of the same soil. The second soil, from Wuchang county, has been under rice cultivation with no record of previous soybean cultivation. All of the soybean rhizobia recovered from this soil, and at higher dilutions of the soil, were typical slow-growing, alkali-producing isolates. The isolates from both soils were grouped by using intrinsic antibiotic resistance, gel immunodiffusion, and fluorescent-antibody procedures. Representative isolates were tested for symbiotic effectiveness with four soybean cultivars (Peking, Davis, Williams, and Ai Jiao Zao) in a pot experiment. There were significant cultivar-rhizobial interactions. Moreover, on each cultivar, there was at least one fast-growing isolate among these new rhizobia that was as effective as the highly effective slow-growing reference strain USDA 110.     

Factors Affecting Phage D29 Infection: A Tool to Investigate Different Growth States of Mycobacteria

Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non-pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay.     

Verification and Rapid Identification of Soybean Rhizobia in Indian Soils

Sixty root nodule isolates of soybean rhizobia indigenous to eight field sites in India were characterized using PCR-RFLP for repeated sequence RSα a 1195-bp DNA fragment, indole acetic acid production, and nitrogenase activity. Site-dependent variations were observed in terms of IAA production and nitrogenase activities. RSα was conserved in slow-growing soybean rhizobia across locations and sites and was absent in other Rhizobiaceae members and other bacterial genera. The results suggest that RSα can be a useful molecular marker for slow-growing soybean rhizobia. The study also showed the low presence of soybean nodulating fast growers in Indian soils.