The effect of moisture potential on growth and survival of root nodule bacteria in peat culture and on seed

Peat from three sources was dried, milled and packed separately in polyethylene bags and sterilized by irradiation. The carrier was impregnated with broth cultures of either Rhizobium leguminosarum bv. trifolii strain WU95, Bradyrhizobium japonicum strain CB1809 or B. lupini strain WU425 and sterile water to provide five moisture potentials in the range > - 1 × 10⁴ - 1 × 10⁶ Pa. The packets were stored at 26°C under conditions which restricted moisture loss. Numbers of root nodule bacteria were counted at intervals up to 12 weeks. No single moisture potential was optimum for all strains in all carriers because of a significant ( P < 0.05) interaction between moisture potential × strain × carrier × time. Where direct comparisons could be made, all strains survived best at - 1 × 10⁴ and/or −3.2 × 10⁴ Pa. Seeds of Trifolium subterraneum and polypropylene beads (used to avoid seed coat toxicity), were inoculated with WU95 prepared in two sources of peat and at each of the above moisture potentials and stored at 15°C. Seed coat toxicity significantly effected the log death rate ( k ) of WU95 on subterraneum clover seed for the period 0–0.25 d ( k 1.796) compared with k - 0.399 for polypropylene beads. In the first 24 h moisture did not affect survival but by 28 d rhizobia grown in Badenoch peat survived best at −3.2 × 10⁴ Pa. In Millicent peat, survival was equally as good at −3.2 × 10⁴ and −1 × 10⁴ Pa.     

The dynamics of Rhizobium leguminosarum biovar trifolii introduced into soil as determined by immunofluorescence and selective plating techniques

Abstract After the introduction of Rhizobium leguminosarum biovar trifolii into a loamy sand and a silt loam, high recovery percentages were determined using quantitative immunofluorescence. Soil type, but not inoculum density between 10⁴ and 10⁸ cells per gram of soil, significantly influenced the recovery percentage of the immunofluorescence technique. Recovery percentages determined using selective plating were independent of either soil type or inoculum density and exceeded those determined by immunofluorescence.
The serological and genetic markers used for detection were stable during 55 days of incubation in phosphate-buffered saline and soil extract solution. After the introduction of R. leguminosarum biovar trifolii into both sterilized soil types, the population increased to 0.5–1×10⁹ cells per gram of soil, but a decline was demonstrated in non-sterile loamy sand and silt loam during incubation of 90 days at 15°C. Starvation of rhizobial cells in the phosphate-buffered saline and soil extract solution, as well as incubation in both soil types, resulted in a significant decrease in mean cell size.     

Competition studies with Rhizobium trifolii in a field experiment

M c L oughlin , T.J. B ordeleau L.M. & D unican , L.K. 1984. Competition studies with Rhizobium trifolii in a field experiment. Journal of Applied Bacteriology 56 , 131–135.
Competition studies were carried out in a field experiment by comparing the ability of antibiotic marked Rhizobium strains inoculated at three inoculum levels in forming nodules against an effective indigenous Rhizobium population of 1.5 times 10⁵/g. Three inoculum strains were assessed. It was seen that by increasing the level of inoculum there was a corresponding increase in the number of nodules formed by the introduced strains. G1067 formed a high proportion (85%) at the highest level, whereas the other two inoculum strains occupied less than 50% of the nodules at the same level. Persistence of the inoculum strain was measured into the second season; G1032 could not be detected, G1006 (another 'foreign' strain) formed 43% and G1067 formed 97% of the nodules sampled. The indigenous Rhizobium population isolated from nodules was shown to be heterogenous, and 15 different intrinsic antibiotic resistance patterns were found. Fifty per cent of the isolates fell in the same group which showed a similar pattern to that of the inoculum strain G1067.     

Rhizobial siderophore as an iron source for clover

Iron uptake by clover plants ( Trifoliuin pratense L. cv. Hruszowska) was studied using radioactive ferric iron (⁵⁵FeCl₂). As shown by autoradiography of non-infected plants, purified rhizobial siderophore isolated from Rhizobium leguminosarum by, trifolii , stimulated the uptake and shoot transport of iron. Addition of rhizobial siderophore into the growth medium of nodulated clover did not affect the iron transport to the shoots. In the absence of the rhizobial siderophore, clover infected by either nitrogen-fixing (Nod⁻ Fix⁺) or nonfixing (Nod⁺ Fix⁻) R. L. trifolii strains took up and transported into the shoots more iron than the non-infected control plants. Nodulated clover reduced Fe(III) more efficiently than the non-infected control plants.     

The construction, detection and use of bioluminescent Rhizobium leguminosarum biovar trifolii strains

A. CRESSWELL, L. SKØT AND A.R. COOKSON. 1994. The gene encoding the firefly luciferase enzyme ( luc ) was introduced to Rhizobium leguminosarum biovar trifolii strains with a view to using the resulting bioluminescent strains to study the survival of genetically-engineered rhizobia in soil microcosms. The genetically-engineered micro-organisms (GEMs) behaved similarly to their parent strains with respect to growth rate in laboratory media and in their symbiotic performance with their host plants. No gene transfer could be detected in laboratory mating experiments. When inoculated onto a non-sterile soil the population of the GEM declined sharply from an initial cell density of 2 times 107⁷ g^-1 soil to approach a stable cell density of approximately 3 times 10² g^-1 after 150 d. Direct photography of bioluminescent rhizobia enabled the detection of colonies as small as 0.1 mm in diameter without the need for transferring colonies onto filter paper. When a Rhizobium strain carrying the luc marker on a plasmid was used as inoculant it was possible to visualize differences in colonization of the rhizosphere of white clover and ryegrass by contact print and colour transparency films. The photographic detection methods described here demonstrate the possibilities of using bioluminescent rhizobia for assessing their survival in soil, and for looking at rhizosphere populations which may be an important site for potential gene transfer.     

A comparison of the fluorescent ELISA and antibiotic resistance identification techniques for use in ecological experiments with Rhizobium trifolii

The fluorescent ELISA technique for the identification of bacteria was compared with antibiotic resistant mutants as marker systems for use with Rhizobium trifolii in root nodules and in soil. With an effective(CP3B) and an ineffective (R4) strain as a mixed 1:1 inoculum, there was a highly significant correlation ( P < 0.001) between the two techniques when the plants were grown at pH 5.5 when the majority of nodules were inhabited by the effective strain. At pH 6.5, where the ineffective strain predominated in the nodules, there was no correlation. The reason was that 85% of R4 nodules had volumes less than 0.1 mm₃ with bacterial numbers obviously below the necessary threshold for detection using the serological method. Both methods were efficient at enumerating rhizobia from soils although the recovery rate from a brown earth soil was significantly higher than from a peat soil. Fluorescent ELISA was able to detect rhizobia at 8.0 times 10₅ cells/ml soil suspension (1 g soil to 10 ml water) in the brown earth soil and at 2.0 times 10₅ cells/ml in the peat soil. The results are discussed in terms of the limitations of both techniques in ecological studies.     

Toxicity of Sodium and Chloride Ions to Rhizobium spp. in Broth and Peat Culture

The growth of a strain of Rhizobium trifolii and of R. meliloti was studied in broth and peat cultures to determine the relative toxicity of Na⁺ and Cl^-. The following salts were added in a range of concentrations: Na₂HPO₄ as a source of Na⁺, CaCl₂.2H₂O as a source of Cl^-, and NaCl. Disodium hydrogen orthophosphate affected the growth rate of both strains in broth culture but not in peat culture. Unexpectedly, calcium chloride was more toxic than NaCl in broth and peat culture. The toxicity of NaCl can be ascribed to the Cl^-. Rhizobium meliloti strains grew on 3·5% NaCl after adaptation during a long period. Rhizobia for soya bean and cowpea grew at 0·5% NaCl and those for clover and pea, at 1·0% NaCl.     

Symbiotic effectiveness, rate of respiration and glutamine synthetase activity of sodium azide-resistant strains of Rhizobium leguminosarum biovar trifolii

Nitrogen fixing efficiency of sodium azide-resistant strains of Rhizobium leguminosarum bv. trifolii was studied in symbiosis with berseem clover plants in chillum jars. Rate of respiration and glutamine synthetase activity were tested in cultured cells and nodules, respectively. It was observed that shoot dry weight and percentage shoot nitrogen were maximum in plants inoculated with strains resistant to 15 μg ml⁻¹ sodium azide. Rate of respiration in cultured cells was lowest in strains resistant to 15 μg ml⁻¹ sodium azide and highest in strains resistant to 5 μg ml⁻¹ sodium azide. A negative correlation was observed between rate of respiration (in cultured cells) and shoot dry weight of host plants. Glutamine synthetase activity was maximum in nodule extracts of host plants inoculated with strains resistant to 5 and 10 μg ml⁻¹ sodium azide, whereas it was minimum for strains resistant to 15 μg ml⁻¹ sodium azide. Hence, resistance to low doses (15 μg ml⁻¹) of sodium azide, together with lower respiratory and glutamine synthetase activities, could be used as a potential method for isolating the symbiotically effective strains of Rh. leguminosarum bv. trifolii.     

The bacterial population of a blanket peat

The number of aerobic bacteria in a blanket peat decreased with depth from 26 times 10⁶/g dry peat in the surface layers to 0.5 times 10⁶/g dry peat at 30–40 cm down the profile, thereafter remaining roughly constant. Obligate psychrophiles comprised <2.5% of this population. Anaerobes were most numerous, 9 times 10⁶/g dry peat at 6–10 cm depth, decreasing to 0.5 times 10⁶/g at 20–30 cm. Calculations indicated that these counts, 10³–10⁴-fold lower than the direct counts, substantially underestimated the active microbial population. Gram negative rods, the predominant aerobes in the surface layers, were replaced by unidentified Bacillus strains at 10–20 cm depth but became increasingly more numerous further down the profile. The Gram negative rods were the most numerous organisms/m² but the Bacillus strains, one third of which were present as spores, made the largest contribution to the biomass/m². Gram positive cocci, Arthrobacter and, infrequently, Nocardia were also isolated. Actinomyces -like forms were the predominant obligate anaerobes and were approximately three times more numerous than clostridia and a curved Gram negative rod.     

Influence of soil pH on the nitrate-reducing microbial populations and their potential to reduce nitrate to NO and N2O

Abstract After the introduction of Rhizobium leguminosarum biovar trifolii into natural loamy sand and silt loam, bacterial numbers increased only directly after inoculation. Thereafter, bacterial numbers decreased until an equilibrium was reached. This decrease was exponential on a log scale and could be described by the function Y = A + B − R ', where Y is the log number of rhizobial cells at time: T ; A represents the lgo of the final population size; B is the difference between the log (initial number of bacteria) and A ; R is the daily reduction factor of Y−A and t is time in days after inoculation. The final population sizes increased with increasing inoculum densities (10⁴−10⁸ bacteria/g soil). In sterilized soil, however, the populations increased up to an equilibrium, which was not affected by the inoculum density.
The final population sizes were higher in silt loam than in loamy sand in natural, as well as in sterilized soil. The final population size was reached earlier in natural silt loam than in loamy sand. Also the growth rate in sterilized soil was higher in silt loam than in loamy sand. The growth rate of low inoculum densities in silt loam was exponential and approximately the same as in yeast extract mannitol broth. The growth rate in loamy sand could be improved by incresing the bulk density of the soil from 1.0 to 1.4 g/cm³.     

Using stable isotopes to determine soil carbon input differences under ambient and elevated atmospheric CO2 conditions

Quantitative estimates of soil C input under ambient (35 Pa) and elevated (60 Pa) CO₂-partial pressure (pCO₂) were determined in a Free-Air Carbon dioxide Enrichment (FACE) experiment. To facilitate ¹³C-tracing, Trifolium repens L. was grown in a soil with an initial δ¹³C distinct by at least 5‰ from the δ¹³C of T. repens grown under ambient or elevated pCO₂. A shift in δ¹³C of the soil organic C was detected after one growing season. Calculated new soil C inputs in soil under ambient and elevated pCO₂ were 2 and 3 t ha^–1, respectively. Our findings suggest that under elevated CO₂ conditions, soil C sequestration may be altered by changes in plant biomass production and quality.     

Bacteria Antagonistic to Pseudomonas tolaasii and their Control of Brown Blotch of the Cultivated Mushroom Agaricus bisporus

S ummary : Pseudomonas tolaasii was isolated from casing peat of healthy and diseased mushroom beds, compost of diseased mushroom beds and from soils round a mushroom farm. It was not isolated from fresh peat or compost from healthy mushroom beds. Three bacteria antagonistic to Ps. tolaasii were isolated from soil and peat. These were a nonfluorescent Pseudomonas sp. (closest to Ps. multivorans ) from soil; and strains of Ps. fluorescens and Enterobacter aerogenes from peat. When the antagonists and the pathogen were added in the ratio of 8 × 10⁷: 10⁶ cells/ml to unsterilized peat and applied to mushroom trays, infection of mushroom sporophores by the pathogen was effectively controlled. In vitro studies failed to show lysis or growth inhibition of Ps. tolaasii by the antagonists.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10³ and 10⁵ cfu/ml and stored at 4°, 10° and 15°C and at room temperature (10°-15°C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C₁ and C₂ were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10⁸ cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10⁷ cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

Direct amplification of nodD from community DNA reveals the genetic diversity of Rhizobium leguminosarum in soil

Sequences of nodD , a gene found only in rhizobia, were amplified from total community DNA isolated from a pasture soil. The polymerase chain reaction (PCR) primers used, Y5 and Y6, match nodD from Rhizobium leguminosarum biovar trifolii , R. leguminosarum biovar viciae and Sinorhizobium meliloti . The PCR product was cloned and yielded 68 clones that were identified by restriction pattern as derived from biovar trifolii [11 restriction fragment length polymorphism (RFLP) types] and 15 clones identified as viciae (seven RFLP types). These identifications were confirmed by sequencing. There were no clones related to S. meliloti nodD . For comparison, 122 strains were isolated from nodules of white clover ( Trifolium repens ) growing at the field site, and 134 from nodules on trap plants of T. repens inoculated with the soil. The nodule isolates were of four nodD RFLP types, with 77% being of a single type. All four of these patterns were also found among the clones from soil DNA, and the same type was the most abundant, although it made up only 34% of the trifolii -like clones. We conclude that clover selects specific genotypes from the available soil population, and that R. leguminosarum biovar trifolii was approximately five times more abundant than biovar viciae in this pasture soil, whereas S. meliloti was rare.     

Limitations of the enzyme-linked immunosorbent assay for routine determination of legume inoculant quality

Peat inoculants containing strains of either Rhizobium or Bradyrhizobium spp. were used to determine correlations between cell numbers and A₄₀₅ values obtained with double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) and indirect ELISA. ELISA values of inoculants containing strains of Rhizobium were weak and non-specific; with Bradyrhizobium spp. strains, readings were higher and cross-reactions negligible when heated inoculant suspensions were allowed to stand for 3 h before ELISA determinations were made. With soybean inoculant, correlation coefficients of r = 0.93 and 0.83 were obtained with DAS and indirect ELISA, respectively. A linear curve relating log cell numbers to A₄₀₅ values was used to determine the reliability of DAS ELISA values obtained over 2 years with tests on commercially produced soybean inoculants. In the range 5 times 10⁸-ca 3 times 10⁹ cells/g inoculant, DAS ELISA estimates closely followed plate counts but no significant correlation was found when inoculants contained >ca 3 times 10⁹ cells/g. With a minimum requirement of 1 times 10⁹ cells/g inoculant, discrepancies between DAS ELISA estimates and plate counts obtained with inoculants produced with gamma-irradiated peat would have resulted in the erroneous rejection or acceptance of 14.5% of all inoculants tested, based on DAS ELISA estimates. With inoculants produced with steam-sterilized peat, which was unfavourable for survival of strain WB1, 70.0% of the inoculants rejected because of low plate counts would have been acceptable on the basis of DAS ELISA estimates.     

Competition among Strains of Rhizobium leguminosarum biovar trifolii and Use of a Diallel Analysis in Assessing Competition

Competition between indigenous Rhizobium leguminosarum biovar trifolii strains and inoculant strains or between mixtures of inoculant strains was assessed in field and growth-room studies. Strain effectiveness under competition was compared with strain performance in the absence of competition. Field inoculation trials were conducted at Elora, Ontario, Canada, with soil containing indigenous R. leguminosarum biovar trifolii. The indirect fluorescent-antibody technique was used for the identification of nodule occupants. Treatments consisted of 10 pure strains, a commercial peat inoculant containing a mixture of strains, and an uninoculated control. Inoculant strains occupied 17.5 to 85% of nodules and resulted in increased dry weight and nitrogen content, as compared with the uninoculated control. None of the strains was capable of completely overcoming resident rhizobia, which occupied, on average, 50% of the total nodules tested. In growth-room studies single commercial strains were mixed in all possible two-way combinations and assessed in a diallel mating design. Significant differences in plant dry weight of red clover were observed among strain combinations. Specific combining ability effects were significant at the 10% level, suggesting that the effectiveness of strain mixtures depended on the specific strain combinations. Strains possessing superior effectiveness and competitive abilities were identified by field and growth-room studies. No relationship was detected between strain effectiveness and competitive ability or between strain recovery and host cultivar. The concentration of indigenous populations was not considered to be a limiting factor in the recovery of introduced strains at this site.     

The Use of Inoculation and Pelleting in the Establishment of White Clover under Mountain Conditions

S ummary . Aberystwyth S.184 and New Zealand Certified Mother white clover ( Trifolium repens ) seed was inoculated with an effective strain of Rhizobium trifolii and pelleted with lime and lime and Gafsa phosphate. When sown in conjunction with S.59 red fescue ( Festuca rubra ) on rotary cultivated Nardus-Festuca mountain pasture treated seed gave a significantly better establishment of clover than the uninoculated, non-pelleted seed of the same varieties.     

AUTOMATED 35S-LABELLED PROTEIN ELECTROPHORESIS FOR IDENTIFICATION OF STRAINS OF RHIZOBIUM LEGUMINOSARUM bv. TRIFOLII

A technique for strain-level identification within a Rhizobium biovar is described, based on automated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of ³⁵S-labelled proteins, offering substantial improvements on existing SDS-PAGE methods particularly in the areas of standardization of electrophoresis conditions and rapidity of positive identification. Gels were analysed with a β-scanner, the beta particle emission data being directly relayed to an IBM PC/AT computer for subsequent manipulation. Analysis of the total protein profiles obtained by this method revealed regions of variability between strains of R. leguminosarum bv. trifolii. Automated comparison of these regions enabled identification of strains. The method was successfully used for identifying root nodule isolates obtained from competition studies between known pairs of R. leguminosarum bv. trifolii strains inoculated onto Trifolium repens.     

A Note on a Highly Sensitive Modified ELISA Technique for Rhizobium Strain Identification

A modification of the ELISA technique using a fluorescent substrate is described and comparisons are made between this highly sensitive technique and the conventional ELISA method for investigations of the Rhizobium /legume symbiosis. The technique can be used to detect Rhizobium spp. both from pure culture and from nodules on Trifolium repens , at a concentration of 10⁴ cells/ml. Tests for cross-reactivity indicated that the technique will facilitate a wide range of experiments which require the identification of Rhizobium strains.     

Survival of Ralstonia solanacearum in Soil under Irrigated Rice Culture and Aquatic Weeds

The survival of Ralstonia solanacearum in naturally infested sandy loam soil under irrigated rice culture was investigated in Sankhu village (1400 m above sea level) in central Nepal. The experimental plot had a previous history of bacterial wilt and a range of 1.5 × 10⁴–3 × 10⁴ colony-forming units (CFU) per g soil was present. The survival of R. solanacearum was monitored in roots of naturally growing aquatic weeds in the rice plot and in soil before and after rice harvest. The incidence of the bacterial infection in the weeds, Dopatrium sp. and Monochoria vaginalis , were 57.5 and 10%, respectively. The bacterial population detected in soil before rice harvest was 1.5 × 10⁴ CFU per g soil whereas a range of 7.5 × 10²–1.5 × 10³ CFU per g was detected after the rice harvest. Biovar typing of R. solanacearum isolated from potato plants, potato tubers, aquatic weeds, and the soil from the experimental plot yielded the diverse biovars 2 A, 3 and 4. This is the first report of the survival of these biovars in soil, which was under continuous flow of irrigation water for 3 months during rice culture.