Habitable pore space and survival ofRhizobium leguminosarum biovartrifolii introduced into soil

The hypothesis that the population size of introduced bacteria is affected by habitable pore space was studied by varying moisture content and bulk density in sterilized, as well as in natural loamy sand and silt loam. The soils were inoculated withRhizobium leguminosarum biovartrifolii and established and maintained at soil water potentials between –5 and –20 kPa (pF 1.7 and 2.3). Rhizobial cells were enumerated when population sizes were expected to be more or less stable. In sterilized soils, the rhizobial numbers were not affected or decreased only slightly when water potentials increased from –20 to –5 kPa. In natural soils, the decrease in rhizobial numbers with increasing water potentials was more pronounced. Bulk density had only minor effects on the population sizes of rhizobia or total bacteria. Soil water retention curves of both soils were used to calculate volume and surface area of pores from different diameter classes, and an estimation of the habitable pore space was made. Combining these values of the theoretical habitable pore space with the measured rhizobial numbers showed that only 0.37 and 0.44% of the habitable pore space was occupied in the sterilized loamy sand and silt loam, respectively. The situation in natural soil is more complicated, since a whole variety of microorganisms is present. Nevertheless, it was suggested that, in general, pore space does not limit proliferation and growth of soil microorganisms.     

Growth and survival of cowpea rhizobia in bauxitic silt loam and sandy clay loam soils

Survival of 4 cowpea Rhizobium strains, IRC291, MI-50A, JRW3 and JRC29, in two soil types (bauxitic silt loam and sandy clay loam) undergoing drying at 30°C and 37°C was examined. While all strains except JRW3 showed a general pattern of increase in their numbers during the first 3 weeks in sterile soils, none of the strains showed any increase in their population in non-sterile soils. Cowpea rhizobia showed better survival in non-sterile bauxitic silt loam than in clay loam soils at 30°C. However, the long-term survival (examined up to 6 months) of rhizobia in both soils was poor at 37°C as compared to 30°C. We also found that cowpea rhizobia survived better in soils undergoing drying than in moist soils at 30°C. Our results suggest that (a) cowpea rhizobia survived better in bauxitic silt loam than in clay loam soil and (b) the low indigenous cowpea rhizobial population in Jamaican soils may be due to their poor long-term survival and weak saprophytic competence.     

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.     

Survival of Cowpea Rhizobia in Soil as Affected by Soil Temperature and Moisture

Successful inoculation of peanuts and cowpeas depends on the survival of rhizobia in soils which fluctuate between wide temperature and moisture extremes. Survival of two cowpea rhizobial strains (TAL309 and 3281) and two peanut rhizobial strains (T-1 and 201) was measured in two soils under three moisture conditions (air-dry, moist (−0.33 bar), and saturated soil) and at two temperatures (25 and 35°C) when soil was not sterilized and at 40°C when soil was sterilized. Populations of rhizobia were measured periodically for 45 days. The results in nonsterilized soil indicated that strain 201 survived relatively well under all environmental conditions. The 35°C temperature in conjunction with the air-dry or saturated soil was the most detrimental to survival. At this temperature, the numbers of strains T-1, TAL309, and 3281 decreased about 2 logs in dry soil and 2.5 logs in saturated soil during 45 days of incubation. In sterilized soil, the populations of all strains in moist soil increased during the first 2 weeks, but decreased rapidly when incubated under dry conditions. The populations did not decline under saturated soil conditions. From these results it appears that rhizobial strains to be used for inoculant production should be screened under simulated field conditions for enhanced survival before their selection for commercial inoculant production.     

Population Dynamics of Rhizobium leguminosarum Tn5 Mutants with Altered Cell Surface Properties Introduced into Sterile and Nonsterile Soils

The influence of cell surface properties on attachment to soil particles and on population dynamics of introduced bacteria was studied in sterilized and nonsterilized loamy sand and silt loam. Rhizobium leguminosarum RBL5523 and three Tn5 mutants (RBL5762, RBL5810, and RBL5811) with altered cell surface properties were used. Cellulose fibrils were not produced by RBL5762. Both RBL5810 and RBL5811 produced 80 to 90% less soluble exopolysaccharides and RBL5811 had, in addition, an altered lipopolysaccharide composition. In sterilized soil the total number of cells as well as the number of particle-associated cells of RBL5523 and RBL5810 were, in general, higher as compared with cell numbers of RBL5762 and RBL5811. Differences between strains in percentage of particle-associated cells in sterilized soil were only found at high inoculum densities, when populations increased little. In the nonsterilized silt loam, final population sizes, as well as numbers of particle-associated cells, of the parental strain (RBL5523) were higher than those of strains with altered cell surface properties after 56 and 112 days of incubation. But in general, differences in survival among the strains were not very marked. The importance of association with soil particles or aggregates for the survival of introduced cells was affirmed by the pronounced increase of the percentage of particle-associated cells during incubation in nonsterilized as well as sterilized soil. However, no clear relation among altered cell surface properties, particle association, and survival was found.     

Soil types with different texture affects development of Rhizoctonia root rot of wheat seedlings

The effect of different soil textures, sandy (97.5% sand, 1.6% silt, 0.9% clay), loamy sand (77% sand, 11% silt, 12% clay) and a sandy clay loam (69% sand, 7% silt, 24% clay), on root rot of wheat caused by Rhizoctonia solani Kühn Anastomosis Group (AG) 8 was studied under glasshouse conditions. The reduction in root and shoot biomass following inoculation with AG-8 was greater in sand than in loamy sand or sandy clay loam. Dry root weight of wheat in the sand, loamy sand and sandy clay loam soils infested with AG-8 was 91%, 55% and 28% less than in control uninfested soils. There was greater moisture retention in the loamy sand and sandy clay loam soils as compared to the sand in the upper 10–20 cm. Root penetration resistance was greater in loamy sand and sandy clay loam than in sand. Root growth in the uninfested soil column was faster in the sand than in the loamy sand and sandy clay loam soils, the roots in the sandy soil being thinner than in the other two soils. Radial spread of the pathogen in these soils in seedling trays was twice as fast in the sand in comparison to the loamy sand which in turn was more than twice that in the sandy clay loam soil. There was no evidence that differences among soils in pathogenicity or soil spread of the pathogen was related to their nutrient status. This behaviour may be related to the severity of the disease in fields with sandy soils as compared to those with loam or clay soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Long-Term Effects of Metal-Rich Sewage Sludge Application on Soil Populations of Bradyrhizobium japonicum

The application of sewage sludge to land may increase the concentration of heavy metals in soil. Of considerable concern is the effect of heavy metals on soil microorganisms, especially those involved in the biocycling of elements important to soil productivity. Bradyrhizobium japonicum is a soil bacterium involved in symbiotic nitrogen fixation with Glycine max, the common soybean. To examine the effect of metal-rich sludge application on B. japonicum, the MICs for Pb, Cu, Al, Fe, Ni, Zn, Cd, and Hg were determined in minimal media by using laboratory reference strains representing 11 common serogroups of B. japonicum. Marked differences were found among the B. japonicum strains for sensitivity to Cu, Cd, Zn, and Ni. Strain USDA 123 was most sensitive to these metals, whereas strain USDA 122 was most resistant. In field studies, a silt loam soil amended 11 years ago with 0, 56, or 112 Mg of digested sludge per ha was examined for total numbers of B. japonicum by using the most probable number method. Nodule isolates from soybean nodules grown on this soil were serologically typed, and their metal sensitivity was determined. The number of soybean rhizobia in the sludge-amended soils was found to increase with increasing rates of sludge. Soybean rhizobia strains from 11 serogroups were identified in the soils; however, no differences in serogroup distribution or proportion of resistant strains were found between the soils. Thus, the application of heavy metal-containing sewage sludge did not have a long-term detrimental effect on soil rhizobial numbers, nor did it result in a shift in nodule serogroup distribution.     

Fractionation and Estimation of Particle-Attached and Unattached Bradyrhizobium japonicum Strains in Soils

Rhizobial cells attached or unattached to soil particles were estimated. Nonsterile soils into which antibiotic-resistant mutants of Bradyrhizobium japonicum had been introduced were fractionated by a centrifugation technique into two fractions: A, which contained mainly rhizobial cells attached to soil particles, and F, which contained mainly rhizobial cells unattached to them. Rhizobial counts decreased in both fractions during incubation of the soil at 30°C, with a concomitant decrease in the proportion of the count of fraction F to that of fraction A. Sonication of fraction A of the soil incubated for more than 3 weeks caused an increase in the rhizobial count. The ratio of the count of fraction A estimated by the plant infection method to that estimated by the dilution plate method increased after 5 days of soil incubation. More than 90% of the indigenous rhizobia in an agricultural field existed in fraction A. These results suggest that the majority of rhizobial cells are attached to soil particles.     

Xiphinema americanum as Affected by Soil Organic Matter and Porosity

The effects of four soil types, soil porosity, particle size, and organic matter were tested on survival and migration of Xiphinema americanum. Survival and migration were significantly greater in silt loam than in clay loam and silty clay soils. Nematode numbers were significantly greater in softs planted with soybeans than in fallow softs. Nematode survival was greatest at the higher of two pore space levels in four softs. Migration of X. americanum through soft particle size fractions of 75-150, 150-250, 250-500, 500-700, and 700-1,000 μ was significantly greater in the middle three fractions, with the least occurring in the smallest fraction. Additions of muck to silt loam and loamy sand soils resulted in reductions in survival and migration of the nematode. The fulvic acid fraction of muck, extracted with sodium hydroxide, had a deleterious effect on nematode activity. I conclude that soils with small amounts of air-filled pore space, extremes in pore size, or high organic matter content are deleterious to the migration and survival of X. americanum, and that a naturally occurring toxin affecting this species may be present in native soft organic matter.     

Growth of Indigenous Rhizobium leguminosarum and Rhizobium meliloti in Soils Amended with Organic Nutrients

The ability of indigenous Rhizobium leguminosarum and Rhizobium meliloti to use organic nutrients as growth substrates in soil was assessed by indirect bacteriophage analysis. A total of 17 organic compounds, including 9 carbohydrates, 3 organic acids, and 5 amino acids, were tested (1,000 μg g⁻¹) in three soils with different cropping histories. Four additional soils were screened with a glucose amendment. Nutrient amendments stimulated growth of indigenous rhizobia, allowing subsequent replication of indigenous bacteriophages. Phage populations were enumerated by plating soil extracts on 19 R. leguminosarum and 9 R. meliloti indicator strains, including root nodule isolates from the soils assayed. On the basis of indirect phage analysis, all soils contained native rhizobia similar to one or more of the indicator strains, although not all indicator strains were detected in soil. All organic compounds stimulated growth of indigenous rhizobia, but the growth response varied for each rhizobial strain depending on the nutrient, the nutrient concentration, and the soil. Indigenous rhizobia readily utilized most organic compounds except phenylalanine, glycine, and aspartic acid. The ability of indigenous rhizobia to utilize a wide range of organic compounds as growth substrates in situ indicates their ability to successfully compete with other soil bacteria for nutrients in these soils.     

Effect of Burkholderia (Pseudomonas) cepacia and soil type on the control of crown rot in wheat

A rifampicin-resistant isolate of Burkholderia (Pseudomonas) cepacia (A3R) reduced crown rot (Fusarium graminearum Group 1) symptoms significantly (P 0.05) in wheat in glasshouse and field experiments and increased grain yield significantly (P 0.05) in one of two field experiments. In glasshouse experiments, applying the bacteria as a soil drench (2.5 × 109 cfu/g soil) was more effective than coating the bacteria on wheat seed (3.4 × 107cfu/seed). In field experiments, the bacteria were applied as a soil drench at the rate of 1.8 x 10¹⁰ cfu/m row. In both the glasshouse and the field, disease severity in the bacteria-inoculated treatments was significantly less in a silt loam than in a sandy loam. The silt loam had a large proportion of fine clay and silt particles (51.7%), which may have favoured the biocontrol activity and survival of the introduced B. cepacia. In a glasshouse experiment, control by B. cepacia was significantly greater in the silt loam than in the sandy loam, which in turn was greater than in a loamy sand. The loamy sand appeared to favour crown rot development but not the activity or survival of the bacterial antagonist. The latter was reflected by the relative populations of the rifampicin-resistant bacteria re-isolated from the various soils during a 5-week period after application of the bacteria (silt loam > sandy loam > loamy sand). This study further confirms that soil type can influence the populations and the level of biocontrol activity of some bacterial antagonists.     

Environmental Factors Influencing Numbers of Rhizobium leguminosarum biovar trifolii and Its Bacteriophages in Two Field Soils

Fluctuations in numbers of Rhizobium leguminosarum biovar trifolii and its bacteriophages in two fields with different soil types were followed during a 17-month period in 1981 and 1982. Mean levels of both phage and rhizobia varied significantly (P < 0.05) on different occasions, with rhizobial levels varying from 1.6 × 10² to 2.0 × 10⁴ cell per g of soil and phage from 0 to 1.7 × 10⁴ PFU/g of soil. Multivariate regression analysis showed rhizobial levels to be significantly and positively related to vegetation height and solar radiation, but not to mean temperature, precipitation, soil matric potential, or soil type. Rhizobiophage concentrations were significantly and positively related to soil matric potential and vegetation height. They were reduced in the silty clay loam soil, although the presence of 34% clay did not prevent phage multiplication and the occurrence of high phage levels.     

Transport of bacterial inoculants through intact cores of two different soils as affected by water percolation and the presence of wheat plants

Abstract Water flow-innduced transport of Burkholderia cepacia strain P2 and Pseudomonas fluorescens strain R2f cells through intact cores of loamy sand and silt loam field soils was measured for two percolation regimes, 0.9 and 4.4 mm h⁻¹, applied daily during 1 hour. For each strain, transport was generally similar between the two water regimes. Translocation of B. cepacia , with 4.4 mm h⁻¹, did occur initially in both soils. In the loamy sand soil, no change in the bacterial distribution occurred during the experiment (51 days). In the silt loam, B. cepacia cell numbers in the lower soil layers were significantly reduced, to levels at or below the limit of detection. Transport of P. fluorescens in both soils also occurred initially and was comparable to that of B. cepacia . Later in the experiment, P. fluorescens was not detectable in the lower soil layers of the loamy sand cores, due to a large decrease in surviving cell numbers. In the silt loam, the inoculant cell distribution did not change with time. Pre-incubation of the inoculated cores before starting percolation reduced B. cepacia inoculant transport in the loamy sand soil measured after 5 days, but not that determined after 54 days. Delayed percolation in the silt loam soil affected bacterial transport only after 54 days. The presence of growing wheat plants overall enhanced bacterial translocation as compared to that in unplanted soil cores, but only with percolating water. Percolation water from silt loam cores appeared the day after the onset of percolation and often contained inoculant bacteria. With loamy sand, percolation water appeared only 5 days after the start of percolation, and no inoculant bacteria were found. The results presented aid in predicting the fate of genetically manipulated bacteria in a field experiment.     

Growth analysis of soil-grown spinach plants at different N-regimes

Spinach plants were grown in pots under controlled conditions in three different soils (a loamy sand, a silt loam at low mineral-N level and a silt loam at the double mineral-N level). The nitrogen uptake pattern varied considerably between the three soil types and was used to validate an equation between the relative growth rate and nitrogen content. This equation is based on the growth response of spinach plants grown hydroponically at equal environmental conditions either at optimum nitrogen supply (complete nutrient solution) or with a relative nitrate addition rate of 0.30 day^–1, 0.225 day^–1 or 0.15 day^–1 effecting an exponential increase in nitrogen uptake. Growth in potted soil was slightly overestimated. Part of this bias was explained by the lower shoot weight ratio observed for the soil grown plants. This was demonstrated by the improvement in growth predictions when using net assimilation rate rather than relative growth rate as the driving variable in the model.     

Influence of soil type on the transfer of plasmid RP4p from Pseudomonas fluorescens to introduced recipient and to indigenous bacteria

Abstract Transfer of plasmid RP4p from introduced Pseudomonas fluorescens to a co-introduced recipient strain or to members of the indigenous bacterial population was studied in four different soils of varying texture planted with wheat. Donor and recipient strains showed good survival in the four soils throughout the experiment. The numbers of transconjugants found in donor and recipient experiments in two soils, Ede loamy sand and Löss silt loam were significantly higher in the rhizosphere than in corresponding bulk soil. In the remaining two soils, Montrond and Flevo silt loam, transconjugant numbers were not significantly higher in the rhizosphere than in the bulk soil.
The combined utilization of a specific bacteriophage eliminate the donor strain and the pat sequence as a specific marker to detect RP4p was found to be very efficient in detecting indigenous transconjugants under various environmental conditions. The numbers of indigenous transconjugants were consistently higher in rhizosphere than bull soil. A significant rhizosphere effect on transconjugant numbers of transconjugants were recovered from Flevo and Montrond silt loam; these soils possess characteristics such as clay or organic matter contents which may be favorable to conjugation.     

A comparison of fine root distribution and water consumption of mature Caragana korshinkii Kom grown in two soils in a semiarid region, China

Water is a key limiting factor for vegetation restoration in the semi-arid areas of China. Caragana korshinkii Kom is a shrub that is widely planted in this region to control soil erosion and land desertification. The objective of this study was to investigate the fine root distribution of mature C. korshinkii and its water consumption, when grown in either silt loam or sandy soils, in order to understand differences between the water cycles of two such soils found in the transition zone between fertile loess hills and desert of the Northern Loess Plateau. Fine root distributions were measured using the trench-profile method. Soil water dynamics were monitored with a neutron probe during two growing seasons. The results showed that fine root area density (FRAD) declined with increasing soil depth in both soils, with 70.7% and 96.6% of the total fine roots being concentrated in the upper 1-m layer of the silt loam and sandy soils, respectively. Water consumption by C. korshinkii in the silt loam was close to that in the sandy soil. Most water consumption in both soil types was from the upper 1-m layer. Little variation in plant available water (PAW) occurred in the 3–6 m soil layer during the whole study period. However, in this layer, the PAW was significantly lower in the silt loam soil than in the sandy soil. Total actual evapotranspiration (ET_a) was slightly higher from the sandy soil plots than from those of the silt loam soil during both growing seasons. Our study indicated that mature C. korshinkii effectively uses about the same amount of water from either the silt loam or sandy soils, but that more soil water at depth was extracted from silt loam soil than from sandy soil.     

A determination of protective microhabitats for bacteria introduced into soil

Abstract Survival studies with rhizobia introduced into loamy sand showed that a kaolinite amendment of the soil improved the survival of Rhizobium , and that bentonite had a very strong positive effect on rhizobial survival. The survival level was significantly higher in soil amended with 10% than with 5% bentonite. The amount of water present in the bentonite amended soil had a significant influence on rhizobial survival; in drier soil, survival levels were highest. For the loamy sand, the loamy sand amended with 5 and 10% bentonite or with 10% kaolinite, the number of rhizobial cells surviving on day 57 after introducing 2.5–5.0×10⁷ cells g⁻¹ dry soil could be described using the distribution of pores from three size classes in a mathematical relationship. Pores with necks < 3 μm and between 3 and 6 μm positively affected the survival of introduced rhizobia whereas pores with necks > 6 μm had a negative effect.     

Transfer of the Pea Symbiotic Plasmid pJB5JI in Nonsterile Soil

Transfer of the pea (Pisum sativum L.) symbiotic plasmid pJB5JI between strains of rhizobia was examined in sterile and nonsterile silt loam soil. Sinorhizobium fredii USDA 201 and HH003 were used as plasmid donors, and symbiotic plasmid-cured Rhizobium leguminosarum 6015 was used as the recipient. The plasmid was carried but not expressed in S. fredii strains, whereas transfer of the plasmid to R. leguminosarum 6015 rendered the recipient capable of nodulating pea plants. Confirmation of plasmid transfer was obtained by acquisition of plasmid-encoded antibiotic resistance genes, nodulation of pea plants, and plasmid profiles. Plasmid transfer in nonsterile soil occurred at frequencies of up to 10⁻⁴ per recipient and appeared to be highest at soil temperatures and soil moisture levels optimal for rhizobial growth. Conjugation frequencies were usually higher in sterile soil than in nonsterile soil. In nonsterile soil, transconjugants were recovered only with strain USDA 201 as the plasmid donor. Increasing the inoculum levels of donor and recipient strains up to 10⁹ cells g of soil⁻¹ increased the number of transconjugants; peak plasmid transfer frequencies, however, were found at the lower inoculum level of 10⁷ cells g of soil⁻¹. Plasmid transfer frequencies were raised in the presence of the pea rhizosphere or by additions of plant material. Transconjugants formed by the USDA 201(pJB5JI) × 6015 mating in soil formed effective nodules on peas.     

Escherichia coli Contamination of Vegetables Grown in Soils Fertilized with Noncomposted Bovine Manure: Garden-Scale Studies

In this study we tested the validity of the National Organic Program (NOP) requirement for a ≥120-day interval between application of noncomposted manure and harvesting of vegetables grown in manure-fertilized soil. Noncomposted bovine manure was applied to 9.3-m² plots at three Wisconsin sites (loamy sand, silt loam, and silty clay loam) prior to spring and summer planting of carrots, radishes, and lettuce. Soil and washed (30 s under running tap water) vegetables were analyzed for indigenous Escherichia coli. Within 90 days, the level of E. coli in manure-fertilized soil generally decreased by about 3 log CFU/g from initial levels of 4.2 to 4.4 log CFU/g. Low levels of E. coli generally persisted in manure-fertilized soil for more than 100 days and were detected in enriched soil from all three sites 132 to 168 days after manure application. For carrots and lettuce, at least one enrichment-negative sample was obtained ≤100 days after manure application for 63 and 88% of the treatments, respectively. The current ≥120-day limit provided an even greater likelihood of not detecting E. coli on carrots (≥1 enrichment-negative result for 100% of the treatments). The rapid maturation of radishes prevented conclusive evaluation of a 100- or 120-day application-to-harvest interval. The absolute absence of E. coli from vegetables harvested from manure-fertilized Wisconsin soils may not be ensured solely by adherence to the NOP ≥120-day limit. Unless pathogens are far better at colonizing vegetables than indigenous E. coli strains are, it appears that the risk of contamination for vegetables grown in Wisconsin soils would be elevated only slightly by reducing the NOP requirement to ≥100 days.