Effect of nematodes on rhizosphere colonization by seed-applied bacteria

There is much interest in the use of seed-applied bacteria for biocontrol and biofertilization, and several commercial products are available. However, many attempts to use this strategy fail because the seed-applied bacteria do not colonize the rhizosphere. Mechanisms of rhizosphere colonization may involve active bacterial movement or passive transport by percolating water or plant roots. Transport by other soil biota is likely to occur, but this area has not been well studied. We hypothesized that interactions with soil nematodes may enhance colonization. To test this hypothesis, a series of microcosm experiments was carried out using two contrasting soils maintained under well-defined physical conditions where transport by mass water flow could not occur. Seed-applied Pseudomonas fluorescens SBW25 was capable of rhizosphere colonization at matric potentials of -10 and -40 kPa in soil without nematodes, but colonization levels were substantially increased by the presence of nematodes. Our results suggest that nematodes can have an important role in rhizosphere colonization by bacteria in soil.     

Nematode-enhanced microbial colonization of the wheat rhizosphere

The mechanisms by which seed-applied bacteria colonize the rhizosphere in the absence of percolating water are poorly understood. Without mass flow, transport of bacteria by growing roots or soil animals, particularly nematodes may be important. We used a sand-based microcosm system to investigate the ability of three species of nematodes (Caenorhabditis elegans, Acrobeloides thornei and a Cruznema sp.) to promote rhizosphere colonization by four strains of beneficial rhizobacteria. In nearly all cases, rhizosphere colonization was substantially increased by the presence of nematodes, irrespective of bacterial or nematode species. Our results suggest that nematodes are important vectors for bacteria rhizosphere colonization in the absence of percolating water.     

Colonization of Wheat Roots by an Exopolysaccharide-Producing Pantoea agglomerans Strain and Its Effect on Rhizosphere Soil Aggregation

The effect of bacterial secretion of an exopolysaccharide (EPS) on rhizosphere soil physical properties was investigated by inoculating strain NAS206, which was isolated from the rhizosphere of wheat (Triticum durum L.) growing in a Moroccan vertisol and was identified as Pantoea aglomerans. Phenotypic identification of this strain with the Biotype-100 system was confirmed by amplified ribosomal DNA restriction analysis. After inoculation of wheat seedlings with strain NAS206, colonization increased at the rhizoplane and in root-adhering soil (RAS) but not in bulk soil. Colonization further increased under relatively dry conditions (20% soil water content; matric potential, −0.55 MPa). By means of genetic fingerprinting using enterobacterial repetitive intergenic consensus PCR, we were able to verify that colonies counted as strain NAS206 on agar plates descended from inoculated strain NAS206. The intense colonization of the wheat rhizosphere by these EPS-producing bacteria was associated with significant soil aggregation, as shown by increased ratios of RAS dry mass to root tissue (RT) dry mass (RAS/RT) and the improved water stability of adhering soil aggregates. The maximum effect of strain NAS206 on both the RAS/RT ratio and aggregate stability was measured at 24% average soil water content (matric potential, −0.20 MPa). Inoculated strain NAS206 improved RAS macroporosity (pore diameter, 10 to 30 μm) compared to the noninoculated control, particularly when the soil was nearly water saturated (matric potential, −0.05 MPa). Our results suggest that P. agglomerans NAS206 can play an important role in the regulation of the water content (excess or deficit) of the rhizosphere of wheat by improving soil aggregation.     

Simulated herbivory effects on rhizosphere organisms in pea (Pisum sativum) depended on phosphate

The effects of simulated aboveground herbivory and phosphate addition to soil on rhizosphere organisms (arbuscular mychorrhiza (AM), Rhizobium spp., bacteria, protozoa and nematodes) were studied in a 2 by 2 factorial designed pot experiment with Pea plants (Pisum sativum). Measurements were performed on 24 day old plants that were still in the nutrient acquisition phase before flowering. AM colonization and bacterial feeding nematodes were stimulated by high simulated her- bivory especially when plants were phosphate deficient. Total number of nematodes was higher with phosphate deficiency. Furthermore, non-significant peak values in soil respiration, total number of nematodes, and bacterial number were observed in phosphate deficient plants with high simulated herbivory. In phosphate amended plants, fast-growing protozoa and bacterial feeding nematodes decreased at high simulated herbivory. These results support the hypothesis that the plant regulates abundances of both AM and free-living rhizosphere organisms and thereby the amount of plant-available nutrients, according to demand via root exudation. Rhizobium spp. was significantly stimulated by phosphate addition but not affected by simulated herbivory. Metabolites produced by rhizosphere bacteria from plants exposed to high simulated herbivory in phosphate amended soil stimulated seed performance. Possible interactions between protozoa and nematodes in relation to production and composition of bacteria in the rhizosphere are discussed.     

Inactivation of gacS Does Not Affect the Competitiveness of Pseudomonas chlororaphis in the Arabidopsis thaliana Rhizosphere

Quorum-sensing-controlled processes are considered to be important for the competitiveness of microorganisms in the rhizosphere. They affect cell-cell communication, biofilm formation, and antibiotic production, and the GacS-GacA two-component system plays a role as a key regulator. In spite of the importance of this system for the regulation of various processes, strains with a Gac⁻ phenotype are readily recovered from natural habitats. To analyze the influence of quorum sensing and the influence of the production of the antibiotic phenazine-1-carboxamide on rhizosphere colonization by Pseudomonas chlororaphis, a gnotobiotic system based on Arabidopsis thaliana seedlings in soil was investigated. Transposon insertion mutants of P. chlororaphis isolate SPR044 carrying insertions in different genes required for the production of N-acyl-homoserine lactones and phenazine-1-carboxamide were generated. Analysis of solitary rhizosphere colonization revealed that after prolonged growth, the population of the wild type was significantly larger than that of the homoserine lactone-negative gacS mutant and that of a phenazine-1-carboxamide-overproducing strain. In cocultivation experiments, however, the population size of the gacS mutant was similar to that of the wild type after extended growth in the rhizosphere. A detailed analysis of growth kinetics was performed to explain this phenomenon. After cells grown to the stationary phase were transferred to fresh medium, the gacS mutant had a reduced lag phase, and production of the stationary-phase-specific sigma factor RpoS was strongly reduced. This may provide a relative competitive advantage in cocultures with other bacteria, because it permits faster reinitiation of growth after a change to nutrient-rich conditions. In addition, delayed entry into the stationary phase may allow more efficient nutrient utilization. Thus, GacS-GacA-regulated processes are not absolutely required for efficient rhizosphere colonization in populations containing the wild type and Gac⁻ mutants.     

Influence of Nematodes on Resource Utilization by Bacteria—an in vitro Study

The positive influence of bacterial feeding nematodes on bacterial mediated processes such as organic matter mineralization and nutrient cycling is widely accepted, but the mechanisms of these interactions are not always apparent. Both transport of bacteria by nematodes, and nutritional effects caused by nematode N excretion are thought to be involved, but their relative importance is not known because of the difficulties in studying these interactions in soil. We developed a simple in vitro assay to study complex nematode/bacterial interactions and used it to conduct a series of experiments to determine the potential influence of nematode movement and nutritional effects on bacterial resource use. The system used bacterial feeding and nonfeeding insect parasitic nematodes, and luminescent bacteria marked with metabolic reporter genes. Both nutritional enhancement of bacterial activity and bacterial transport were observed and we hypothesize that in nature, the relative importance of transport is likely to be greater in bulk soil, whereas nematode excretion may have greater impact in the rhizosphere. In both cases, the ability of nematodes to enhance bacterial resource utilization has implications for soil components of biogeochemical cycling.     

Nitrogen loss by anaerobic oxidation of ammonium in rice rhizosphere

Anaerobic oxidation of ammonium (anammox) is recognized as an important process for nitrogen (N) cycling, yet its role in agricultural ecosystems, which are intensively fertilized, remains unclear. In this study, we investigated the presence, activity, functional gene abundance and role of anammox bacteria in rhizosphere and non-rhizosphere paddy soils using catalyzed reporter deposition–fluorescence in situ hybridization, isotope-tracing technique, quantitative PCR assay and 16S rRNA gene clone libraries. Results showed that rhizosphere anammox contributed to 31–41% N₂ production with activities of 0.33–0.64 nmol N₂ g⁻¹ soil h⁻¹, whereas the non-rhizosphere anammox bacteria contributed to only 2–3% N₂ production with lower activities of 0.08–0.26 nmol N₂ g⁻¹ soil h⁻¹. Higher anammox bacterial cells were observed (0.75–1.4 × 10⁷ copies g⁻¹ soil) in the rhizosphere, which were twofold higher compared with the non-rhizosphere soil (3.7–5.9 × 10⁶ copies g⁻¹ soil). Phylogenetic analysis of the anammox bacterial 16S rRNA genes indicated that two genera of ‘Candidatus Kuenenia'' and ‘Candidatus Brocadia'' and the family of Planctomycetaceae were identified. We suggest the rhizosphere provides a favorable niche for anammox bacteria, which are important to N cycling, but were previously largely overlooked.     

Competition between Pseudomonas fluorescens Ag1 and Alcaligenes eutrophus JMP134 (pJP4) during colonization of barley roots

Abstract: To use deliberately released beneficial microorganisms in the rhizosphere, we need a better understanding of the process of root colonization by seed-borne or soil-borne inocula. In this study, we determine the survival of Pseudomonas fluorescens Ag1 and Alcaligenes eutrophus JMP134, their colonization ability as affected by substrates, and the relative importance of migration versus competition for colonization of the root. Ag1 and the 2,4-dichlorophenoxy-acetic acid (2,4-D) degrader JMP134 were inoculated in sterile barley rhizosphere systems. After inoculation of seeds with individual strains, comparable population sizes were established in the rhizosphere as determined by immunofluorescence microscopic total cell counts. Both strains were motile and able to colonize the entire root system without percolating water to stimulate passive transport. Comparing immunofluorescence microscopic cell counts with colony-forming units demonstrated that a subpopulation of A. eutrophus JMP134 closely associated with the root was non-culturable in contrast to the population in rhizosphere soil. Hence, the sole use of culture-dependent methods may give misleading information about the distribution of bacteria in the rhizosphere. Colonization studies with both strains showed that co-inoculation of Ag1 and JMP134 caused a decrease of the population size of JMP134 if 2,4-D was not added to the soil as a specific carbon source for this strain. Thus, competition for limited carbon sources might influence the composition of the bacterial community in the rhizosphere. We also found that the presence of an established inoculum in the soil reduced subsequent root colonization by a seed-inoculated strain, probably by filling available niches, also indicating that competition from other bacteria may be an important factor determining the distribution of seed-borne inocula. This factor may be just as important for the distribution of bacteria as migration.     

Involvement of Nitrate Reductase and Pyoverdine in Competitiveness of Pseudomonas fluorescens Strain C7R12 in Soil

Involvement of nitrate reductase and pyoverdine in the competitiveness of the biocontrol strain Pseudomonas fluorescens C7R12 was determined, under gnotobiotic conditions, in two soil compartments (bulk and rhizosphere soil), with the soil being kept at two different values of matric potential (−1 and −10 kPa). Three mutants affected in the synthesis of either the nitrate reductase (Nar⁻), the pyoverdine (Pvd⁻), or both (Nar⁻ Pvd⁻) were used. The Nar⁻ and Nar⁻ Pvd⁻ mutants were obtained by site-directed mutagenesis of the wild-type strain and of the Pvd⁻ mutant, respectively. The selective advantage given by nitrate reductase and pyoverdine to the wild-type strain was assessed by measuring the dynamic of each mutant-to-total-inoculant (wild-type strain plus mutant) ratio. All three mutants showed a lower competitiveness than the wild-type strain, indicating that both nitrate reductase and pyoverdine are involved in the fitness of P. fluorescens C7R12. The double mutant presented the lowest competitiveness. Overall, the competitive advantages given to C7R12 by nitrate reductase and pyoverdine were similar. However, the selective advantage given by nitrate reductase was more strongly expressed under conditions of lower aeration (−1 kPa). In contrast, the selective advantage given by nitrate reductase and pyoverdine did not differ in bulk and rhizosphere soil, indicating that these bacterial traits are not specifically involved in the rhizosphere competence but rather in the saprophytic ability of C7R12 in soil environments.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

Chloroplast Response to Low Leaf Water Potentials: III. Differing Inhibition of Electron Transport and Photophosphorylation

Cyclic and noncyclic photophosphorylation and electron transport by photosystem 1, photosystem 2, and from water to methyl viologen (“whole chain”) were studied in chloroplasts isolated from sunflower (Helianthus annus L. var Russian Mammoth) leaves that had been desiccated to varying degrees. Electron transport showed considerable inhibition at leaf water potentials of −9 bars when the chloroplasts were exposed to an uncoupler in vitro, and it continued to decline in activity as leaf water potentials decreased. Electron transport by photosystem 2 and coupled electron transport by photosystem 1 and the whole chain were unaffected at leaf water potentials of −10 to −11 bars but became progressively inhibited between leaf water potentials of −11 and −17 bars. A low, stable activity remained at leaf water potentials below −17 bars. In contrast, both types of photophosphorylation were unaffected by leaf water potentials of −10 to −11 bars, but then ultimately became zero at leaf water potentials of −17 bars. Although the chloroplasts isolated from the desiccated leaves were coupled at leaf water potentials of −11 to −12 bars, they became progressively uncoupled as leaf water potentials decreased to −17 bars. Abscisic acid and ribonuclease had no effect on chloroplast photophosphorylation. The results are generally consistent with the idea that chloroplast activity begins to decrease at the same leaf water potentials that cause stomatal closure in sunflower leaves and that chloroplast electron transport begins to limit photosynthesis at leaf water potentials below about −11 bars. However, it suggests that, during severe desiccation, the limitation may shift from electron transport to photophosphorylation.     

Enhanced Mineralization of [U-14C]2,4-Dichlorophenoxyacetic Acid in Soil from the Rhizosphere of Trifolium pratense

Enhanced biodegradation in the rhizosphere has been reported for many organic xenobiotic compounds, although the mechanisms are not fully understood. The purpose of this study was to discover whether rhizosphere-enhanced biodegradation is due to selective enrichment of degraders through growth on compounds produced by rhizodeposition. We monitored the mineralization of [U-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in rhizosphere soil with no history of herbicide application collected over a period of 0 to 116 days after sowing of Lolium perenne and Trifolium pratense. The relationships between the mineralization kinetics, the number of 2,4-D degraders, and the diversity of genes encoding 2,4-D/α-ketoglutarate dioxygenase (tfdA) were investigated. The rhizosphere effect on [¹⁴C]2,4-D mineralization (50 μg g⁻¹) was shown to be plant species and plant age specific. In comparison with nonplanted soil, there were significant (P < 0.05) reductions in the lag phase and enhancements of the maximum mineralization rate for 25- and 60-day T. pratense soil but not for 116-day T. pratense rhizosphere soil or for L. perenne rhizosphere soil of any age. Numbers of 2,4-D degraders in planted and nonplanted soil were low (most probable number, <100 g⁻¹) and were not related to plant species or age. Single-strand conformational polymorphism analysis showed that plant species had no impact on the diversity of α-Proteobacteria tfdA-like genes, although an impact of 2,4-D application was recorded. Our results indicate that enhanced mineralization in T. pratense rhizosphere soil is not due to enrichment of 2,4-D-degrading microorganisms by rhizodeposits. We suggest an alternative mechanism in which one or more components of the rhizodeposits induce the 2,4-D pathway.     

Exploiting Genotypic Diversity of 2,4-Diacetylphloroglucinol-Producing Pseudomonas spp.: Characterization of Superior Root-Colonizing P. fluorescens Strain Q8r1-96

The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat. Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis. One RAPD group made up 50% of the total population of DAPG-producing Pseudomonas spp. Both short- and long-term studies indicated that this dominant genotype, exemplified by P. fluorescens Q8r1-96, is highly adapted to the wheat rhizosphere. Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed⁻¹ or soil⁻¹) to establish high rhizosphere population densities (10⁷ CFU g of root⁻¹) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing P. fluorescens strains. Q8r1-96 maintained a rhizosphere population density of approximately 10⁵ CFU g of root⁻¹ after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles. In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all. After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels. We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops. By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.     

Phosphorus acquisition from phytate depends on efficient bacterial grazing, irrespective of the mycorrhizal status of Pinus pinaster

Background and aims

Phosphorus from phytate, although constituting the main proportion of organic soil P, is unavailable to plants. Despite the well-known effects of rhizosphere trophic relationships on N mineralization, no work has been done yet on P mineralization. We hypothesized that the interactions between phytate-mineralizing bacteria, mycorrhizal fungi and bacterial grazer nematodes are able to improve plant P use from phytate.

Methods

We tested this hypothesis by growing Pinus pinaster seedlings in agar containing phytate as P source. The plants, whether or not ectomycorrhizal with the basidiomycete Hebeloma cylindrosporum, were grown alone or with a phytase-producing bacteria Bacillus subtilis and two bacterial-feeder nematodes, Rhabditis sp. and Acrobeloides sp. The bacteria and the nematodes were isolated from ectomycorrhizal roots and soil from P. pinaster plantations.

Results

Only the grazing of bacteria by nematodes enhanced plant P accumulation. Although plants increased the density of phytase-producing bacteria, these bacteria alone did not improve plant P nutrition. The seedlings, whether ectomycorrhizal or not, displayed a low capacity to use P from phytate.

Conclusions

In this experiment, the bacteria locked up the phosphorus, which was delivered to plant only by bacterial grazers like nematodes. Our results open an alternative route for better utilization of poorly available organic P by plants.     

Rhizosphere Colonization and Control of Meloidogyne spp. by Nematode-trapping Fungi

The ability of nematode-trapping fungi to colonize the rhizosphere of crop plants has been suggested to be an important factor in biological control of root-infecting nematodes. In this study, rhizosphere colonization was evaluated for 38 isolates of nematode-trapping fungi representing 11 species. In an initial screen, Arthrobotrys dactyloides, A. superba, and Monacrosporium ellipsosporum were most frequently detected in the tomato rhizosphere. In subsequent pot experiments these fungi and the non-root colonizing M. geophyropagum were introduced to soil in a sodium alginate matrix, and further tested both for establishment in the tomato rhizosphere and suppression of root-knot nematodes. The knob-forming M. ellipsosporum showed a high capacity to colonize the rhizosphere both in the initial screen and the pot experiments, with more than twice as many fungal propagules in the rhizosphere as in the root-free soil. However, neither this fungus nor the other nematode-trapping fungi tested reduced nematode damage to tomato plants.     

Bacterial Activity in the Rhizosphere Analyzed at the Single-Cell Level by Monitoring Ribosome Contents and Synthesis Rates

The growth activity of Pseudomonas putida cells colonizing the rhizosphere of barley seedlings was estimated at the single-cell level by monitoring ribosomal contents and synthesis rates. Ribosomal synthesis was monitored by using a system comprising a fusion of the ribosomal Escherichia coli rrnBP1 promoter to a gene encoding an unstable variant of the green fluorescent protein (Gfp). Gfp expression in a P. putida strain carrying this system inserted into the chromosome was strongly dependent on the growth phase and growth rate of the strain, and cells growing exponentially at rates of ≥0.17 h⁻¹ emitted growth rate-dependent green fluorescence detectable at the single-cell level. The single-cell ribosomal contents were very heterogeneous, as determined by quantitative hybridization with fluorescently labeled rRNA probes in P. putida cells extracted from the rhizosphere of 1-day-old barley seedlings grown under sterile conditions. After this, cells extracted from the root system had ribosomal contents similar to those found in starved cells. There was a significant decrease in the ribosomal content of P. putida cells when bacteria were introduced into nonsterile bulk or rhizosphere soil, and the Gfp monitoring system was not induced in cells extracted from either of the two soil systems. The monitoring system used permitted nondestructive in situ detection of fast-growing bacterial microcolonies on the sloughing root sheath cells of 1- and 2-day-old barley seedlings grown under sterile conditions, which demonstrated that it may be possible to use the unstable Gfp marker for studies of transient gene expression in plant-microbe systems.     

Population Dynamics of Soil Pseudomonads in the Rhizosphere of Potato (Solanum tuberosum L.)

Rhizosphere population dynamics of seven Pseudomonas fluorescens and Pseudomonas putida strains isolated from rhizospheres of various agricultural plants were studied on potato (Solanum tuberosum L.) in field soil under controlled environmental conditions. Rhizosphere populations of two strains (B10 and B4) were quantitatively related to initial seed piece inoculum levels when plants were grown at −0.3 bar matric potential. At a given inoculum level, rhizosphere populations of strain B4 were consistently greater than those of strain B10. In vivo growth curves on 4-cm root tip-proximal segments indicated that both strains grew at similar rates in the potato rhizosphere, but large populations of strain B10 were not maintained at 24°C after 7 h, whereas those of strain B4 were maintained for at least 40 h. Although both strains grew more rapidly in the rhizosphere at 24°C than at 12°C, their rhizosphere populations after seed piece inoculation were generally greater at 12 or 18°C, indicating that in vivo growth did not solely determine rhizosphere populations in these studies. In vitro osmotolerance of seven Pseudomonas strains (including strains B4 and B10) was correlated with their abilities to establish stable populations in the rhizosphere of potato. Stability of rhizosphere populations of the Pseudomonas strains studied here was maximized at low (i.e., 12°C) soil temperatures. These results indicate that Pseudomonas strains differ in their capacity to maintain stable rhizosphere populations in association with potato. This capacity, distinct from the ability to grow in the rhizosphere, may limit the establishment of rhizosphere populations under some environmental conditions.     

Prey Range Characterization, Ribotyping, and Diversity of Soil and Rhizosphere Bdellovibrio spp. Isolated on Phytopathogenic Bacteria

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 × 10² to 6 × 10³ and 2.8 × 10² to 2.3 × 10⁴ PFU g⁻¹. A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.     

Sensitivity of Nematode Life-History Groups to Ions and Osmotic Tensions of Nitrogenous Solutions

Guild designation of nematodes of similar trophic function and life-history strategy provides a basis for using nematode faunal analyses in an integrative assessment of soil food web condition. Omnivorous and predaceous nematodes, categorized at the upper end of a colonizer-persister (c-p) continuum of nematode functional guilds are generally not abundant in cropped soil. These nematodes are more sensitive to heavy metal concentrations than those in other c-p groups, but whether sensitivity to agrochemicals contributes to the observed low abundance of high c-p groups in cropped soils is less well understood. An exposure assay in solution was used to compare the sensitivity of nematodes representing various guilds obtained from field soils and from laboratory culture to several nitrogen sources. Nematodes in c-p groups 4 and 5 were more sensitive to nitrogen solutions than nematodes representing lower c-p groups. There were both osmotic and specific ion effects—the latter most evident in exposure of nematodes to NaNO₂ and (NH₄)₂SO₄. The RC₅₀ (concentration resulting in nematode recovery of one half of that of distilled water) for (NH₄)₂SO₄ was < 0.052 M-N for c-p groups 4 and 5 compared to much greater values (0.34 to 0.81 M-N) for c-p groups 1 to 3. In non-ionic polyethylene glycol (PEG) solutions, osmotic tensions of 0.40 to 0.43 MPa reduced the recovery of exposed nematodes by half (RT₅₀; water potential of solution resulting in nematode recovery of one half of that of distilled water) for c-p groups 4 and 5 compared to > 1.93 MPa for c-p groups 1 to 3. RT₅₀ values for urea solutions, also non-ionic, were greater than for PEG. Caenorhabditis elegans N2 (c-p 1) and Meloidogyne javanica (c-p 3) reared on solid medium and in hydroponic culture, respectively, were slightly more sensitive to specific ion and osmotic effects than nematodes of similar c-p groups obtained from soil. The greater sensitivity of c-p 4 and 5 nematodes to nitrogen solutions suggests that fertilizers may contribute to the low abundance of these nematodes in annual cropping systems. This study supports the use of nematode faunal analyses as indicators of chemical stress in soil.     

Stomatal and nonstomatal regulation of water use in cotton, corn, and sorghum

Stomata of corn (Zea mays L.) and sorghum (Sorghum bicolor L.) responded to changes in leaf water potential during the vegetative growth phase. During reproductive growth, leaf resistances were minimal and stomata were no longer sensitive to bulk leaf water status even when leaf water potentials approached −27 bars. Stomata of corn, cotton (Gossypium hirsutum L.), and sorghum appear to respond to changes in the humidity deficit between the leaf and air and in this manner, regulated transpirational flux to some degree. Distinct differences in water transport efficiency were observed in the three species. Under nonlimiting soil water conditions, sorghum exhibited the greatest efficiency of water transport while under limiting soil moisture conditions, cotton appeared most efficient. Corn was the least efficient with respect to nonstomatal regulation of water use. Differences in drought tolerance among the three species are partially dependent on stomatal regulation of water loss, but efficiency of the water transport system may be more related to drought adaptation. This is particularly important since stomata of all three species did not respond to bulk leaf water status during a large portion of the growing season.