Fluorescent Pseudomonas influences palisade mesophyll development and spatial root development in Phaseolus vulgaris

Three strains of plant growth promoting fluorescent Pseudomonads (HPR6, RRLJ008 and RRLJ134) were studied for their effect on growth and yield of French bean (Phaseolus vulgaris L.) under field conditions. The effect of these strains on nature of root development and leaf palisade tube length were also examined. The strains induced positive response on growth and physiological parameters resulting in higher yield in P. vulgaris. Strain HPR6 produced the most promising results in thickening of leaf palisade layer, spreading of lateral roots and production of root hairs. The increase in specific leaf weight (SLW), net assimilation rate (NAR) and relative growth rate (RGR) by these strains were 68%, 152% and 167%, respectively. The growth and yield parameters were also significantly improved compared to the uninoculated control. Antibiotic resistant mutant strains demonstrated that these bacteria effectively colonized the rhizosphere of French bean. The results suggest that the strains could be developed for field application on a large scale.     

Tn5 insertion mutants of Pseudomonas sp. 267 defective in siderophore production and their effect on clover (Trifolium pratense) nodulated with Rhizobium leguminosarum bv. trifolii

Pseudomonas sp. strain 267 isolated from soil promoted growth of different plants under field conditions and enhanced symbiotic nitrogen fixation in clover under gnotobiotic conditions. This strain produced pyoverdine-like compound under low-iron conditions and secreted vitamins of the B group. The role of fluorescent siderophore production in the beneficial effect of strain 267 on nodulated clover plants was investigated. Several non-fluorescent (Pvd^-) Tn5 insertion mutants of Pseudomonas sp. strain 267 were isolated and characterized. The presence of Tn5 insertions was confirmed by Southern analysis of EcoRI digested genomic DNA of each derivative strain. The siderophore-negative mutants were compared to the parental strain with respect to their growth promotion of nodulated clover infected with Rhizobium leguminosarum bv. trifolii 24.1. We found that all isolated Pvd^- mutants stimulated growth of nodulated clover plants in a similar manner to the parental strain. No consistent differences were observed between strain 267 and Pvd^- derivatives strains with respect to their plant growth promotion activity under gnotobiotic conditions.Dr Deryto died in august 1994     

Enhancement of symbiotic nitrogen fixation by vitamin-secreting fluorescent Pseudomonas

Fluorescent Pseudomonas sp. strain 267 promotes growth of nodulated clover plants under gnotobiotic conditions. In the growth conditions (60 M FeCl₃), the production of siderophores of the pseudobactin-pyoverdin group was repressed. Plant growth enhancement results from secretion of B vitamins by Pseudomonas sp. strain 267. This was proven by stimulation of clover growth by naturally auxotrophic strains of Rhizobium leguminosarum bv. trifolii and marker strains E. coli thi^- and R. meliloti pan^- in the presence of the supernatant of Pseudomonas sp. strain 267. The addition of vitamins to the plant medium increased symbiotic nitrogen fixation by the clover plants.     

Selection of Portuguese Rhizobium leguminosarum bv. trifolii strains for production of legume inoculants

From several native clover species, growing in six different soil types, 170 Rhizobium leguminosarum biovar trifolii strains were isolated, covering the central and southern regions of Portugal. The effectiveness of the strains varied from ineffective to highly effective on T. subterraneum cv. Clare and on T. fragiferum cv. Palestine, with a predominance of medium and high effectiveness on both host plants. The effectiveness was not influenced by provenence (soil or plant), except for the strains from the rankers soils and for the strains isolated from T. pratense, that were ineffective or medium effective on T. subterraneum.Selected strains were evaluated for effectiveness on T. subterraneum cv. Clare, using the commercial strain TA1 as reference. Several of the isolated strains were more effective than TA1, indicating that local strains may be used to produce better inoculants.     

Studies on the nodulation of strain-specific resistant pea lines using single,mixed and delayed inoculation by Rhizobium leguminosarum

Symbiotic interactions between peas and Rhizobium leguminosarum were investigated by inoculating four pea lines, three of which are strain-specific resistant to the European strain 311d, with various combinations of two strains of Rhizobium, 311d and Tom⁺⁺. The strains were almost equally good to infect the susceptible European cultivar Hero when added singly inoculated. After mixed inoculation (1:1 proportion) strain analysis by ELISA revealed that the nodules were preferentially formed by 311d, although some Tom⁺⁺ nodules were also found mainly on the upper part of the root. Our conclusion is that Tom⁺⁺ is less compatible in comparison with 311d. In addition, we found that as the Hero plants emerged, they were becoming more resistant towards infection with not adapted bacteria. The strain-specific resistant lines from Afghanistan belong to two different systems: Afgh. I, completely resistant to 311d and highly nodulating with Tom⁺⁺, and Afgh. III, incompletely resistant to 311d and poorly nodulating with Tom⁺⁺. Mixed inoculations resulted in nodule depressions, as compared to single inoculations with Tom⁺⁺ ranging from 87% to 14%. The ability of 311d to block infection sites on the roots were found to depend on the degree of symbiotic adaptation between Afgh. I and Tom⁺⁺, respectively Afgh. III and Tom⁺⁺. Strain analysis after double strain inoculation of Afgh. I plants revealed that some nodules were induced by strain 311d. Thus, the presence of Tom⁺⁺ in this case influences the degree of host resistance. However, in Afgh. III plants the resistance towards nodulation were unaffected by the presence of Tom⁺⁺. We suggest that the degree of symbiotic adaptation may change the barrier of resistance towards infection.     

Plant protection and rhizosphere colonization of barley by seed inoculated herbicide degrading Burkholderia (Pseudomonas) cepacia DBO1(pRO101) in 2,4-D contaminated soil

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterium, Burkholderia cepacia (formerly Pseudomonas cepacia) DBO1(pRO101) was coated on non-sterile barley (Hordeum vulgare) seeds, which were planted in two non-sterile soils amended with varying amounts of 2,4-D herbicide. In the presence of 10 or 100 mg 2,4-D per kg soil B. cepacia DBO1(pRO101) readily colonized the root at densities up to 10⁷ CFU per cm root. In soil without 2,4-D the bacterium showed weak root colonization. The seeds coated with B. cepacia DBO1(pRO101) were able to germinate and grow in soils containing 10 or 100 mg kg^–1 2,4-D, while non-coated seeds either did not germinate or quickly withered after germination. The results suggest that colonization of the plant roots by the herbicide-degrading B. cepacia DBO1(pRO101) can protect the plant by degradation of the herbicide in the rhizosphere soil. The study shows that the ability to degrade certain pesticides should be considered, when searching for potential plant growth-promoting rhizobacteria. The role of root colonization by xenobiotic degrading bacteria is further discussed in relation to bioremediation of contaminated soils.     

Inoculation of pea by application of Rhizobium in the planting furrow

Summary Selected streptomycin resistant strains ofRhizobium leguminosarum suspended in nutrient broth were added to the planting furrow immediately before the sowing of pea. The nodule occupancy by a strain isolated from Risø soil (Risø la) was increased from 74 to 90%, when the inoculum rate was increased from 3.7×10⁶ to 3.7×10⁸ cells per cm row. The experimental soil contained 10³ to 10⁴ cells ofR. leguminosarum per gram. An almost inefficient strain isolated from Risø soil (SV10) was less competitive with respect to nodulation on two pea cultivars than an efficient Risø strain (SV15) and an efficient non-Risø strain (R1045). The nodule occupancy by the introduced strains varied between pea cultivars.Irrespective of the generally high nodulation by the efficient strains introduced to the soil, the pea seed yield, compared to pea nodulated by the indigenous population, was not significantly increased. Neither were two commercial inoculants, applied in rates corresponding to 3 times the recommended rate, able to increase the yield. This suggests that the indigenous populations ofR. leguminosarum were sufficient in number and nitrogen fixing capacity to ensure an optimal pea crop. However, some inoculation treatments slightly increased the seed N concentration and total N accumulation, indicating that it may be possible to select or develop bacterial strains that may increase the yield.     

Glucose uptake by free living and bacteroid forms of Rhizobium leguminosarum

Free living cells of Rhizobium leguminosarum contain a constitutive glucose uptake system, except when they are grown on succinate, which appears to prevent its formation. Bacteroids isolated from Pisum sativum L fail to accumulate glucose although they actively take up ¹⁴C-succinate. Glucose uptake in free living cells is an active process since uptake was inhibited by azide, cyanide, dinitrophenol and carbonyl-m-chlorophenyl hydrazone but not by fluoride or arsenate. The non-metabolizable analogue -methyl glucose was extracted unchanged from cells, showing that it was not phosphorylated during its transport. Galactose also appears to the transported via the glucose uptake system. Organic acids, amino acids and polyols had no effect on the actual uptake of glucose. The K _m for -methyl glucose uptake was 2.9×10^-4 M.     

Evidence for two uptake systems in Rhizobium leguminosarum for hydroxy-aromatic compounds metabolized by the 3-oxoadipate pathway

Rhizobium leguminosarum biovar viciae and Rhizobium leguminosarum biovar trifolii have separate uptake systems for 4-hydroxybenzoate and protocatechuate. The 4-hydroxybenzoate uptake system (pobP) is inhibited by a range of compounds with substitution at the 4-position on the aromatic ring whereas the uptake system for protocatechuate (pcaP) is markedly inhibited only by other dihydroxybenzoic acids. The rate of 4-hydroxybenzoate uptake is very low in Rhizobium leguminosarum and Rhizobium trifolii grown on protocatechuate but mutants defective in 4-hydroxybenzoate uptake transport protocatechuate at rates similar to the wild-type grown under similar conditions.     

Selection of Rhizobium leguminosarum bv. viceae strains for inoculation of Pisum sativum L. cultivars: Analysis of symbiotic efficiency and nodulation competitiveness

From an analysis of 481 Rhizobium leguminosarum bv. viceae strains with 7 pea cultivars in pot and field experiments, we demonstrated that effective strains could be isolated from a rich medium-acid grey forest soil of the Oröl area (Central region of the European part of Russia) but not from a poor acid podzolic soil of the St. Petersburg area (North-West Russia). The proportion of the isolates significantly increasing N accumulation in pea plants (10.2%) is higher than that of strains increasing the shoot dry mass (4.6%) in the pot experiments. The mean values of the increase for N accumulation (33.8%) upon inoculation are also higher than for shoot mass (27.0%) in these experiments. N accumulation in the inoculated pea plants in the pot experiments was significantly correlated with seed yield and seed N accumulation in field experiments, while for shoot dry mass these correlations were either weak or not significant. Two-factor analysis of variance demonstrated that the contribution of plant cultivars to the variation of the major symbiotic efficiency parameters is higher (30.8–31.6%) and contributions of cultivar-strain specificity is lower (5.4–8.8%) than the contributions of strain genotypes (13.4–14.9%). We identified an ineffective R. leguminosarum bv. viceae strain 50 which can be used as a tester for assessing the nodulation competitiveness of the effective strains by an indirect method (analysis of dry mass and N accumulation in pea plants inoculated with the mixture of the tested effective strains and the tester strain). The relative competitive ability (RCA) determined by this method was 75.7–82.8% for strain 52 but only 10.5–13.8% for strain 250a; this difference was confirmed by a direct method (use of the streptomycin-resistant mutants). Results of screening of the diverse collection of 53 effective R. leguminosarum bv. viceae strains by the indirect method permits us to divide them into 3 groups (32 high-competitive, 10 medium-competitive and 11 low-competitive strains) but reveals no correlation between the competitiveness and symbiotic efficiency. N accumulation in the pea shoots is demonstrated to be a much more suitable criterion than the shoot mass for selection either of the highly-effective or of highly-competitive (by the indirect estimation) R. leguminosarum bv. viceae strains in the pot experiments.     

Growth promotion of maize and lettuce by phosphate-solubilizing Rhizobium leguminosarum biovar. phaseoli

Rhizobium leguminosarum bv. phaseoli strains P31 and R1, Serratia sp. strain 22b, Pseudomonas sp. strain 24 and Rhizopus sp. strain 68 were examined for their plant growth-promoting potential on lettuce and forage maize. All these phosphate solubilizing microorganisms (PSM) were isolated from Québec soils. The plants were grown in field conditions in three sites having high to low amounts of available P. In site 1 (very fertile soil), strains R1 and 22b tended to increase the dry matter yield of lettuce shoots (p≤0.10). Lettuce inoculated with rhizobia R1 had a 6% higher P concentration (p≤0.10) than the uninoculated control. In site 2 (poorly fertile soil), the dry matter of lettuce shoots was significantly increased (p≤0.05) by inoculation with strain P31 and 24 plus 35 kg ha^-1 P-superphosphate, or with strain 68 plus 70 kg ha^-1 P-superphosphate. In site 3 (moderately fertile soil), the dry matter of maize shoots was significantly increased (p≤0.05) by inoculation with strain 24 plus 17.5 kg ha^-1 P-superphosphate, or with strain P31 plus 35 kg ha^-1 P-superphosphate. Inoculation with PSM did not affect lettuce P uptake in the less fertile soil in site 2. In site 3 with the moderately fertile soil, maize plants inoculated with strain R1 had 8% higher P concentration than the uninoculated control (p≤0.01), and 6% with strains P31 and 68 (p≤0.05). The results clearly demonstrate that rhizobia specifically selected for P solubilization function as plant growth promoting rhizobacteria with the nonlegumes lettuce and maize. The P solubilization effect seems to be the most important mechanism of plant growth promotion in moderately fertile and very fertile soils when P uptake was increased with rhizobia and other PSM.     

Parallel variation in isoenzyme and nitrogen fixation markers in a Rhizobium population

Twenty isolates of Rhizobium leguminosarum bv. viceae were isolated at random from one field and examined for symbiotic plasmid fragment length polymorphisms and for isoenzyme patterns. The latter are most probably chromosome markers. With one exception both methods separated the isolates into the same 13 different groups. The largest group was represented 7 times according to isoenzymes and 8 times according to RFLP. This fixed non-random association of plasmid and chromosomal genotypes is consistent with a clonal population structure; it indicates limited exchange of plasmids under natural conditions. Seventeen isolates of 11 groups were highly effective and 2 isolates in one group almost ineffective.     

Inoculation of Vicia sativa subsp. nigra roots with Rhizobium leguminosarum biovar viciae results in release of nod gene activating flavanones and chalcones

Flavonoids released by roots of Vicia sativa subsp. nigra (V. sativa) activate nodulation genes of the homologous bacterium Rhizobium leguminosarum biovar viciae (R. l. viciae). Inoculation of V. sativa roots with infective R. l. viciae bacteria largely increases the nod gene-inducing ability of V. sativa root exudate (A.A.N. van Brussel et al., J Bact 172: 5394–5401). The present study showed that, in contrast to sterile roots and roots inoculated with R. l. viciae cured of its Sym plasmid, roots inoculated with R. l. viciae harboring its Sym plasmid released additional nod gene-inducing flavonoids. Using ¹H-NMR, the structures of the major inducers released by inoculated roots, 6 flavanones and 2 chalcones, were elucidated. Roots extracts of (un)inoculated V. sativa contain 4 major non-inducing, most likely glycosylated, flavonoids. Therefore, the released flavonoids may either derive from the root flavonoids or inoculation with R. l. viciae activates de novo flavonoid biosynthesis.     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.     

A simple assay for fluorescent siderophores produced by Pseudomonas species and an efficient isolation of pseudobactin

Several iron binding metabolites (siderophores) of Pseudomonas fluorescens B10 (JL-3133) have been detected using C₁₈ reverse phase HPLC coupled with photodiode array detection methods. This analysis utilized a volatile mobile phase of 90% 20 mm NH₄HCO₃/10% MeOH, pH 6.5. It has been shown to be applicable to other P. fluorescens strains for the detection of related metabolites. Direct scale-up of the analytical HPLC conditions allowed for the efficient preparative isolation of pseudobactin, the principle siderophore produced by P. fluorescens B10 (JL-3133).     

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

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

Degradation of mandelate and 4-hydroxymandelate by Rhizobium leguminosarum biovar trifolii TA1

Rhizobium leguminosarum biovar trifolii TA1 grows on 4-hydroxymandelate and enzymes involved in its catabolism are inducible. Strain TA1 does not grown on mandelate or cis, cis-muconate, but spontaneous mutants capable of growth on these substrates were isolated. Enzymes involved in mandelate degradation were also inducible. The presence of intermediates of the mandelate and hydroxymandelate pathways resulted in a significant decrease in some of the enzymes involved in their degradation. Succinate and acetate, end products of the pathways, and glucose caused reductions in the levels of enzymes in the mandelate and hydroxymandelate pathways.     

Effect of pH on competition for nodule occupancy by type I and type II strains ofRhizobium leguminosarum bv.phaseoli

The effect of soil pH on the competitive abilities of twoRhizobium leuminosarum bv.phaseoli type I and one type II strains was examined in a nonsterile soil system.Phaseolus vulgaris seedlings, grown in unlimed (pH 5.2) or limed (pH 7.6) soil, were inoculated with a single-strain inoculum containing 1 × 10⁶ cells mL^–1 of one of the three test strains or with a mixed inoculum (1:1, type I vs. type II) containing the type II strain CIAT 899 plus one type I strain (TAL 182 or CIAT 895). At harvest, nodule occupants were determined. In a separate experiment, a mixed suspension (1:1, type I vs. type II) of CIAT 899 paired with either TAL 182 or CIAT 895 was used to inoculateP. vulgaris seedlings grown in sterile, limed or unlimed soil. The numbers of each strain in the rhizosphere were monitored for 10 days following inoculation. The majority of nodules (> 60%) formed on plants grown in acidic soil were occupied by CIAT 899, the type II strain. This pattern of nodule occupancy changed in limed soil. When CIAT 899 was paired with TAL 182, the type I strain formed 78% of the nodules. The number of nodules formed by CIAT 899 and CIAT 895 (56% and 44%, respectively) were not significantly different. The observed patterns of nodule occupancy were not related to the relative numbers or specific growth rates of competing strains in the host rhizosphere prior to nodulation. The results indicate that soil pH can influence which symbiotype ofR. leguminosarum bv.phaseoli will competitively nodulateP. vulgaris.     

Biological control of bacterial wilt caused by Pseudomonas solanacearum in India with antagonistic bacteria

From among 125 strains of fluorescent and 52 strains of nonfluorescent bacteria initially screened in the laboratory for their antibiosis towards the bacterial wilt pathogen, Pseudomonas solanacearum, strain Pfcp of Pseudomonas fluorescens and strains B33 and B36 of Bacillus spp., were chosen and evaluated further in greenhouse and field tests. Pfcp treated banana (Musa balbisiana), eggplant and tomato plants were protected from wilt upto 50, 61 and 95% in greenhouse and upto 50, 49 and 36% respectively in field. Protection afforded by the Bacillus strains was lower. In bacteria-treated plants which were subsequently inoculated with P. solanacearum plant height and biomass values increased and were close to those of nontreated and noninoculated control plants.     

Potential for plant growth promotion in groundnut (Arachis hypogaea L.) cv. ALR-2 by co-inoculation of sulfur-oxidizing bacteria and Rhizobium

The use of Rhizobium inoculant for groundnut is a common practice in India. Also, co-inoculation of Rhizobium with other plant growth-promoting bacteria received considerable attention in legume growth promotion. Hence, in the present study we investigated effects of co-inoculating the sulfur (S)-oxidizing bacterial strains with Rhizobium, a strain that had no S-oxidizing potential in groundnut. Chemolithotrophic S-oxidizing bacterial isolates from different sources by enrichment isolation technique included three autotrophic (LCH, SWA5 and SWA4) and one heterotrophic (SGA6) strains. All the four isolates decreased the pH of the growth medium through oxidation of elemental S to sulfuric acid. Characterization revealed that these isolates tentatively placed into the genus Thiobacillus. Clay-based pellet formulation (2.5 x 10(7) cf ug(-1) pellet) of the Thiobacillus strains were developed and their efficiency to promote plant growth was tested in groundnut under pot culture and field conditions with S-deficit soil. Experiments in pot culture yielded promising results on groundnut increasing the plant biomass, nodule number and dry weight, and pod yield. Co-inoculation of Thiobacillus sp. strain LCH (applied at 60 kg ha(-1)) with Rhizobium under field condition recorded significantly higher nodule number, nodule dry weight and plant biomass 136.9 plant(-1), 740.0mg plant(-1) and 15.0 g plant(-1), respectively, on 80 days after sowing and enhanced the pod yield by 18%. Also inoculation of S-oxidizing bacteria increased the soil available S from 7.4 to 8.43 kg ha(-1). These results suggest that inoculation of S-oxidizing bacteria along with rhizobia results in synergistic interactions promoting the yield and oil content of groundnut, in S-deficit soils.