Rhizosphere Isolates Research Articles

Soil amendment effects of rape ( Brassica napus) residues on pea rhizosphere bacteria

Rhizosphere bacteria were isolated from pea ( Pisum sativum) plants grown in soil amended with a green manure of high-glucosinolate rape ( Brassica napus ‘Dwarf Essex’), and from pea plants grown in soil without amendment. Total viable counts of bacteria and fluorescent Psuedomonas spp., and proportionate representation of actinomycetes and Gram-negative bacteria in the pea rhizosphere community were not significantly different for amended and non-amended pea. Carbon substrate utilization patterns obtained from Biolog GN microplates were compared. Rhizosphere isolates from the two treatments, that differed by less than 50% in utilization of specific carbon substrates, were considered to use those substrates in a similar manner. Cluster analysis was used to evaluate relationships of strains according to their abilities to utilize the different carbon substrates. Non-amended pea and amended pea isolates used 88% of the carbon substrates in a similar manner, and cluster patterns were similar for the amended and non-amended pea strains. The number of nodules formed on amended and non-amended pea roots by Rhizobium leguminosarum were not significantly different. Amendment of soil with rape residues did not appear to significantly alter the pea rhizosphere population and community attributes measured.

A Method for Selection and Characterization of Rhizosphere-Competent Bacteria of Chickpea

A greenhouse assay was developed to evaluate the root-colonizing capability of the native chickpea rhizospheric bacterial population. In this assay system, screening time was reduced on two counts. First, spontaneous chromosomal rifampicin-resistant (Rifr) strains were directly inoculated to seeds without any check for the stability of the mutation, and second, no attempts were made to taxonomically identify all the strains being screened for chickpea rhizosphere competence. Only two chickpea rhizosphere-competent Rifr strains from the group of six good chickpea rhizosphere colonizers forming 10(7) to 10(8) colony-forming units (cfu)/g root were taxonomically identified as Pseudomonas fluorescens NB13R and Pseudomonas spp. NB49R, after screening 49 bacteria. Both the strains showed no difference from their corresponding wild-type strains P. fluorescens NB13 and Pseudomonas spp. NB49 in terms of chickpea rhizosphere competence. Isogenic or equally rhizospheric competitive second non-isogenic bacterial isolate, when present in tenfold higher amount, pre-empted the colonization of the soil by the bacterium, which was present in smaller ratio. These findings indicate that the isogenic or equally rhizospheric competitive second non-isogenic Rifr strains should be compared for their survival and competition with that of the isogenic parent and with each other for specific ecological niche, before using a mixture of isolates, for stable and consistent biological seed treatment to control soilborn pathogens or pests or to promote plant growth.

Fluorescent pseudomonad populations in modified rhizosphere atmospheres

Populations of four isolates of fluorescent pseudomonads in the rhizosphere of tomato ( Lycopersicon esculentum cv. Earlypak) and cucumber ( Cucumis sativus cv. Straight 8) seedlings were measured 48 h after exposure of roots to four soil atmospheres containing O 2-to-CO 2 concentrations of 210-to-0.3 (210 ml 1 −1 O 2, 0.3 ml 1 −1 CO 2, ambient concentrations), 180-to-30, 150-to-60 or 120-to-90. While populations varied depending on the bacterial isolate, the plant genus and the composition of the soil atmosphere, they were generally greater under modified soil atmospheres (atmospheres containing lower-than-ambient concentrations of O 2 and higher-than-ambient concentrations of CO 2) than under a soil atmosphere containing the ambient concentration of O 2 and CO 2. The populations of three of four test isolates in tomato rhizosphere and of two of three isolates in cucumber rhizosphere were between 0.44 and 1.38-fold higher under test modified soil atmospheres than under the ambient atmosphere. The differences were significant ( P < 0.05). Populations of indigenous fluorescent pseudomonads were also significantly ( P < 0.05) greater under modified soil atmospheres than under the ambient atmosphere. These results suggest that the reported inconsistency in biocontrol activity of several fluorescent pseudomonad isolates may in part be due to the effect of soil atmospheric composition on the ability of these bacteria to establish in the rhizosphere.

Utilization of heterologous siderophores and rhizosphere competence of fluorescentPseudomonasspp.

In this study, the potential of different Pseudomonas strains to utilize heterologous siderophores was compared with their competitiveness in the rhizosphere of radish. This issue was investigated in interactions between Pseudomonas putida WCS358 and Pseudomonas fluoresceins WCS374 and in interactions between strain WCS358 and eight indigenous Pseudomonas strains capable of utilizing pseudobactin 358. During four successive plant growth cycles of radish, strain WCS358 significantly reduced rhizosphere population densities of the wild-type strain WCS374 by up to 30 times, whereas derivative strain WCS374(pMR), harboring the siderophore receptor PupA for ferric pseudobactin 358, maintained its population density. Studies involving interactions between strain WCS358 and eight different indigenous Pseudomonas strains demonstrated that despite the ability of these indigenous isolates to utilize pseudobactin 358, their rhizosphere population densities were significantly reduced by strain WCS358 by up to 20 times. Moreover, rhizosphere colonization by WCS358 was not affected by any of these indigenous strains, even though siderophore-mediated growth inhibition of WCS358 by a majority of these strains was demonstrated in a plate bioassay. In conclusion, it can be stated that siderophore-mediated competition for iron is a major determinant in interactions between WCS358 and WCS374 in the rhizosphere. Moreover, our findings support the common assumption that cloning of siderophore receptor genes from one Pseudomonas strain into another can confer a competitive advantage in interactions in the rhizosphere. Interactions between WCS358 and the selected indigenous rhizosphere isolates, however, indicate that other traits also contribute to the rhizosphere competence of fluorescent Pseudomonas spp.Key words: siderophore, siderophore receptors, root colonization, fluorescent Pseudomonas.

Molecular characterization of rhizosphere and clinical isolates of Burkholderia cepacia

Four Burkholderia cepacia strains isolated from the rhizosphere and pathological samples of infected human patients were characterized at the molecular level by different methodologies, including the determination of 16S ribosomal rDNA sequence, restriction endonuclease analysis of total DNA, random amplified polymorphic DNA fingerprinting and Southern hybridization with gene probes for nitrogen fixation and siderophore synthesis. The results indicate that the four strains cluster together within genus Burkholderia, but differ from one another. The DNA from the four strains hybridized to the nifA gene probe from Klebsiella penumoniae, and an appreciable homology with the nifHDK structural genes of Azospirillum brasilense was demonstrated for one rhizosphere strain. Although the four isolates produced an ornibactin-like siderophore, they did not give hybridization with the pvdA probe for hydroxamate biosynthesis from Pseudomonas aeruginosa.

Detection and differentiation of microbial siderophores by isoelectric focusing and chrome azurol S overlay

Siderophores are microbial, low molecular weight iron-chelating compounds. Fluorescent Pseudomonads produce different, strain-specific fluorescent siderophores (pyoverdines) as well as non-fluorescent siderophores in response to low iron conditions. We present an isoelectric focusing method applicable to unpurified as well as to purified pyoverdine samples where the fluorescent siderophores are visualized under UV illumination. Siderophores from different Pseudomonas sp., amongst which are P. aeruginosa, P. fluorescens and P. putida, including egg yolk, rhizospheric and clinical isolates as well as some derived Tn5 mutants were separated by this technique. Different patterns could be observed for strains known to produce different siderophores. The application of the chrome azurol S assay as a gel overlay further allows immediate detection of non-fluorescent siderophores or possibly degradation products with residual siderophore activity. The method was also applied to other microbial siderophores such as deferrioxamine B.

Induced Resistance in the Biocontrol of Pythium aphanidermatum by Pseudomonas spp. on Cucumber

AbstractPlants of Cucumis sativus cv. Corona were grown in rockwool, with the root systems split into two separate pots. B pots were treated with water or a cell suspension of Pseudomonas corrugata isolate 13 or P. fluorescens biovar C isolate 15, two rhizosphere isolates effective as biocontrol agents against Pythium aphanidermatum. The P pot was treated with water or a suspension of zoospores of P. aphanidermatum applied at the same time (simultaneous treatment) or 1 week after B pot treatments. Most of the bacterial treatments reduced disease incidence and disease severity. Simultaneous treatment with isolate 15 increased shoot dry weight, leaf area, and number of fruit of inoculated plants in one trial. Some treatments with isolate 13 or 15 also increased root volume or root dry weight in B or P pots, compared to the inoculated plants where the B pot was treated with water only (Pythium control). Lower population densities of P. aphanidermatum in P pots were detected in some treatments to which bacteria were applied to the B pot, but only in the early stages of the experiment. This is the first report of systemic‐induced resistance against a root pathogen in cucumber obtained by application of Pseudomonas spp. to a root system spatially separated from the pathogen‐inoculated root.

Sterile fungi from zoysiagrass rhizosphere as plant growth promoters in spring wheat

Eleven out of 18 sterile fungal isolates and an isolate each of Penicillium sp. and Trichoderma sp. from the zoysiagrass rhizosphere were effective in enhancing the growth of two wheat varieties in greenhouse conditions. They enhanced the top length and top dry biomass of plants significantly and induced the plants to produce long earheads and more seeds. Notable among isolates were GS6-1, GS6-2, GS7-3, GS7-4, GS8-6, GS10-1, GS10-2, and GU23-3, which enhanced the growth by several times, resulting in a conspicuous growth promotion effect that differed depending on the variety. Penicillium and Trichoderma species were less effective than sterile isolates in enhancing growth. Seven of the 11 effective sterile isolates from the zoysiagrass rhizosphere (as determined under greenhouse conditions) and a wheat rhizosphere isolate (K-17) were further tested under field conditions. Most of the isolates except K-17 enhanced the growth of one variety, whereas a few isolates enhanced the growth of the other variety. However, at least four isolates each increased yields of both varieties. Isolate GS6-1, which was very effective under greenhouse conditions in promoting overall growth, was less effective under field conditions; however, the yield components were not affected. The efficiency of the plant growth promoting isolates depended upon the wheat variety and soil nutrient conditions in addition to their inherent growth promotion abilities.Key words: plant growth promoting fungi (PGPF), sterile fungi, wheat growth promotion, yield components.

Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads.

Tn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141. Pyoverdine negative (Pvd-) mutants were detected by the absence of fluorescence on King's B medium and by their inability to grow in the presence of the iron chelator EDDHA [ethylenediamine di(o-hydroxyphenylacetic acid)]. In P. fluorescens ATCC 17400 and three rhizosphere isolates (one P. putida and two P. fluorescens), the percentage of Pvd- mutants ranged between 0 and 0.54%. In a P. chlororaphis rhizosphere isolate, this percentage was higher (4%). In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization. In Pvd- mutants of P. fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants. In P. aeruginosa PAO1, where 13.1% of the mutants were Pvd-, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization. However, the Pvd- phenotype was generally stable in these mutants. The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P. aeruginosa PAO1 and Kmr mutants were recovered, 89% of which were Pvd- and confirmed to be P. aeruginosa by PCR amplification of the P. aeruginosa lipoprotein gene. The mini-Tn10 insertions were also found to be unstable in PAO1.

Purification of a root-adhesive outer membrane protein of root-colonizingPseudomonas fluorescens

The major outer membrane protein (MOMP) from the rhizosphere isolate Pseudomonas fluorescens OE 28.3 adsorbs strongly and selectively to plant roots. A rapid purification procedure was developed for this protein, involving differential solubilization of the 33-kDa protein from the isolated cell envelope, and preparative SDS-PAGE followed by electro-elution. The purified protein was devoid of lipopolysaccharides, occurred in monomeric form, but was heat-modifiable.

Bacterial characteristics important to rhizosphere competence

Ten bacteria isolated from the soybean rhizosphere and 10 from soil were compared with respect to several characteristics that have been suggested as important to successful root colonization. Rhizosphere and soil isolates differed significantly in their ability to move along soybean roots. Rhizosphere bacteria were able to colonize root segments farther from the point of inoculation in greater numbers than soil bacteria. Rhizosphere and soil bacteria did not differ significantly in rates and extents of growth in either exudates collected from germinating seeds or inorganic salts solution. However, 9 of 10 rhizosphere isolates but only 5 of 10 soil isolates grew quickly or had high cell yields or both when grown in exudates. The extent of adherence to root tips did not differ significantly between rhizosphere and soil bacteria. A significant difference was not evident between rhizosphere and soil isolates in chcmotactic response to exudates. tolerance of low osmotic potentials or the agglutination of cells by exudutes. The results suggest that mobility along the root is important to successful rhizosphere colonization, but none of the other characteristics individually is sufficient to ensure rhizosphere competence.

Use of 15N isotope dilution for quantification of N2 fixation associated with roots of kallar grass (Leptochloa fusca (L.))

Leptochloa fusca (L.) Kunth (kallar grass) has previously been found to exhibit high rates of nitrogen fixation. A series of experiments to determine the level of biological nitrogen fixation using 15N isotopic dilution were carried out in nutrient solution and saline soil. In the nutrient solution, E. coli inoculated plants were taken as non-nitrogen-fixing control. It was observed that nearly 60%–80% of the plant N was derived from atmospheric fixation. Estimations based on the N difference method gave much lower values (18%–35%). In experiments with saline soil which was initially sterilized with chloroform fumigation, a mixed culture of N2-fixing rhizospheric isolates from kallar grass roots was inoculated and planted to kallar grass. Uninoculated treatments were regarded as controls. The soil was previously labelled with 15N by adding cellulose and (15NH4)2SO4. The results of these studies showed fixation values of 6%–32% when estimated by 15N dilution, whereas by the N difference method 54% of the plant N was estimated to be derived from fixation. This discrepancy is due to the increase in root proliferation due to inoculation, which results in greater uptake of soil N. The distribution of 15N in different fractions of the soil-N indicted isotopic dilution due to bacterial fixation of atmospheric N2.

Antigenic relationship of N2-fixing Pseudomonas strain H8 to various known cultures and rice rhizosphere isolates studied by indirect enzyme-linked immunosorbent assay (ELISA)

The serological relationship of several, mainly Gram-negative, bacteria including type cultures and previously isolated rhizosphere bacteria, to Pseudomonas sp. strain H8, a N2-fixing rhizosphere isolate from Maahas soil, was examined by indirect enzyme-linked immunosorbent assay. None cross-reacted significantly with H8 antibody except some of those rhizosphere bacteria previously found cross-reactive by fluorescent antibody test. Several new rhizosphere isolates from wetland rice grown in various fields showed significant immunoreactivity, and most of the reactive bacteria reduced acetylene. However, the cultural characteristics and acetylene reduction activity of most of these isolates were different from those of H8. None of the rhizosphere isolates from dryland rice cross-reacted. In a pot experiment, bacterial cultures similar to H8 were found only in Maahas soil, but none reacted with antibodies against strains H8 and KLH76 (another N2-fixing pseudomonad from rice root). Other isolates showing reaction with these antibodies were, however, neither culturally similar to these strains nor N2 fixing. The data suggest that Pseudomonas sp. H8 is not distributed widely in soils other than that from which it was originally isolated.

Interactions of the microflora from nodulation problem and non-problem soils towards Rhizobium spp on agar culture

Microorganisms (348 fungi, 388 actinomycetes and 319 bacteria) were isolated from a nodulation problem soil, a non-problem virgin soil, a cultivated problem soil and the rhizosphere of clover plants grown in the problem soil. Rhizobium trifolii TA 1 which failed to establish in problem soils was inhibited on laboratory media by a greater number of these soil microorganisms than the better soil colonizing R. trifolii (WU95 and WU290) and R. lupini (WU425). R. lupini was not inhibited or stimulated on agar by many soil or rhizosphere isolates. R. meliloti showed greater stimulation than either R. trifolii or R. lupini and was inhibited by relatively few soil microorganisms so that its poor soil survival was thought to be due to chemical or physical soil conditions rather than to biotic factors. The greatest incidence of rhizobial inhibitors, mainly associated with TA 1, was found among the isolates from the clover rhizosphere. There was a reduction in the relative numbers of rhizobial inhibitors isolated from the cultivated soil compared with the virgin problem soils, a result possibly due to the changed soil environment changing with cultivation, altered vegetation and the addition of superphosphate. Inhibitors of rhizobia were more frequent amongst the bacteria than fungi or actinomycetes. Strong stimulation was more commonly shown by fungi than by actinomycetes or bacteria. The interaction on agar between rhizobia and the soil microflora is related to soil colonization and persistence.

Vitamin production in relation to phosphate solubilization by soil bacteria

Studies were conducted to determine the relationship of vitamin production and ability to dissolve bicalcium phosphate in bacteria isolated from rhizosphere, rhizoplane and control soil. Production of vitamin B 12, riboflavin, niacin, pantothenate and biotin was determined using a bioassay procedure that may be useful for large-scale screening of bacteria for vitamin production. Among isolates producing one or more vitamins, 76.4, 37.5 and 57.5% of control, rhizosphere and rhizoplane isolates, respectively, solubilized phosphate. However, phosphate-solubilizing isolates from rhizosphere and rhizoplane were more active vitamin producers than solubilizing isolates from control soil, and non-solubilizing isolates from any of the three regions. Production of vitamin B 12, riboflavin and niacin by rhizosphere isolates and of riboflavin by rhizoplane isolates was also correlated with ability to solubilize phosphate, but similar relationships were not observed for control isolates.

Rhizosphere Microflora in Relation to Soil Conditions. Part I: Comparison of Bacteria in Soil, Rhizosphere and Rhizoplane

Wheat was grown on two adjacent plots, one fertilized with organic matter (“organic” plot and the other with ammonium sulphate (“inorganic” plot). On four occasions during the growing season bacteria were isolated from soil, rhizosphere and rhizoplane. Principal coordinate analysis of the bacterial isolates demonstrated that the isolate groups from the “organic” and “inorganic” soils were distinctly different. This difference became more pronounced for rhizosphere soil isolates and still more for the rhizoplane. Auf zwei aneinander grenzenden Parzellen wurde Weizen angezogen. Eine der Parzellen wurde mit organischem Material versorgt („organische“ Parzelle), die andere mit Ammoniumsulfat gedüngt („anorganische“ Parzelle). Während der Vegetationsperiode wurden aus dem Boden, der Rhizosphäre und der Rhizoplane viermal Bakterien isoliert. Mit Hilfe einer Koordinationsanalyse konnte gezeigt werden, daß sich die Gruppen der Isolate aus den „organischen“ und „anorganischen“ Böden deutlich voneinander unterscheiden. Diese Unterschiede erwiesen sich bei den Isolaten aus der Rhizosphäre auffallender, mehr noch bei den Isolaten aus der Rhizoplane.

Rhizosphere microflora ofCajanus cajan in relation to Fusarium wilt resistance

Studies were made to find out the factors responsible for resistance inCajanus cajan (L.) Millsp. variety C-11-6 against wilt caused byFusarium udum Butl. It was thought to be either due to associated antagonistic microflora in the rhizosphere or due to biochemical constituents in the plant itself. Screening of the rhizosphere isolates revealed the absence of any potent antagonists againstF. udum suggesting that the resistance in C-11-6 variety is not due to antagonistic organisms in the rhizosphere.

Interactions between Rhizobium japonicum and Soybean Rhizosphere Bacteria1

AbstractThe success of soybean inoculation is influenced by the survival of Rhizobium japonicum as a free‐living soil bacterium. One factor affecting survival is the interaction of R. japonicum with other rhizosphere bacteria. The objective of this study was to observe interactions of R. japonicum and soybean rhizosphere bacteria with techniques of increasing complexity with and without the soybean host (Glycine max (L.) Merrill). Eight of nine rhizosphere isolates inhibited R. japonicum on agar. The degree of inhibition was dependent upon the strain of rhizobia, the bacterial isolate, and the agar medium. The interactions of R. japonicum and rhizosphere bacteria with the soybean host (Glycine max (L.) Merrill) were evaluated by observing soybean nodulation, plant weight, and plant elemental analysis. Nodulation was not affected by rhizosphere bacteria when plants were grown in vermiculite or in sterile soil. One rhizosphere isolate, which failed to inhibit R. japonicum on agar, caused severe soybean tap root injury under gnotobiotic growth in soil. Even with the root damage, nodulation was unaffected with this bacterium‐R. japonicum combination.

Studies on the rhizosphere microflora of onion plants in relation to temperature changes

A study was made of the changes in the rhizosphere microflora of onion seedlings grown under controlled conditions at either 25, 18 or 16°C. In plants grown at the lower temperatures there was a very small rhizosphere effect for the bacteria compared with that for plants grown at 25°C. A series of tests on bacteria isolated from both rhizosphere and root-free soil showed the rhizosphere isolates from seedlings grown at 25°C to be more active physiologically than those from seedlings grown at the lower temperatures. In particular, the ammonifying bacteria were greatly increased in the rhizosphere of plants grown at 25°C. Numbers of rhizosphere fungi were also greater at the higher temperature but the rhizosphere effect was not as great as that for bacteria. Contrary to expectation the greatest rhizosphere effect did not occur at the optimum temperature for growth of the onions.

Studies on rhizosphere organisms and molybdenum concentration in plants—I. Identification of rhizosphere isolates to generic level

Over 1500 bacteria have been identified to generic level within the bounds of Skerman's key (Skerman, Guide to the Identification of the Genera of Bacteria, 1967). A method of recording the characteristics of each isolate on a computer card is presented, together with a procedure for obtaining identification with the aid of a computer.