In vitro adhesion of n(2)-fixing enteric bacteria to roots of grasses and cereals

Nitrogen-fixing Klebsiella and Enterobacter strains isolated from several plants were assayed for fimbriae and for adhesion to plant roots in vitro. All eight Klebsiella strains formed type 3 fimbriae, and five strains also formed type 1 fimbriae; all 21 Enterobacter strains had type 1 fimbriae. Three strains of Klebsiella carrying either type 1, type 3, or no fimbriae were used as model organisms in developing an in vitro adhesion test. Adhesion was assayed with bacterial cells labeled with [H]leucine. Fifteen N(2)-fixing strains and the three model strains were compared for adhesion to the roots of seven grasses and five cereals. Type 3-fimbriated Klebsiella strains adhered better than the other strains, and type 3 fimbriae appeared to be major adhesins for the Klebsiella strains. Although variations between plants were observed, no host specificity for bacterial adhesion was found.     

Adhesion of fimbriated nitrogen-fixing enteric bacteria to roots of grasses and cereals

Summary The role of fimbriae in enterobacterial adhesion to roots of grasses and cereals is discussed. All nitrogen-fixing enteric bacteria isolated in Finland had fimbriae. AllEnterobacter isolates had mannose-binding type-1 fimbriae, whereas most of theKlebsiella isolates had both type-1 and type-3 fimbriae. The strains were isolated from a total of ten different grass species, and no specific association was found between grass species and bacterial fimbriation, biogroup or serogroup. Purified, radiolabeled fimbriae bound to roots ofPoa pratensis in vitro, and bacterial adhesion was inhibited by Fab fragments specific for fimbriae.Klebsiella strains carrying type-3 fimbriae adhered to roots of various grasses and cereals more efficiently than type-1- or nonfimbriated strains, and it was concluded that type-3 fimbriae are the major adhesions ofKlebsiella. Immunofluorescence studies revealed that the bacteria preferentially adhered to root hairs, and to a lesser extent, to the zone of elongation and the root cap mucilage. No strict host specificity in enterobacterial adhesion was observed.     

Associative Nitrogen Fixation by Klebsiella spp.: Adhesion Sites and Inoculation Effects on Grass Roots

Adhesion sites on grass roots for Klebsiella strains carrying type 3 or type 1 fimbriae or both were determined. Adhesion of the strains to the roots of Poa pratensis and Festuca rubra was highly localized; the bacteria adhered strongly to root hairs and with a markedly lower efficiency to the surface of the zone of elongation and to the root cap mucilage. No adhesion to the epidermal cells between root hairs was observed. The adhesion sites were identical for the type 3- and 1-fimbriated bacteria and for P. pratensis, F. rubra, and Trifolium pratense. Inoculation of P. pratensis seedlings with Klebsiella pneumoniae strain As resulted in morphological changes in plant roots. The roots of infected plants were heavily covered with root hairs, which often were deformed and branched.     

Type 1 fimbria-mediated adhesion of enteric bacteria to grass roots

Type 1 fimbriae of Klebsiella pneumoniae and Enterobacter agglomerans mediated bacterial adhesion to the roots of bluegrass, Poa pratensis. Purified, radiolabeled fimbriae bound to grass roots in vitro; binding was inhibited by alpha-methyl-d-mannoside or Fab fragments to the fimbriae. Anti-type 1 fimbriae Fab fragments and alpha-methyl-d-mannoside also inhibited adhesion of type 1-fimbriated bacteria to P. pratensis roots. It is proposed that associative nitrogen fixation by Klebsiella and Enterobacter strains also involves type 1 fimbriae, in addition to the type 3 fimbriae of Klebsiella spp. (T. K. Korhonen, E. Tarkka, H. Ranta, and K. Haahtela, J. Bacteriol. 155:860-865, 1983).     

Type 3 fimbriae of Klebsiella sp.: molecular characterization and role in bacterial adhesion to plant roots.

Type 3 fimbriae of Klebsiella were purified and characterized. The fimbriae were 4 to 5 nm in diameter and 0.5 to 2 microns long. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the fimbrillin had an apparent molecular weight of 23,500, and it differed from enterobacterial type 1 fimbrillins in its amino acid composition. Hydrophobic amino acids comprised 33.6% of all amino acids in the fimbrillin, which lacked cystine, phenylalanine, and arginine. Serologically, the type 3 fimbriae were also distinct from the type 1 fimbriae. Purified type 3 fimbriae agglutinated tannin-treated human blood group O erythrocytes; this confirms the role of type 3 fimbriae as hemagglutinins. Purified 125I-labeled type 3 fimbriae bound to the roots of Poa pratensis, and this binding could be inhibited by Fab fragments to the purified fimbriae. Anti-type 3 fimbriae Fab fragments also inhibited bacterial adhesion to plant roots. These results demonstrate that type 3 fimbriae mediate adhesion of klebsiellas to plant roots. Eight nitrogen-fixing strains of Klebsiella also produced type 3 fimbriae when grown under anaerobic nitrogen fixation conditions. It is proposed that type 3 fimbriae are involved in the establishment of the plant-bacterium association concerning nitrogen-fixing Klebsiella strains.     

Screening of plant growth promoting Rhizobacteria isolated from sunflower (Helianthus annuus L.)

Background and Aims

This study was aimed at assessing the diversity of putatively diazotrophic rhizobacteria associated with sunflower (Helianthus annuus L.) cropped in the south of Brazil, and to examine key plant growth promotion (PGP) characteristics of the isolates for the purposes of increasing plant productivity.

Methods

299 strains were isolated from the roots and rhizosphere of sunflower cultivated in five different areas using N-free media. 16S rDNA PCR-RFLP and 16S rRNA partial sequencing were used for identification and the Shannon index was used to evaluate bacterial diversity. Production of siderophores and indolic compounds (ICs), as well phosphate solubilization activities of each isolate were also evaluated in vitro. On the basis of multiple PGP activities, eight isolates were selected and tested for their N-fixation ability, and their capacity as potential PGPR on sunflower plants was also assessed.

Results

All except three Gram-positive strains (phylum Actinobacteria) belonged to the Gram-negative Proteobacteria subgroups [Gamma (167), Beta (78), and Alpha (50)] and the family Flavobacteriaceae (1)]. Shannon indexes ranged from 0.96 to 2.13 between the five sampling sites. Enterobacter and Burkholderia were the predominant genera isolated from roots and rhizosphere, respectively. Producers of siderophores and ICs were widely found amongst the isolates, but only 19.8% of them solubilized phosphate. About 8% of the isolates exhibited all three PGP traits, and these mostly belonged to the genus Burkholderia. Four isolates were able to stimulate the growth of sunflower plants under gnotobiotic conditions.

Conclusions

Enterobacter and Burkholderia were the dominant rhizospheric bacterial genera associated with sunflower plants. Inoculation with isolates belonging to the genera Achromobacter, Chryseobacterium, Azospirillum, and Burkholderia had a stimulatory effect on plant growth.     

Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation

The influence of type 1 fimbriae, mannose-sensitive structures, on biofilm development and maturation has been examined by the use of three isogenic Escherichia coli K12 strains: wild type, fimbriated, and non-fimbriated. Experiments with the three strains were done in minimal medium or Luria–Bertani broth supplemented with different concentrations of d-mannose. The investigation consisted of: (1) characterizing the bacterial surface of the three strains with respect to hydrophilicity and surface charge, (2) investigating the effect of type 1 fimbriae on bacterial adhesion rate and reversibility of initial adhesion on glass surfaces, and (3) verifying the role of type 1 fimbriae and exopolysaccharides (EPS) in biofilm maturation. The results suggest that type 1 fimbriae are not required for the initial bacterial adhesion on glass surfaces as the non-fimbriated cells had higher adhesion rates and irreversible deposition. Type 1 fimbriae, however, are critical for subsequent biofilm development. It was hypothesized that in the biofilm maturation step, the cells synthesize mannose-rich EPS, which functions as a ‘conditioning film’ that can be recognized by the type 1 fimbriae.     

Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria

Several bacterial isolates were recovered from surface-sterilized root nodules of Arachis hypogaea L. (peanut) plants growing in soils from Córdoba, Argentina. The 16S rDNA sequences of seven fast-growing strains were obtained and the phylogenetic analysis showed that these isolates belonged to the Phylum Proteobacteria, Class Gammaproteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. After storage, these strains became unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were even found colonizing pre-formed nodules. Analysis of symbiotic genes showed that the nifH gene was only detected for the Klebsiella-like isolates and the nodC gene could not be amplified by PCR or be detected by Southern blotting in any of the isolates. The results obtained support the idea that these isolates are opportunistic bacteria able to colonize nodules induced by rhizobia.     

Molecular characterization and identification of plant growth promoting endophytic bacteria isolated from the root nodules of pea (Pisum sativum L.)

Root nodule accommodates various non-nodulating bacteria at varying densities. Present study was planned to identify and characterize the non-nodulating bacteria from the pea plant. Ten fast growing bacteria were isolated from the root nodules of cultivated pea plants. These bacterial isolates were unable to nodulate pea plants in nodulation assay, which indicate the non-rhizobial nature of these bacteria. Bacterial isolates were tested in vitro for plant growth promoting properties including indole acetic acid (IAA) production, nitrogen fixation, phosphate solubilization, root colonization and biofilm formation. Six isolates were able to produce IAA at varying level from 0.86 to 16.16 μg ml^?1, with the isolate MSP9 being most efficient. Only two isolates, MSP2 and MSP10, were able to fix nitrogen. All isolates were able to solubilize inorganic phosphorus ranging from 5.57 to 11.73 μg ml^?1, except MSP4. Bacterial isolates showed considerably better potential for colonization on pea roots. Isolates MSP9 and MSP10 were most efficient in biofilm formation on polyvinyl chloride, which indicated their potential to withstand various biotic and abiotic stresses, whereas the remaining isolates showed a very poor biofilm formation ability. The most efficient plant growth promoting agents, MSP9 and MSP10, were phylogenetically identified by 16S rRNA gene sequence analysis as Ochrobactrum and Enterobacter, respectively, with 99 % similarity. It is suggested the potential endophytic bacterial strains, Ochrobactrum sp. MSP9 and Enterobacter sp. MSP10, can be used as biofertilizers for various legume and non-legume crops after studying their interaction with the host crop and field evaluation.     

Effect of Acetate upon the Formation of Acetoin in Klebsiella and Enterobacter and its Possible Practical Application in a Rapid Voges-Proskauer Test

Acetate stimulates the formation of acetoin during 1-h incubation of Voges-Proskauer-positive strains of Klebsiella and Enterobacter. Of these organisms, 124 of 126 strains were recognized as positive in the presence of acetate, and 106 were recognized as positive in its absence.     

Differential Effects of Plant Growth-Promoting Rhizobacteria on Maize Growth and Cadmium Uptake

Maize is a plant known for food, feed, and energy value, but being a greater biomass, it may also be utilized to extract pollutants from soil. Plant growth-promoting rhizobacteria (PGPR) may act as biofertilizer to improve plant health and indirectly may enhance metal extraction. This study focuses on five bacterial strains isolated from the vegetable (Bitter gourd) rhizosphere irrigated with industrial effluent and characterized for various plant growth-promoting activities. Based on 16S rRNA gene sequencing, bacterial strains belonging to the genera, Bacillus (CIK-517, CIK-519), Klebsiella (CIK-518), Leifsonia (CIK-521), and Enterobacter (CIK-521R), were tested for their ability to promote maize growth in axenic conditions. Results showed negative and positive regulation of maize growth by the exogenous application of Cd and PGPR, respectively. Seed germination assays revealed significant reduction in relative seedling growth of maize cultivars upon Cd exposure (0–80 mg Cd L^?1). The tested strains showed tolerance to Cd (1.78–4.45 mmol L^?1) and were positive for catalase, oxidase, phosphate solubilization, exopolysaccharide (EPS), and auxin production, whereas CIK-518, CIK-519, and CIK-521R were negative for EPS, phosphate solubilization, and oxidase activities, respectively. Bacterial strains significantly increased shoot/root growth and their dry biomass in normal and Cd-contaminated soil as compared to their respective controls. None of the strains showed significant effects on relative water content or membrane permeability; however, Cd uptake significantly increased in plant tissues upon bacterial inoculation. Bacterial strains CIK-518 and CIK-521R are effective colonizers and thus can be potential inoculants to promote maize growth and Cd extraction/stabilization in Cd-contaminated soil.     

Antibiotic Resistance and Its Transfer Among Clinical and Nonclinical Klebsiella Strains in Botanical Environments

A total of 183 isolates of Klebsiella from drinking water, market vegetables, wood, sawdust, industrial effluents, and human and animal origin were examined for susceptibility to 10 antibacterial agents. Incidence of resistance to two or more antibiotics tested was: 65% of the human clinical isolates, 59% among bovine mastitis, and 24% among the nonclinical isolates. The five different multiple resistance patterns among nonclinically derived Klebsiella were also found among the human and bovine mastitis isolates. Statistical analyses revealed that patterns of resistance among Klebsiella isolates from drinking water, market vegetables, and industrial effluents were highly correlated with each other and with resistance patterns of human clinical isolates. Antibiotic resistance was transferred between Klebsiella growing in two habitat-simulated environments (growing radish plants and aqueous sawdust suspensions). Transconjugants were detected in 5 of 21 and 6 of 21 mating pairs, respectively. Average transconjugants/donor ranged from 10⁻³ to 10⁻⁶ in Penassay broth, from 10⁻⁶ to 10⁻⁷ on radish plants, and from 10⁻⁵ to 10⁻⁸ in sawdust suspensions. Although antibiotic resistance transfer under simulated environmental conditions can occur, regrowth of clinical strains is probably the major cause for the widespread occurrence of antibiotic-resistant Klebsiella in the nonclinical environment.     

Antagonistic bacteria of composted agro-industrial residues exhibit antibiosis against soil-borne fungal plant pathogens and protection of tomato plants from Fusarium oxysporum f.sp. radicis-lycopersici

Rhizospheric and root-associated/endophytic (RAE) bacteria were isolated from tomato plants grown in three suppressive compost-based plant growth media derived from the olive mill, winery and Agaricus bisporus production agro-industries. Forty-four (35 rhizospheric and 9 RAE) out of 329 bacterial strains showed in vitro antagonistic activity against at least one of the soil-borne fungal pathogens, Fusarium oxysporum f.sp. radicis-lycopersici (FORL), F. oxysporum f.sp. raphani, Phytophthora cinnamomi, P. nicotianae and Rhizoctonia solani. The high percentage of total isolates showing antagonistic properties (13%) and their common chitinase and β-glucanase activities indicate that the cell wall constituents of yeasts and macrofungi that proliferate in these compost media may have become a substrate that favours the establishment of antagonistic bacteria to soil-borne fungal pathogens. The selected bacterial strains were further evaluated for their suppressiveness to tomato crown and root rot disease caused by FORL. A total of six rhizospheric isolates, related to known members of the genera Bacillus, Lysinibacillus, Enterobacter and Serratia and one RAE associated with Alcaligenes faecalis subsp. were selected, showing statistically significant decrease of plant disease incidence. Inhibitory effects of extracellular products of the most effective rhizospheric biocontrol agent, Enterobacter sp. AR1.22, but not of the RAE Alcaligenes sp. AE1.16 were observed on the growth pattern of FORL. Furthermore, application of cell-free culture extracts, produced by Enterobacter sp. AR1.22, to tomato roots led to plant protection against FORL, indicating a mode of biological control action through antibiosis.     

Klebsiella Biotypes Among Coliforms Isolated from Forest Environments and Farm Produce

Samples of water, soil, needles, and bark were collected from three different forest environments and from a pulp and paper mill. In addition, samples of fresh produce were obtained from a local supermarket. All samples were examined for total and fecal coliforms. The counts obtained from the forestrelated samples did not correlate with sample type or location. When 123 isolates were identified biochemically, 71% were Klebsiella, 19% Enterobacter, 8% Citrobacter, and 2% Escherichia. All the Citrobacter, 75% of the Enterobacter, and 65% of the Klebsiella were negative for growth in elevated coliform (EC) broth. All the Escherichia were EC positive. The counts obtained from the fresh produce were generally higher than the forest counts, but the distribution of biotypes was similar. Of the 146 isolates examined 64% were Klebsiella, 14% were Escherichia, 14% were Enterobacter, and 8% were Citrobacter. All the Enterobacter and Citrobacter were EC negative, whereas 25% of the Klebesiella and 80% of the Escherichia were EC positive.     

Distribution of Endophytic Bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. from Soils Contaminated by Polycyclic Aromatic Hydrocarbons

The distributions of endophytic bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. grown in soils contaminated with different levels of polycyclic aromatic hydrocarbons (PAHs) were investigated with polymerase chain reaction followed by denaturing gradient gel electrophoresis technology (PCR-DGGE) and cultivation methods. Twelve types of PAHs, at concentrations varying from 0.16 to 180 mg·kg⁻¹, were observed in the roots and shoots of the two plants. The total PAH concentrations in Alopecurus aequalis Sobol obtained from three different PAH-contaminated stations were 184, 197, and 304 mg·kg⁻¹, and the total PAH concentrations in Oxalis corniculata L. were 251, 346, and 600 mg·kg⁻¹, respectively. The PCR-DGGE results showed that the endophytic bacterial communities in the roots and shoots of the two plants were quite different, although most bacteria belonged to Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. A total of 68 endophytic bacterial strains were isolated from different tissues of the two plants and classified into three phyla: Firmicutes, Proteobacteria and Bacteroidetes. In both plants, Bacillus spp. and Pseudomonas spp. were the dominant cultivable populations. With an increase in the PAH pollution level, the diversity and distribution of endophytic bacteria in the two plants changed correspondingly, and the number of cultivable endophytic bacterial strains decreased rapidly. Testing of the isolated endophytic bacteria for tolerance to each type of PAH showed that most isolates could grow well on Luria-Bertani media in the presence of different PAHs, and some isolates were able to grow rapidly on a mineral salt medium with a single PAH as the sole carbon and energy source, indicating that these strains may have the potential to degrade PAHs in plants. This research provides the first insight into the characteristics of endophytic bacterial populations under different PAH pollution levels and provides a species resource for the isolation of PAH-degrading endophytic bacteria.     

Characterization of Phragmites cummunis rhizosphere bacterial communities and metabolic products during the two stage sequential treatment of post methanated distillery effluent by bacteria and wetland plants

This study deals with the characterization of rhizosphere bacterial communities and metabolic products produced during the two stage sequential treatment of post methanated distillery effluent by bacteria and constructed wetland plants. Results showed that bacterial treatment followed by wetland plants (Phragmites cummunis) resulted 94.5% and 96.0% reduction in BOD and COD values, respectively. The PCR-RFLP analysis showed the presence of Stenotrophomonas, Enterobacter, Pantoea, Acinetobacter and Klebsiella sp., as dominant rhizosphere bacterial communities which play an important role in degradation and decolorization of PMDE in wetland treatment system. Further, the LC-MS-MS and other spectrophotometric analysis have shown that most of the pollutants detected in untreated PMDE were diminished from bacteria and wetland plant treated PMDE indicating that bacteria and wetland plant rhizosphere microbes utilized them as carbon, nitrogen and energy source. While, methylbenzene, furfuryl alcohol, and 4-vinyl-2-methoxyphenol were detected as metabolites in bacteria and hexadecanol in wetland plant rhizosphere treated PMDE.     

Characterization and identification of compost bacteria based on 16S rRNA gene sequencing

The aim of this study was to isolate and characterize bacteria from the compost of fruit and vegetable waste (FVW) for plant growth-promoting (PGP) activities and investigate the pro-active influence of bacterial isolates on wheat growth. Fourteen bacterial strains (RHC-1 to RHC-14) were isolated and purified in tryptic soya agar (TSA). In addition to being biochemically characterized, these bacterial strains were also tested for their PGP traits, such as phosphate (P)-solubilization, nifH gene amplification, indole-3-acetic acid (IAA) quantification and the production of ammonia, oxidase and catalase. Based on 16S rRNA gene sequencing, these bacterial strains were identified as belonging to species of Bacillus, Lysinibacillus, Lysobacter, Staphylococcus, Enterobacter, Pseudomonas and Serratia. All bacterial strains solubilized tri-calcium phosphate and produced IAA. Two bacterial strains RHC-8 (Enterobacter sp.) and RHC-13 (Pseudomonas sp.) solubilized the maximum amount of tri-calcium phosphate, i.e. 486 and 464 μg/ml, respectively. P-solubilization was associated with a significant drop in the pH of the broth culture from an initial pH of 7 to pH 4.43. In addition to P-solubilization and IAA production, six bacterial strains also carried the nifH gene and were further evaluated for their effect on wheat (Triticum aestivum) growth under controlled conditions. All six bacterial strains enhanced wheat growth as compared to uninoculated control plants. Two of the bacterial strains, RHC-8 and RHC-13, identified as Enterobacter aerogenes and Pseudomonas brenneri, respectively, were assessed as potential PGP rhizobacteria due to exhibiting characteristics of four or more PGP traits and enhancing wheat growth though their specific mechanism of action.     

Enhanced Growth of Wheat and Soybean Plants Inoculated with Azospirillum brasilense Is Not Necessarily Due to General Enhancement of Mineral Uptake

The capacity of Azospirillum brasilense to enhance the accumulation of K⁺, P, Ca²⁺, Mg²⁺, S, Na⁺, Mn²⁺, Fe²⁺, B, Cu²⁺, and Zn²⁺ in inoculated wheat and soybean plants was evaluated by using two different analytical methods with five A. brasilense strains originating from four distinct geographical regions. A Pseudomonas isolate from the rhizosphere of Zea mays seedlings was included as a control. All A. brasilense strains significantly improved wheat and soybean growth by increasing root and shoot dry weight and root surface area. The degree of plant response to inoculation varied among the different strains of A. brasilense. All strains were capable of colonizing roots, but the best root colonizer, Pseudomonas sp., had no effect on plant growth. The numbers of organisms of Brazilian strains Sp-245 and Sp-246 colonizing roots were similar regardless of the host plant. Numbers of organisms for the other strains were directly dependent on the host plant. The main feature characterizing mineral accumulation in inoculated plants was that all inoculation treatments changed the mineral balance of the plants, but in an inconsistent manner. Enhancement of mineral uptake by plants also varied among strains to a great extent and was directly dependent on the strain-plant combination; i.e., a strain capable of increasing accumulation of a particular ion in one plant species or cultivar often lacked the ability to do so in another. Minerals in inoculated plants were not evenly distributed in different plant tissues, and the changes varied among groups of plants within each bacterial strain inoculation treatment. We suggest that, although A. brasilense strains are capable of changing the mineral balance and content of plants, it is unlikely that this ability is a general mechanism responsible for plant improvement by A. brasilense.     

<Emphasis Type="Italic">Enterobacter</Emphasis> spp.: A new evidence causing bacterial wilt on mulberry

Thirty-six pathogenetic bacterial strains were isolated from wilted mulberry plants in Hangzhou, Zhejiang province of China. The six representative strains were confirmed to be involved in more than one Enterobacter species by common bacteriological test, electron microscope observation, hypersensitive reaction, Koch’s postulates, physiological and biochemical test, biolog, fatty acid methyl esters analysis (FAMEs), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), 16s rRNA sequences analysis, and comparative analysis with 7 type strains and 3 reference strains. This is the first report on mulberry disease caused by Enterobacter spp. in the world providing new evidence on induction of the plant disease in this genus. The results are not only important in the mulberry disease management but also have significant scientific value for further studies of opportunistic human pathogens and environmental strains in Enterobacter.     

Plant Growth-Promoting Rhizobacteria Inoculation to Enhance Vegetative Growth,Nitrogen Fixation and Nitrogen Remobilisation of Maize under Greenhouse Conditions

Plant growth-promoting rhizobacteria (PGPR) may provide a biological alternative to fix atmospheric N₂ and delay N remobilisation in maize plant to increase crop yield, based on an understanding that plant-N remobilisation is directly correlated to its plant senescence. Thus, four PGPR strains were selected from a series of bacterial strains isolated from maize roots at two locations in Malaysia. The PGPR strains were screened in vitro for their biochemical plant growth-promoting (PGP) abilities and plant growth promotion assays. These strains were identified as Klebsiella sp. Br1, Klebsiella pneumoniae Fr1, Bacillus pumilus S1r1 and Acinetobacter sp. S3r2 and a reference strain used was Bacillus subtilis UPMB10. All the PGPR strains were tested positive for N₂ fixation, phosphate solubilisation and auxin production by in vitro tests. In a greenhouse experiment with reduced fertiliser-N input (a third of recommended fertiliser-N rate), the N₂ fixation abilities of PGPR in association with maize were determined by ¹⁵N isotope dilution technique at two harvests, namely, prior to anthesis (D₅₀) and ear harvest (D₆₅). The results indicated that dry biomass of top, root and ear, total N content and bacterial colonisations in non-rhizosphere, rhizosphere and endosphere of maize roots were influenced by PGPR inoculation. In particular, the plants inoculated with B. pumilus S1r1 generally outperformed those with the other treatments. They produced the highest N₂ fixing capacity of 30.5% (262 mg N₂ fixed plant⁻¹) and 25.5% (304 mg N₂ fixed plant⁻¹) of the total N requirement of maize top at D₅₀ and D₆₅, respectively. N remobilisation and plant senescence in maize were delayed by PGPR inoculation, which is an indicative of greater grain production. This is indicated by significant interactions between PGPR strains and time of harvests for parameters on N uptake and at. % ¹⁵N_e of tassel. The phenomenon is also supported by the lower N content in tassels of maize treated with PGPR, namely, B. pumilus S1r1, K. pneumoniae Fr1, B. subtilis UPMB10 and Acinetobacter sp. S3r2 at D₆₅ harvest. This study provides evidence that PGPR inoculation, namely, B. pumilus S1r1 can biologically fix atmospheric N₂ and provide an alternative technique, besides plant breeding, to delay N remobilisation in maize plant for higher ear yield (up to 30.9%) with reduced fertiliser-N input.