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Objective: Maize is an important crop for fodder, food and feed industry. The present study explores the plant-microbe interactions as alternative eco-friendly sustainable strategies to enhance the crop yield. Methodology: Bacterial diversity was studied in the rhizosphere of maize by culture-dependent and culture-independent techniques by soil sampling, extraction of DNA, amplification of gene of interest, cloning of desired fragment and library construction. Results: Culturable bacteria were identified as Achromobacter, Agrobacterium, Azospirillum, Bacillus, Brevibacillus, Bosea, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus, Stenotrophomonas and Xanthomonas genera. For culture-independent approach, clone library of 16S ribosomal RNA gene was assembled and 100 randomly selected clones were sequenced. Majority of the sequences were related to Firmicutes (17%), Acidobacteria (16%), Actinobacteria (17%), Alpha-Proteobacteria (7%), Delta-proteobacteria (4.2%) and Gemmatimonadetes (4.2%) However, some of the sequences (30%) were novel that showed no homologies to phyla of cultured bacteria in the database. Diversity of diazotrophic bacteria in the rhizosphere investigated by analysis of PCR-amplified nifH gene sequence that revealed abundance of sequences belonging to genera Azoarcus (25%), Aeromonas (10%), Pseudomonas (10%). The diazotrophic genera Azotobacter, Agrobacterium and Zoogloea related nifH sequences were also detected but no sequence related to Azospirillum was found showing biasness of the growth medium rather than relative abundance of diazotrophs in the rhizosphere. Conclusion: The study provides a foundation for future research on focussed isolation of the Azoarcus and other diazotrophs found in higher abundance in the rhizosphere. Keywords: Azospirillum, Diazotrophs, 16S rRNA, nifH, Metagenomics

Microbes from hypersaline environments are useful in biotechnology as sources of novel enzymes and proteins. The current study aimed to characterize halophilic bacteria from the rhizosphere of halophytes (Salsola stocksii and Atriplex amnicola), non-rhizospheric, and brine lake-bank soils collected from Khewra Salt Mine and screening of these bacterial strains for industrially important enzymes. A total of 45 bacterial isolates from the rhizosphere of Salsola, 38 isolates from Atriplex, 24 isolates from non-rhizospheric, and 25 isolates from lake-bank soils were identified by using 16S rRNA gene analysis. Phylogenetic analysis showed that bacterial strains belonging to Bacillus, Halobacillus, and Kocuria were dominant in the rhizosphere of halophytes (Salsola and Atriplex), and Halobacillus and Halomonas were dominating genera from non-rhizospheric and lake-bank soils. Mostly identified strains were moderately halophilic bacteria with optimum growth at 1.5–3.0 M salt concentrations. Most of the bacterial exhibited lipase, protease, cellulase, amylase, gelatinase, and catalase activities. Halophilic and halotolerant Bacilli (AT2RP4, HL1RS13, NRS4HaP9, and LK3HaP7) identified in this study showed optimum lipase, protease, cellulase, and amylase activities at 1.0–1.5 M NaCl concentration, pH 7–8, and temperature 37 °C. These results indicated that halophilic and halotolerant bacteria can be used for bioconversion of organic compounds to useful products under extreme conditions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00044-y) contains supplementary material, which is available to authorized users.

The present study was carried out to evaluate the diversity of rhizobia associated with nodules of mung bean in Pakistan, because this information is necessary for inoculum development. Based on sequence analysis of 16S rRNA gene of thirty-one bacteria, 11 were assigned to genus Bradyrhizobium, 17 to Ensifer, and 3 to Rhizobium. Phylogenetic analyses on the basis of 16S-23S ITS region, atpD, recA, nifH, and nodA of representative strains revealed that B. yuanmingense is the predominant species distributed throughout different mung bean–growing areas. Among the fast-growing rhizobia, Ensifer aridi was predominant in Faisalabad, Layyah, and Rawalpindi, while E. meliloti in Thal desert. Sequence variations and phylogeny of nifH and nodA genes suggested that these genes might have been co-evolved with the housekeeping genes and maintained by vertical gene transfer in rhizobia detected in the present study. Host infectivity assay revealed the successful nodulation of host by rhizobia related to genera Bradyrhizobium, Ensifer and Rhizobium. Among all, Bradyrhizobium and Ensifer spp. inoculation exhibited a significantly higher number of nodules (11–34 nodules plant(−1)) and nitrogenase activity (nodule ARA 60–110 μmol g(−1) h(−1)). Contrary to the previous studies, our data reveal that B. yuanmingense and E. aridi are predominant species forming effective nodules in mung bean in Pakistan. Furthermore, to the best of our knowledge, this is the first report showing the effective symbiosis of E. aridi, E. meliloti, and Rhizobium pusense with mung bean. The diversity of rhizobia in different habitats revealed in the present study will contribute towards designing site-specific inocula for mung bean. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-020-00397-9.

Bacterial plasmids carry genes that code for additional traits such as osmoregulation, CO2 fixation, antibiotic and heavy metal resistance, root nodulation and nitrogen fixation. The main objective of the current study was to identify plasmid-conferring osmoregulatory genes in bacteria isolated from rhizospheric and non-rhizospheric soils of halophytes (Salsola stocksii and Atriplex amnicola). More than 55% of halophilic bacteria from the rhizosphere and 70% from non-rhizospheric soils were able to grow at 3 M salt concentrations. All the strains showed optimum growth at 1.5–3.0 M NaCl. Bacterial strains from the Salsola rhizosphere showed maximum (31%) plasmid elimination during curing experiments as compared to bacterial strains from the Atriplex rhizosphere and non-rhizospheric soils. Two plasmid cured strains Bacillus HL2HP6 and Oceanobacillus HL2RP7 lost their ability to grow in halophilic medium, but they grew well on LB medium. The plasmid cured strains also showed a change in sensitivity to specific antibiotics. These plasmids were isolated and transformed into E. coli strains and growth response of wild-type and transformed E. coli strains was compared at 1.5–4 M NaCl concentrations. Chromosomal DNA and plasmids from Bacillus filamentosus HL2HP6 were sequenced by using high throughput sequencing approach. Results of functional analysis of plasmid sequences showed different proteins and enzymes involved in osmoregulation of bacteria, such as trehalose, ectoine synthetase, porins, proline, alanine, inorganic ion transporters, dehydrogenases and peptidases. Our results suggested that plasmid conferring osmoregulatory genes play a vital role to maintain internal osmotic balance of bacterial cells and these genes can be used to develop salt tolerant transgenic crops.

In Rhizobium-legume symbiosis, the nodule is the most frequently studied compartment, where the endophytic/symbiotic microbiota demands critical investigation for development of specific inocula. We identified the bacterial diversity within root nodules of mung bean from different growing areas of Pakistan using Illumina sequencing of 16S rRNA gene. We observed specific OTUs related to specific site where Bradyrhizobium was found to be the dominant genus comprising of 82-94% of total rhizobia in nodules with very minor fraction of sequences from other rhizobia at three sites. In contrast, Ensifer (Sinorhizobium) was single dominant genus comprising 99.9% of total rhizobial sequences at site four. Among non-rhizobial sequences, the genus Acinetobacter was abundant (7-18% of total sequences), particularly in Bradyrhizobium-dominated nodule samples. Rhizobia and non-rhizobial PGPR isolated from nodule samples include Ensifer, Bradyrhizobium, Acinetobacter, Microbacterium and Pseudomonas strains. Co-inoculation of multi-trait PGPR Acinetobacter sp. VrB1 with either of the two rhizobia in field exhibited more positive effect on nodulation and plant growth than single-strain inoculation which favors the use of Acinetobacter as an essential component for development of mung bean inoculum. Furthermore, site-specific dominance of rhizobia and non-rhizobia revealed in this study may contribute towards decision making for development and application of specific inocula in different habitats.

Phosphorus (P) is an essential plant nutrient, but often limited in soils for plant uptake. A major economic constraint in the rice production is excessive use of chemical fertilizers to meet the P requirement. Bioaugmentation of phosphate solubilizing rhizobacteria (PSB) can be used as promising alternative. In the present study 11 mineral PSB were isolated from Basmati rice growing areas of Pakistan. In broth medium, PSB solubilized tricalcium phosphate (27–354 μg mL−1) with concomitant decrease in pH up to 3.6 due to the production of different organic acids, predominantly gluconic acid. Of these, 4 strains also have ability to mineralize phytate (245–412 μg mL−1). Principle component analysis showed that the gluconic acid producing PSB strains (Acinetobacter sp. MR5 and Pseudomonas sp. MR7) have pronounced effect on grain yield (up to 55%), plant P (up to 67%) and soil available P (up to 67%), with 20% reduced fertilization. For simultaneous validation of gluconic acid production by MR5 and MR7 through PCR, new specific primers were designed to amplify gcd, pqqE, pqqC genes responsible for glucose dehydrogenase (gcd) mediated phosphate solubilization. These findings for the first time demonstrated Acinetobacter soli as potent P solubilizer for rice and expands our knowledge about genus specific pqq and gcd primers. These two gcd containing PSB Acinetobacter sp. MR5 (DSM 106631) and Pseudomonas sp. MR7 (DSM 106634) submitted to German culture collection (DSMZ), serve as global valuable pool to significantly increase the P uptake, growth and yield of Basmati rice with decreased dependence on chemical fertilizer in P deficit agricultural soils.

The rhizosphere microbiome plays a significant role in the life of plants in promoting plant survival under adverse conditions. However, limited information is available about microbial diversity in saline environments. In the current study, we compared the composition of the rhizosphere microbiomes of the halophytes Urochloa, Kochia, Salsola, and Atriplex living in moderate and high salinity environments (Khewra salt mines; Pakistan) with that of the non-halophyte Triticum. Soil microbiomes analysis using pyrosequencing of 16S rRNA gene indicated that Actinobacteria were dominant in saline soil samples whereas Proteobacteria predominated in non-saline soil samples. Firmicutes, Acidobacteria, Bacteriodetes and Thaumarchaeota were predominant phyla in saline and non-saline soils, whereas Cyanobacteria, Verrucomicrobia, Gemmatimonadetes and the unclassified WPS-2 were less abundant. Sequences from Euryarchaeota, Ignavibacteriae, and Nanohaloarchaeota were identified only from the rhizosphere of halophytes. Dominant halophilic bacteria and archaea identified in this study included Agrococcus, Armatimonadetes gp4, Halalkalicoccus, Haloferula and Halobacterium. Our analysis showed that increases in soil salinity correlated with significant differences in the alpha and beta diversity of the microbial communities across saline and non-saline soil samples. Having a complete inventory of the soil bacteria from different saline environments in Pakistan will help in the discovery of potential inoculants for crops growing on salt-affected land.

Xanthomonas oryzae pv. oryzae (Xoo) is widely prevalent and causes Bacterial Leaf Blight (BLB) in Basmati rice grown in different areas of Pakistan. There is a need to use environmentally safe approaches to overcome the loss of grain yield in rice due to this disease. The present study aimed to develop inocula, based on native antagonistic bacteria for biocontrol of BLB and to increase the yield of Super Basmati rice variety. Out of 512 bacteria isolated from the rice rhizosphere and screened for plant growth promoting determinants, the isolate BRp3 was found to be the best as it solubilized 97 μg/ mL phosphorus, produced 30 μg/mL phytohormone indole acetic acid and 15 mg/ L siderophores in vitro. The isolate BRp3 was found to be a Pseudomonas aeruginosa based on 16S rRNA gene sequencing (accession no. HQ840693). This bacterium showed antagonism in vitro against different phytopathogens including Xoo and Fusarium spp. Strain BRp3 showed consistent pathogen suppression of different strains of BLB pathogen in rice. Mass spectrometric analysis detected the production of siderophores (1-hydroxy-phenazine, pyocyanin, and pyochellin), rhamnolipids and a series of already characterized 4-hydroxy-2-alkylquinolines (HAQs) as well as novel 2,3,4-trihydroxy-2-alkylquinolines and 1,2,3,4-tetrahydroxy-2-alkylquinolines in crude extract of BRp3. These secondary metabolites might be responsible for the profound antibacterial activity of BRp3 against Xoo pathogen. Another contributing factor toward the suppression of the pathogen was the induction of defense related enzymes in the rice plant by the inoculated strain BRp3. When used as an inoculant in a field trial, this strain enhanced the grain and straw yields by 51 and 55%, respectively, over non-inoculated control. Confocal Laser Scanning Microscopy (CLSM) used in combination with immunofluorescence marker confirmed P. aeruginosa BRp3 in the rice rhizosphere under sterilized as well as field conditions. The results provide evidence that novel secondary metabolites produced by BRp3 may contribute to its activity as a biological control agent against Xoo and its potential to promote the growth and yield of Super Basmati rice.

In the present study, the relative distribution of endophytic rhizobia in field-collected root nodules of the promiscuous host mung bean was investigated by sequencing of 16S ribosomal RNA (rRNA) and nifH genes, amplified directly from the nodule DNA. Co-dominance of the genera Bradyrhizobium and Ensifer was indicated by 32.05 and 35.84% of the total retrieved 16S rRNA sequences, respectively, and the sequences of genera Mesorhizobium and Rhizobium comprised only 0.06 and 2.06% of the recovered sequences, respectively. Sequences amplified from rhizosphere soil DNA indicated that only a minor fraction originated from Bradyrhizobium and Ensifer strains, comprising about 0.46 and 0.67% of the total retrieved sequences, respectively. 16S rRNA gene sequencing has also identified the presence of several non-rhizobial endophytes from phyla Proteobacteria, Actinobacteria, Bacteroides, and Firmicutes. The nifH sequences obtained from nodules also confirmed the co-dominance of Bradyrhizobium (39.21%) and Ensifer (59.23%) strains. The nifH sequences of the genus Rhizobium were absent, and those of genus Mesorhizobium comprised only a minor fraction of the sequences recovered from the nodules and rhizosphere soil samples. Two bacterial isolates, identified by 16S rRNA gene sequence analysis as Bradyrhizobium strain Vr51 and Ensifer strain Vr38, successfully nodulated the original host (mung bean) plants. Co-dominance of Bradyrhizobium and Ensifer strains in the nodules of mung bean indicates the potential role of the host plant in selecting specific endophytic rhizobial populations. Furthermore, successful nodulation of mung bean by the isolates showed that strains of both the genera Bradyrhizobium and Ensifer can be used for production of inoculum.

Rhizosphere bacteria play a vital role in plant growth, pathogen control, biodegradation and rhizosphere signaling. A motile, rod-shaped bacterium, Pv2Z2, isolated from the nodules of the common bean grown in Tanzanian soil was characterized using a polyphasic approach. The traits assessed included the production of indole-3-acetic acid and N-acyl homoserine lactone (AHL) molecules, solubilization of insoluble phosphate and zinc compounds and biodegradation of a number of toxic compounds. The 16S rRNA sequence of Pv2Z2 (EU399793) showed 99 % homology to Ochrobactrum anthropi isolates (Accession no. AJ867292, AJ867291, AJ867290) from soil samples of wheat root. Phylogenetic analysis showed relatedness to nodulating strain Ochrobactrum cytisi rather than to the clinical/pathogenic type strain of O. anthropi. Moreover, it showed unique fingerprints in the randomly amplified polymorphic DNA (RAPD) and two primers-RAPD assays which were different from those of the pathogenic type strain of O. anthropi. The bacterium produced 6.68 μg/mL-1 indoleacetic acid in the presence of tryptophan, released 25.7 μg/mL-1 phosphorus from inorganic tri-calcium phosphate in the Pikoviskaya’s medium and solubilized zinc sulphate and zinc oxide in the LG1 medium. The production of AHLs (e.g. 3O-C7-HSL, 3OH-C7-HSL) was detected with biosensor strains CV026 using reverse phase thin layer chromatography. The bacterium was able to grow in minimal salt medium supplemented with 100 mg/L each of phenol, 2-bromophenol, 2,4-diamino phenol hydrochloride, 3,4-dimethoxy benzyl alcohol and 4-methoxy benzyl alcohol. Phenol degradation was recorded up to a level of 94 % within 12 days. Inoculation of common bean plants resulted in a significant increase in plant height, fresh/dry weight and nitrogen uptake as compared to non-inoculated plants. The data suggest that the plant growth-promoting and biodegradation potential of this bacterium may be exploited on a large scale. The capacity to produce AHL molecules by members of the Ochrobactrum genus has not been previously reported and needs to be explored in detail.

Multifaceted phosphate solubilizing bacteria (PSB) promote growth of plants by acting both as biofertilizer and biopesticide. The present study aimed to use native beneficial rhizobacteria to suppress rice Bacterial Leaf Blight (BLB) pathogen as well as to combat the plant in P deficiency without reduction in crop yield. In present investigation, of 50 PSB isolated from rice rhizosphere, three bacterial strains inhibited the BLB pathogen (18–25 mm), solubilized phosphorus (up to197 µg mL−1) produced IAA (1.7–14 µg mL−1). Gluconic acid produced by phosphate solubilizing antagonistic bacteria and molecular confirmation through amplification of glucose dehydrogenase genes (gcd) showed that gluconic acid may have dual role in P solubilization as well as BLB suppression. Plate germination assay showed that increase in vigor index (27%), root diameter (29%), root length volume-1 (57%) and root projection area (43%) were observed in inoculated seedlings as compared to uninoculated control. Plant inoculation studies under net house conditions showed BLB disease suppression (up to 56%) along with increase in plant P contents (up to 0.47% in straw and 1.26% in seed) in treatments inoculated with Pseudomonas spp. MR11, MR34 and Bacillus sp. MR42. Increase in the activity of defense related enzymes Phenylalanine ammonia lyase (PAL: 5.165 activity as µmole cinnamic acid g−1. f. wt.), Catalase (CAT: 2.88 activity g−1. f. wt.), Peroxidase (POD: 2.4 activity g−1. f. wt.), β, 1–3 glucanase (283 activity g−1. f. wt.) and Polyphenol oxidase (PPO: 65 activity g−1. f. wt.) were observed in response to inoculation of P-solubilizing antagonistic bacteria. BOX PCR finger printing and viable cell count coupled with Fluorescent in situ hybridization showed the competence and survival of inoculated bacteria in rhizosphere and phyllosphere. The findings of the current research work strongly encouraged the use of PSB strains for BLB suppression in phosphorus deficient soils.

Plant-bacteria partnership is a promising approach for remediating soil contaminated with organic pollutants. The colonization and metabolic activity of an inoculated microorganism depend not only on environmental conditions but also on the physiological condition of the applied microorganisms. This study assessed the influence of different inoculum pretreatments on survival, gene abundance and catabolic gene expression of an applied strain (Pantoea sp. strain BTRH79) in the rhizosphere of ryegrass vegetated in diesel contaminated soil. Maximum bacterium survival, gene abundance and expression were observed in the soil inoculated with bacterial cells that had been pregrown on complex medium, and hydrocarbon degradation and genotoxicity reduction were also high in this soil. These findings propose that use of complex media for growing plant inocula may enhance bacterial survival and colonization and subsequently the efficiency of pollutant degradation.

The present study was conducted to evaluate the potential of rice rhizosphere associated antagonistic bacteria for growth promotion and disease suppression of bacterial leaf blight (BLB). A total of 811 rhizospheric bacteria were isolated and screened against 3 prevalent strains of BLB pathogen Xanthomonas oryzae pv. oryzae (Xoo) of which five antagonistic bacteria, i.e., Pseudomonas spp. E227, E233, Rh323, Serratia sp. Rh269 and Bacillus sp. Rh219 showed antagonistic potential (zone of inhibition 1–19 mm). Production of siderophores was found to be the common biocontrol determinant and all the strains solubilized inorganic phosphate (82–116 μg mL-1) and produced indole acetic acid (0.48–1.85 mg L-1) in vitro. All antagonistic bacteria were non-pathogenic to rice, and their co-inoculation significantly improved plant health in terms of reduced diseased leaf area (80%), improved shoot length (31%), root length (41%) and plant dry weight (60%) as compared to infected control plants. Furthermore, under pathogen pressure, bacterial inoculation resulted in increased activity of defense related enzymes including phenylalanine ammonia-lyase and polyphenol oxidase, along with 86% increase in peroxidase and 53% increase in catalase enzyme activities in plants inoculated with Pseudomonas sp. Rh323 as well as co-inoculated plants. Bacterial strains showed good colonization potential in the rice rhizosphere up to 21 days after seed inoculation. Application of bacterial consortia in the field resulted in an increase of 31% in grain yield and 10% in straw yield over non-inoculated plots. Although, yield increase was statistically non-significant but was accomplished with overall saving of 20% chemical fertilizers. The study showed that Pseudomonas sp. Rh323 can be used to develop dual-purpose inoculum which can serve not only to suppress BLB but also to promote plant growth in rice.

We report the isolation of nitrogen fixing, phytohormone producing bacteria from sugarcane and their beneficial effects on the growth of micropropagated sugarcane plantlets. Detection of the nitrogen fixing bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) method indicated the presence of up to 106 bacteria per gram dry weight of stem and 107 bacteria per gram dry weight of root of field-grown sugarcane. Two nitrogen fixing bacterial isolates were obtained from stem (SC11, SC20) and two from the roots (SR12, SR13) of field-grown plants. These isolates were identified as Enterobacter sp. strains on the basis of their morphological characteristics and biochemical tests. The isolate SC20 was further characterized by 16S rRNA sequence analysis, which showed high sequence similarity to the sequence of Enterobacter cloacae and Klebsiella oxytoca. All the isolates produced the phytohormone indoleacetic acid (IAA) in pure culture and this IAA production was enhanced in growth medium containing tryptophan. The bacterial isolates were used to inoculate micro-propagated sugarcane in vitro where maximum increase in the root and shoot weight over control was observed in the plantlets inoculated with strain SC20. By using the15N isotope dilution technique, maximum nitrogen fixation contribution (28% of total plant nitrogen) was detected in plantlets inoculated with isolate SC20.

The present study deals with the isolation of plant growth promoting rhizobacteria (PGPR) from rice (variety NIAB IRRI-9) and the beneficial effects of these inoculants on two Basmati rice varieties. Nitrogen-fixing activity (acetylene-reduction activity) was detected in the roots and submerged shoots of field-grown rice variety NIAB IRRI-9. Estimation of the population size of diazotrophic bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) in roots and shoots indicated about 105–106counts/g dry weight at panicle initiation and grain filling stages. Four bacterial isolates from rice roots and shoots were obtained in pure culture which produced phytohormone indoleacetic acid (IAA) in the growth medium. Among these, three isolates S1, S4, and R3 reduced acetylene to ethylene in nitrogen-free semi-solid medium. Morphological and physiological characteristics of the isolates indicated that three nitrogen-fixing isolates S1, S4, and R3 belonged to the genus Enterobacter, while the non-fixing isolate R8 belonged to the genus Aeromonas. 16S rRNA sequence of one isolate from root (R8) and one isolate from shoot (S1) was obtained which confirmed identification of the isolates as Aeromonas veronii and Enterobacter cloacae, respectively. The 1517-nucleotide-long sequence of the isolate R8 showed 99% similarity with Aeromonas veronii (accession No. AF099023) while partial 16S rRNA sequence (two stretches of total 1271 nucleotide length) of S1 showed 97% similarity with the sequence of Enterobacter cloacae (accession No. AJ251469). The seedlings of two rice varieties Basmati 385 and Super Basmati were inoculated with the four bacterial isolates from rice and one Azospirillum brasilense strain Wb3, which was isolated from wheat. In the rice variety Basmati 385, maximum increase in root area and plant biomass was obtained in plants inoculated with Enterobacter S1 and Azospirillum Wb3, whereas in the rice variety Super Basmati, inoculation with Enterobacter R3 resulted in maximum increase of root area and plant biomass. Nitrogen fixation was quantified by using15N isotopic dilution method. Maximum fixation was observed in Basmati 385 with the inoculants Azospirillum Wb3 and Enterobacter S1 where nearly 46% and 41% of the nitrogen was derived from atmosphere (%Ndfa), respectively. In general, higher nitrogen fixation was observed in variety Basmati 385 than in Super Basmati, and different bacterial strains were found more effective as inoculants for the rice varieties Basmati 385 and Super Basmati.Key words: phytohormones, nitrogen fixation, Enterobacter, Aeromonas.

Identification and quantification of the plant growth hormones indoleacetic acid and gibberellic acid, produced by plant growth-promoting rhizobacteria (PGPR), was carried out by using high-pressure liquid chromatography (HPLC). The PGPR strains were isolated from roots of rice, wheat and kallar grass and belonged to the genera Azoarcus, Azospirillum, Enterobacter, Pseudomonas and Zoogloea. For these studies, bacteria were grown in liquid nitrogen free malate (NFM) or combined carbon medium (CCM) containing tryptophan and combined nitrogen. Some Azospirillum strains produced both indoleacetic acid and gibberellic acid, while none of the Enterobacter spp. tested produced these growth hormones. Azoarcus strain K-1 produced higher amounts of gibberellic acid and Azospirillum strain ER-2 produced higher amounts of indoleacetic acid. Indoleacetic acid production increased with the age of bacterial cultures while a decrease in the production of gibberellic acid was noted at later growth stages. Pure indoleacetic acid and gibberellic acid in the concentration range 1–2 µg/ml increased root area and plant biomass of rice and wheat. Among PGPR strains tested, Pseudomonas 96–51 and its extract containing growth hormones increased root area, root length and plant biomass of rice and wheat.

Three bacterial isolates were obtained from the roots of rice, wheat and kallar grass. These isolates fix nitrogen in pure culture as confirmed by acetylene reduction assay in semi-solid nitrogen-free growth media. Production of growth hormones by these isolates was confirmed by colorimetric method. The isolates from wheat (strain Wb-3) and rice (strain N-4) were identified as Azospirillum brasilense and A. lipoferum, respectively, on the basis of their morphology, carbon utilization pattern and biochemical tests. The isolate K-1 from kallar grass, previously identified as Azospirillum brasilense, was confirmed to belong to a newly named genus Azoarcus (Reinhold-Hurek et al., 1993) by using 16S rRNA-targeted oligonucleotide probes and some morphological and physiological characteristics.

A mixture of five plant growth-promoting rhizobacterial strains belonging to the genera Azospirillum, Azoarcus, Pseudomonas and Zoogloea was used to inoculate rice seedlings growing in microplots. The population of these bacteria, as well as the indigenous bacterial population in the rice rhizosphere, was determined at 4-week intervals throughout the growth season. In rhizosphere soil a continuous increase in total bacterial population (CCM-plate counts) in both inoculated and non-inoculated plots was noted until 3 months after transplantation, while the maximum population of diazotrophic bacteria (ARA-based MPN counts) was observed 1 month after transplantation. A larger number of bacteria was found associated with plant roots (107/g) as compared to those present in rhizosphere soil (106/g). The total bacterial population (plate counts) in plant roots showed a continuous increase until harvest, while the population of diazotrophs (MPN counts) started declining 1 month after transplantation. Considerably higher levels of nitrogenase activity (ARA) were noted in the roots of inoculated plants as compared to those of non-inoculated plants, where only a small number of diazotrophic bacteria were detected. The population of one Azospirillum strain (N-4) used as inoculum was determined by using selective media containing antibiotics, morphological characteristics, reaction with fluorescent antibodies and ARA of re-isolates. The maximum number of cells (1.4×105/g) of this strain were detected in the roots 1-month after seedling transplantation from the nursery, constituting about 0.4% of the total bacterial population and 2% of the diazotrophic population.

Halophyte microbiome contributes significantly to plant performance and can provide information regarding complex ecological processes involved in osmoregulation of these plants. The objective of this study is to investigate the microbiomes associated with belowground (rhizosphere), internal (endosphere) and aboveground (phyllosphere) tissues of halophyte (Salsola stocksii) through metagenomics approach. Plant samples were collected from Khewra Salt Mines. The metagenomic DNA from soil, root and shoot samples was isolated with the help of FastDNA spin kit. Through PCR, the 16S rRNA gene from four different Salsola plants and wheat plants was amplified and cloned in InsTAclone PCR cloning kit. Metagenomic analyses from rhizosphere, endosphere and phyllosphere of Salsola showed that approximately 29% bacteria were uncultured and unclassified. Proteobacteria and Actinobacteria were the most abundant phyla in Salsola and wheat. However, Firmicutes, Acidobacteria, Bacteriodetes, Planctomycetes, Cyanobacteria, Thermotogae, Verrucomicrobia, Choroflexi and Euryarchaeota were predominant groups from halophyte whereas Actinobacteria, Proteobacteria, Firmicutes, Cyanobacteria, Acidobacteria, Bacteriodetes, Planctomycetes and Verrucomicrobia were predominant phyla of wheat samples. Diversity and differences of microbial flora of Salsola and wheat suggested that functional interactions between plants and microorganisms contribute to salt stress tolerance.