Inoculation With Azospirillum spp. Acts as the Liming Source for Improving Growth and Nitrogen Use Efficiency of Potato.

Nitrogen (N) is one of the limiting factors for plant growth, and it is mainly supplied exogenously by fertilizer application. It is well documented that diazotrophic rhizobacteria improve plant growth by fixing atmospheric N in the soil. The present study investigates the nitrogen-fixing potential of two Azospirillum spp. strains using the 15N isotope-dilution method. The two diazotrophic strains (TN03 and TN09) native to the rhizosphere of potato belong to the genus Azospirillum (16S rRNA gene accession numbers LN833443 and LN833448, respectively). Both strains were able to grow on an N-free medium with N-fixation potential (138-143 nmol mg-1 protein h-1) and contained the nifH gene. Strain TN03 showed highest indole acetic acid (IAA) production (30.43 µg/mL), while TN09 showed highest phosphate solubilization activity (249.38 µg/mL) while both diazotrophs showed the production of organic acids. A 15N dilution experiment was conducted with different fertilizer inputs to evaluate the N-fixing potential of both diazotrophs in pots. The results showed that plant growth parameters and N contents increased significantly by the inoculations. Moreover, reduced 15N enrichment was found compared to uninoculated controls that received similar N fertilizer levels. This validates the occurrence of N-fixation through isotopic dilution. Strain TN09 showed higher N-fixing potential than TN03 and the uninoculated controls. Inoculation with either strain also showed a remarkable increase in plant growth under field conditions. Thus, there were remarkable increases in N use efficiency, N uptake and N utilization levels. Confocal laser scanning and transmission electron microscopy showed that TN03 is an ectophyte, i.e., present outside root cells or within the grooves of root hairs, while TN09 is an endophyte, i.e., present within root cells, forming a strong association withroot it. This study confirms that diazotrophic Azospirillum spp. added to potato systems can improve plant growth and N use efficiency, opening avenues for improvement of potato crop growth with reduced input of N fertilizer.

First report of diazotrophic Brevundimonas spp. as growth enhancer and root colonizer of potato.

Rhizobacteria contain various plant-beneficial traits and their inoculation can sustainably increase crop yield and productivity. The present study describes the growth-promoting potential of Brevundimonas spp. isolated from rhizospheric soil of potato from Sahiwal, Pakistan. Four different putative strains TN37, TN39, TN40, and TN44 were isolated by enrichment on nitrogen-free malate medium and identified as Brevundimonas spp. based on their morphology, 16S rRNA gene sequence, and phylogenetic analyses. All strains contained nifH gene except TN39 and exhibited nitrogen fixation potential through acetylene reduction assay (ARA) except TN40. Among all, the Brevundimonas sp. TN37 showed maximum ARA and phosphate solubilization potential but none of them exhibited the ability to produce indole acetic acid. Root colonization studies using transmission electron microscopy and confocal laser scanning microscopy showed that Brevundimonas sp. TN37 was resident over the root surface of potato; forming sheets in the grooves in the rhizoplane. TN37, being the best among all was further evaluated in pot experiment using potato cultivar Kuroda in sterilized sand. Results showed that Brevundimonas sp. TN37 increased growth parameters and nitrogen uptake as compared to non-inoculated controls. Based on the results obtained in this study, it can be suggested that Brevundimonas spp. (especially TN37) possess the potential to improve potato growth and stimulate nitrogen uptake. This study is the first report of Brevundimonas spp. as an effective PGPR in potato.

Growth stimulatory effect of AHL producing Serratia spp. from potato on homologous and non-homologous host plants.

Plant growth promoting rhizobacteria are known to improve plant performance by developing healthy and productive interactions with the host plants. These associations may be symbiotic or asymbiotic depending upon the genetic potential of the resident microbe and promiscuity of the host. Present study describes the potential of two Serratia spp. strains for promotion of plant growth in homologous as well as non-homologous hosts. The strains KPS-10 and KPS-14; native to potato rhizosphere belong to genus Serratia based on 16S rRNA gene sequences (accession no. LN831934 and LN831937 respectively) and contain multiple plant growth promoting properties along-with the production of quorum sensing acyl homoserine lactone (AHL) molecules. Both Serratia spp. strains showed solubilization of inorganic tri-calcium phosphate while KPS-14 also exhibited phytase activity (1.98 10-10 kcat). KPS-10 showed higher P-solubilization activity (128.5 µg/mL), IAA production (8.84 µg/mL), antifungal activity and also showed the production of two organic acids i.e., gluconic acid and lactic acid. Both strains produced three common AHLs: C6-HSL, 3oxo-C10-HSL, 3oxo-C12-HSL while some strain-specific AHLs (3OH-C5-HSL, 3OH-C6-HSL, C10-HSL specific to KPS-10 and 3OH-C6-HSL, C8-HSL, 3oxo-C9-HSL, 3OH-C9-HSL specific to KPS-14). Strains showed roots and rhizosphere colonization of potato and other non-homologous hosts up to one month. In planta AHLs-detection confirmed a likely role of AHLs during seedling growth and development where both extracted AHLs or bacteria inoculated roots showed extensive root hair. A significant increase in root/shoot lengths, root/ shoot fresh weights, root/shoot dry weights was observed by inoculation in different hosts. PGP-characteristics along with the AHLs-production signify the potential of both strains as candidate for the development of bio-inoculum for potato crop in specific and other crops in general. This inoculum will not only reduce the input of chemical fertilizer to the environment but also improve soil quality and plant growth.

Functional characterization of potential PGPR exhibiting broad-spectrum antifungal activity.

This study describes the biocontrol potential of rhizobacteria against a range of fungal phytopathogens. Out of 227 bacteria isolated from the rhizosphere of maize, rice, wheat, potato, sunflower and soybean crops cultivated in different agro-ecological regions of Pakistan, 48 exhibited >60 % antifungal activity against Fusarium oxysporum, Fusarium moniliforme, Rhizoctonia solani, Colletotrichum gloeosporioides, Colletotrichum falcatum, Aspergillus niger, and Aspergillus flavus. The rhizobacteria inhibiting >65 % pathogen growth were selected for detailed molecular and in planta studies most of which were identified as Pseudomonas and Bacillus species based on 16S rRNA gene sequence analysis. Antifungal metabolites produced by these rhizobacteria analyzed through LCMS were identified as antibiotics (iturin, surfactins, fengycin, DAPG, Phenazine, etc.), cell wall degrading enzymes (protease, chitinase, and cellulase), plant growth promotion enzymes and hormones (indole-3-acetic acid, ACC-deaminase, phosphates, nitrogen fixation), N-acyl-homoserine lactones and siderophores. The growth room experiment validated the potential of these bacteria as biofertilizer and biopesticide agents. Of all, P. aeruginosa strain FB2 and B. subtilis strain RMB5 showed significantly higher potential as antagonistic plant-beneficial bacteria effective against a range of fungal phytopathogens. Both these bacteria can be used to develop a dual-purpose bacterial inoculum as biopesticide and biofertilizer. Rest of the antagonistic PGPR may be exploited for disease control in less-infested soils.

Enterobacter sp. strain Fs-11 adapted to diverse ecological conditions and promoted sunflower achene yield, nutrient uptake, and oil contents.

Plant growth-promoting rhizobacteria are under extensive investigation to supplement the chemical fertilizers due to cost-effective and eco-friendly nature. However, their consistency in heterogeneous soil and diverse ecological settings is unclear. The current study presents in vitro and field evaluation of pre-characterized PGPR strain Enterobacter sp. Fs-11 (GenBank accession # GQ179978) in terms of its potential to enhance sunflower yield and oil contents under diverse environmental conditions. Under in vitro conditions, strain Fs-11 showed optimal growth at a range of temperature (15 to 40 °C) and pH values (6.5 to 8.5). Extracellular and intracellular localizations of the strain Fs-11 in sunflower root cortical cells through transmission electron microscopy confirmed its epiphytic and endophytic colonization patterns, respectively. In field experiments, conducted at three different agro-climatic locations, inoculation of strain Fs-11 at 50% reduced NP fertilizer resulted in a significant increase in growth, achene yield, nutrient uptake, and oil contents. Inoculation also responded significantly in terms of increase in mono- and polyunsaturated fatty acids (oleic and linoleic acids, respectively) without rising saturated fatty acid (palmitic and stearic acids) contents. We concluded that Enterobacter sp. Fs-11 is a potential candidate for biofertilizer formulations to supplement chemical fertilizer requirements of sunflower crop under diverse climatic conditions.

Pseudomonas sp. AF-54 containing multiple plant beneficial traits acts as growth enhancer of Helianthus annuus L. under reduced fertilizer input.

Plant growth promoting rhizobacteria (PGPR) are capable to increase the growth and yield of crops in eco-friendly and sustainable manner. To evaluate the response of sunflower towards inoculation with PGPR, a sunflower root associated bacterium AF-54 isolated from Diyar Gali Himalayan Mountain region, Azad Jammu and Kashmir (AJK), identified as Pseudomonas sp. by 16S rRNA sequence analysis and was characterized using polyphasic approach. The bacterium produced 23.9 µgmL-1 indole-3-acetic acid in tryptophan-supplemented medium, showed 44.28 nmoles mg-1 protein h-1 nitrogenase activity through acetylene reduction assay and released 48.80 µg mL-1 insoluble phosphorus in Pikovskaya's broth. During P-solubilization, the pH of the Pikovskaya's medium decreased from 7 to 3.04 due to the production of acetic acid, malic acid and gluconic acid. Pseudomonas sp. AF-54 showed metabolic versatility by utilizing 79 carbon sources from BIOLOG GN2 plates and resistance to many antibiotics. Furthermore, it inhibited the growth of Fusarium oxysporum in dual culture assay. To evaluate the plant-inoculation response, series of experiments conducted in hydroponic, sterilized soil and fields at AJK, and Faisalabad where inoculated plants with reduced fertilizer showed a significant increase in growth, yield, oil contents and achene NP uptake as compared to non-inoculated control. AF-54 showed extensive root colonization in sterilized and non-sterile conditions documented through yfp-labeling and fluorescent in situ hybridization coupled with confocal laser scanning microscopy. This study concludes that the Pseudomonas sp. strain AF-54 containing multiple plant growth promoting traits can be a potential candidate for biofertilizer production to enhance sunflower crop yield with reduced application of chemical (NP) fertilizers.

Functional characterization of a soybean growth stimulator Bradyrhizobium sp. strain SR-6 showing acylhomoserine lactone production.

A soybean nodule endophytic bacterium Bradyrhizobium sp. strain SR-6 was characterized for production of acyl homoserine lactones (AHLs) as quorum sensing molecules. Mass spectrometry analysis of AHLs revealed the presence of C6-HSL, 3OH-C6-HSL, C8-HSL, C10-HSL, 3oxoC10-HSL, 3oxo-C12-HSL and 3OH-C12-HSL which are significantly different from those reported earlier in soybean symbionts. Purified AHL extracts significantly improved wheat and soybean seedling growth and root hair development along with increased soybean nodulation under axenic conditions. A positive correlation was observed among in vivo nitrogenase and catalase enzyme activities of the strain SR-6. Transmission electron microscopic analysis showed the cytochemical localization of catalase activity within the bacteroids, specifically attached to the peribacteroidal membrane. Root and nodule colonization proved rhizosphere competence of SR-6. The inoculation of SR-6 resulted in increased shoot length (13%), plant dry matter (50%), grain weight (16%), seed yield (20%) and N-uptake (14%) as compared to non-inoculated soybean plants. The symbiotic bacterium SR-6 has potential to improve soybean growth and yield in sub-humid climate of Azad Jammu and Kashmir region of Pakistan. The production and mass spectrometric profiling of AHLs as well as in vivo cytochemical localization of catalase enzyme activity in soybean Bradyrhizobium sp. have never been reported earlier elsewhere before our these investigations.

Differential Response of Potato Toward Inoculation with Taxonomically Diverse Plant Growth Promoting Rhizobacteria.

Rhizosphere engineering with beneficial plant growth promoting bacteria offers great promise for sustainable crop yield. Potato is an important food commodity that needs large inputs of nitrogen and phosphorus fertilizers. To overcome high fertilizer demand (especially nitrogen), five bacteria, i.e., Azospirillum sp. TN10, Agrobacterium sp. TN14, Pseudomonas sp. TN36, Enterobacter sp. TN38 and Rhizobium sp. TN42 were isolated from the potato rhizosphere on nitrogen-free malate medium and identified based on their 16S rRNA gene sequences. Three strains, i.e., TN10, TN38, and TN42 showed nitrogen fixation (92.67-134.54 nmol h(-1)mg(-1) protein), while all showed the production of indole-3-acetic acid (IAA), which was significantly increased by the addition of L-tryptophan. Azospirillum sp. TN10 produced the highest amount of IAA, as measured by spectrophotometry (312.14 µg mL(-1)) and HPLC (18.3 µg mL(-1)). Inoculation with these bacteria under axenic conditions resulted in differential growth responses of potato. Azospirillum sp. TN10 incited the highest increase in potato fresh and dry weight over control plants, along with increased N contents of shoot and roots. All strains were able to colonize and maintain their population densities in the potato rhizosphere for up to 60 days, with Azospirillum sp. and Rhizobium sp. showing the highest survival. Plant root colonization potential was analyzed by transmission electron microscopy of root sections inoculated with Azospirillum sp. TN10. Of the five test strains, Azospirillum sp. TN10 has the greatest potential to increase the growth and nitrogen uptake of potato. Hence, it is suggested as a good candidate for the production of potato biofertilizer for integrated nutrient management.

Characterization of free and conjugated phenolic compounds in fruits of selected wild plants.

A gas chromatography-mass spectrometric (GC-MS) method was utilized for the separation, and systematic characterization of phenolic compounds as trimethylsilyl derivatives in fruits of wild plants including Olive, Jujube and Common Fig. Both the free and conjugate phenolic acids (rarely determined before and several are reported first time here) were characterized. A baseline separation of the 20 phenolics was achieved in 25 min with standard calibration curves linear over the concentration range from the detection limits to 20 µg/mL. Total of fourteen phenolic acids were identified in wild Olive fruit, eight in wild Jujube fruit and ten in wild Common Fig fruit, out of which 2,4-dihydroxybenzoic acid and trans-cinnamic acid were dominant in these fruits with concentration of 87.02, 5.25 and 14.16 mg/kg and 32.43, 5.77 and 11.70 mg/kg (dry weight), respectively. The results of this study support the utilization of the tested wild fruits as a potential source of valuable phenolics for functional food and nutraceutical applications.

A reliable method for the selection and confirmation of transconjugants of plant growth-promoting bacteria especially plant-associated Burkholderia spp.

Selectable markers, e.g., antibiotic resistance, for conjugation experiments are not always effective for slow-growing plant growth promoting bacteria such as Burkholderia. We used PCAT medium containing Congo Red for selecting Burkholderia transconjugants. This method allows for the reliable selection of transconjugants of these novel plant growth-promoting bacteria.

Cultivation-Based and Molecular Assessment of Bacterial Diversity in the Rhizosheath of Wheat under Different Crop Rotations.

A field study was conducted to compare the formationand bacterial communities of rhizosheaths of wheat grown under wheat-cotton and wheat-rice rotation and to study the effects of bacterial inoculation on plant growth. Inoculation of Azospirillum sp. WS-1 and Bacillus sp. T-34 to wheat plants increased root length, root and shoot dry weight and dry weight of rhizosheathsoil when compared to non-inoculated control plants, and under both crop rotations. Comparing both crop rotations, root length, root and shoot dry weight and dry weight of soil attached with roots were higher under wheat-cotton rotation. Organic acids (citric acid, malic acid, acetic acid and oxalic acid) were detected in rhizosheaths from both rotations, with malic acid being most abundant with 24.8±2 and 21.3±1.5 µg g(-1) dry soil in wheat-cotton and wheat-rice rotation, respectively. Two sugars (sucrose, glucose) were detected in wheat rhizosheath under both rotations, with highest concentrations of sucrose (4.08±0.5 µg g(-1) and 7.36±1.0 µg g(-1)) and glucose (3.12±0.5 µg g(-1) and 3.01± µg g(-1)) being detected in rhizosheaths of non-inoculated control plants under both rotations. Diversity of rhizosheath-associated bacteria was evaluated by cultivation, as well as by 454-pyrosequencing of PCR-tagged 16S rRNA gene amplicons. A total of 14 and 12 bacterial isolates predominantly belonging to the genera Arthrobacter, Azospirillum, Bacillus, Enterobacter and Pseudomonaswere obtained from the rhizosheath of wheat grown under wheat-cotton and wheat-rice rotation, respectively. Analysis of pyrosequencing data revealed Proteobacteria, Bacteriodetes and Verrucomicrobia as the most abundant phyla in wheat-rice rotation, whereas Actinobacteria, Firmicutes, Chloroflexi, Acidobacteria, Planctomycetes and Cyanobacteria were predominant in wheat-cotton rotation. From a total of 46,971 sequences, 10.9% showed ≥97% similarity with 16S rRNA genes of 32 genera previously shown to include isolates with plant growth promoting activity (nitrogen fixation, phosphate-solubilization, IAA production). Among these, the most predominant genera were Arthrobacter, Azoarcus, Azospirillum, Bacillus, Cyanobacterium, Paenibacillus, Pseudomonas and Rhizobium.

Isolation and characterization of a ß-propeller gene containing phosphobacterium Bacillus subtilis strain KPS-11 for growth promotion of potato (Solanum tuberosum L.).

Phosphate-solubilizing and phytate-mineralizing bacteria collectively termed as phosphobacteria provide a sustainable approach for managing P-deficiency in agricultural soils by supplying inexpensive phosphate to plants. A phosphobacterium Bacillus subtilis strain KPS-11 (Genbank accession no. KP006655) was isolated from potato (Solanum tuberosum L.) rhizosphere and characterized for potato plant growth promoting potential. The strain utilized both Ca-phosphate and Na-phytate in vitro and produced 6.48 µg mL(-1) indole-3-acetic acid in tryptophan supplemented medium. P-solubilization after 240 h was 66.4 µg mL(-1) alongwith the production of 19.3 µg mL(-1) gluconic acid and 5.3 µg mL(-1) malic acid. The extracellular phytase activity was higher (4.3 × 10(-10) kat mg(-1) protein) than the cell-associated phytase activity (1.6 × 10(-10) kat mg(-1) protein). B. subtilis strain KPS-11 utilized 40 carbon sources and showed resistance against 20 chemicals in GENIII micro-plate system demonstrating its metabolic potential. Phytase-encoding gene ß-propeller (BPP) showed 92% amino acid similarity to BPP from B. subtilis (accession no.WP_014114128.1) and 83% structural similarity to BPP from B. subtilis (accession no 3AMR_A). Potato inoculation with B. subtilis strain KPS-11 increased the root/shoot length and root/shoot weight of potato as compared to non-inoculated control plants. Moreover, rifampicin-resistant derivative of KPS-11 were able to survive in the rhizosphere and on the roots of potato up to 60 days showing its colonization potential. The study indicates that B. subtilis strain KPS-11 can be a potential candidate for development of potato inoculum in P-deficient soils.

Isolation and characterization of plant growth-promoting rhizobacteria from wheat rhizosphere and their effect on plant growth promotion.

The present study was conducted to characterize the native plant growth promoting (PGP) bacteria from wheat rhizosphere and root-endosphere in the Himalayan region of Rawalakot, Azad Jammu and Kashmir (AJK), Pakistan. Nine bacterial isolates were purified, screened in vitro for PGP characteristics and evaluated for their beneficial effects on the early growth of wheat (Triticum aestivum L.). Among nine bacterial isolates, seven were able to produce indole-3- acetic acid in tryptophan-supplemented medium; seven were nitrogen fixer, and four were able to solubilize inorganic phosphate in vitro. Four different morphotypes were genotypically identified based on IGS-RFLP fingerprinting and representative of each morphotype was identified by 16S rRNA gene sequencing analysis except Gram-positive putative Bacillus sp. Based on 16S rRNA gene sequence analysis, bacterial isolates AJK-3 and AJK-9 showing multiple PGP-traits were identified as Stenotrophomonas spp. while AJK-7 showed equal homologies to Acetobacter pasteurianus and Stenotrophomonas specie. Plant inoculation studies indicated that these Plant growth-promoting rhizobacteria (PGPR) strains provided a significant increase in shoot and root length, and shoot and root biomass. A significant increase in shoot N contents (up to 76%) and root N contents (up to 32%) was observed over the un-inoculated control. The study indicates the potential of these PGPR for inoculums production or biofertilizers for enhancing growth and nutrient content of wheat and other crops under field conditions. The study is the first report of wheat associated bacterial diversity in the Himalayan region of Rawalakot, AJK.

Isolation and identification of indigenous plant growth promoting rhizobacteria from Himalayan region of Kashmir and their effect on improving growth and nutrient contents of maize (Zea mays L.).

Introduction and exploitation of plant growth promoting rhizobacteria (PGPR) in agro-ecosystems enhance plant-microbes interactions that may affect ecosystems sustainability, agricultural productivity, and environmental quality. The present study was conducted to isolate and identify PGPRs associated with maize (Zea mays L.) from twenty sites of Himalayan region of Hajira-Rawalakot, Azad Jammu and Kashmir (AJK), Pakistan. A total of 100 isolates were isolated from these sites, out of which eight (HJR1, HJR2, HJR3, HJR4, HJR5, MR6, HJR7, HJR8) were selected in vitro for their plant growth promoting ability (PGPA) including phosphorus solubilization, indole-3-acetic acid (IAA) production and N2 fixation. The 16S rRNA gene sequencing technique was used for molecular identity and authentication. Isolates were then further tested for their effects on growth and nutrient contents of maize (Z. mays L.) under pouch and pot conditions. The 16S rRNA gene sequencing and phylogenetic analysis identified these isolates belong to Pseudomonas and Bacillus genera. The isolates promoted plant growth by solubilizing soil P which ranged between 19.2 and 35.6 µg mL(-1). The isolates HJR1, HJR2, HJR3, and HJR5 showed positive activity in acetylene reduction assay showing their N2-fixation potential. All eight isolates showed the potential to produce IAA in the range of 0.9-5.39 µg mL(-1) and promote plant growth. Results from a subsequent pot experiment indicated PGPRs distinctly increased maize shoot and root length, shoot and root dry weight, root surface area, leaf surface area, shoot and root N and P contents. Among the eight isolates, HR3 showed a marked P-solubilizing activity, plant growth-promoting attributes, and the potential to be developed as a biofertilizers for integrated nutrient management strategies.

Genetic, physiological and biochemical characterization of Bacillus sp. strain RMB7 exhibiting plant growth promoting and broad spectrum antifungal activities.

BACKGROUND: Plant growth promoting rhizobacteria (PGPR) are functionally diverse group of bacteria having immense potential as biofertilizers and biopesticides. Depending upon their function, they may serve as partial replacements for chemical fertilizer or pesticides as an eco-friendly and cost-effective alternatives as compared to their synthetic counterparts. Therefore, isolation, characterization and practical evaluation of PGPRs having the aforementioned multifaceted beneficial characteristics, are essentially required. This study describes the detailed polyphasic characterization of Bacillus sp. strain RMB7 having profound broad spectrum antifungal activity and plant growth promoting potential. RESULTS: Based on 16S rRNA gene sequencing, strain RMB7 was identified as Bacillus specie. This strain exhibited the production of 8 mg. L(-1)of indole-3-acetic acid (IAA) in tryptophan-supplemented medium. It was able to solubilize 50.6 mg. L(-1) tri-calcium phosphate, reduced 601ηmol acetylene h(-1)/vial and inhibited >70% growth of nine fungal phytopathogens tested in vitro. Under natural pathogen pressure, inoculation with strain RMB7 and RMB7-supernatant conferred resistance by arugula plant against Pythium irregulare with a concurrent growth improvement over non-inoculated plants. The T-RFLP analysis based on 16S rRNA gene showed that inoculation with RMB7 or its supernatant have a major impact on the indigenous rhizosphere bacterial population. Mass spectrometric analysis revealed the production of lipopeptide surfactins as well as iturin A presence in crude extract of RMB7. PCR-amplification further confirmed the presence of genes involved in the biosynthesis of these two bioactive lipopeptide compounds. CONCLUSIONS: The data show that Bacillus sp. strain RMB7 has multifaceted beneficial characteristics. It may be an ideal plant growth promoting as well as biocontrol agent, for its integrated use in disease and nutrient management strategies.

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.

Antimicrobial activity of the bioactive components of essential oils from Pakistani spices against Salmonella and other multi-drug resistant bacteria.

BACKGROUND: The main objective of this study was the phytochemical characterization of four indigenous essential oils obtained from spices and their antibacterial activities against the multidrug resistant clinical and soil isolates prevalent in Pakistan, and ATCC reference strains. METHODS: Chemical composition of essential oils from four Pakistani spices cumin (Cuminum cyminum), cinnamon (Cinnamomum verum), cardamom (Amomum subulatum) and clove (Syzygium aromaticum) were analyzed on GC/MS. Their antibacterial activities were investigated by minimum inhibitory concentration (MIC) and Thin-Layer Chromatography-Bioautographic (TLC-Bioautographic) assays against pathogenic strains Salmonella typhi (D1 Vi-positive), Salmonella typhi (G7 Vi-negative), Salmonella paratyphi A, Escherichia coli (SS1), Staphylococcus aureus, Pseudomonas fluorescens and Bacillus licheniformis (ATCC 14580). The data were statistically analyzed by using Analysis of Variance (ANOVA) and Least Significant Difference (LSD) method to find out significant relationship of essential oils biological activities at p <0.05. RESULTS: Among all the tested essential oils, oil from the bark of C. verum showed best antibacterial activities against all selected bacterial strains in the MIC assay, especially with 2.9 mg/ml concentration against S. typhi G7 Vi-negative and P. fluorescens strains. TLC-bioautography confirmed the presence of biologically active anti-microbial components in all tested essential oils. P. fluorescens was found susceptible to C. verum essential oil while E. coli SS1 and S. aureus were resistant to C. verum and A. subulatum essential oils, respectively, as determined in bioautography assay. The GC/MS analysis revealed that essential oils of C. cyminum, C. verum, A. subulatum, and S. aromaticum contain 17.2% cuminaldehyde, 4.3% t-cinnamaldehyde, 5.2% eucalyptol and 0.73% eugenol, respectively. CONCLUSIONS: Most of the essential oils included in this study possessed good antibacterial activities against selected multi drug resistant clinical and soil bacterial strains. Cinnamaldehyde was identified as the most active antimicrobial component present in the cinnamon essential oil which acted as a strong inhibitory agent in MIC assay against the tested bacteria. The results indicate that essential oils from Pakistani spices can be pursued against multidrug resistant bacteria.

Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11.

An Enterobacter sp. Fs-11 was isolated from sunflower rhizosphere, identified on the basis of 16S rRNA gene sequence analysis (GeneBank accession no. GQ179978) and studied for its root colonization and growth promotion ability in sunflower. Morphologically, it was rod shaped Gram-negative, motile bacterium, producing 4.5 µg mL(-1) indole acetic acid in tryptophan-supplemented medium. It utilized 27 out of 95 substrates in BIOLOG GN2 micro plate system. It was able to convert insoluble tri-calcium phosphate to soluble phosphorus up to 43.5 µg mL(-1) with decrease in pH of the medium up to 4.5 after 10 days incubation at 28 ± 2 °C in the Pikovskaya's broth. High performance liquid chromatography of cell free supernatant showed that Fs-11 produced malic acid and gluconic acid (2.43 and 16.64 µg mL(-1), respectively) in Pikovskaya's broth. Analysis of 900 bp fragment of pyrroloquinoline quinine pqqE gene sequence showed 98 % homology with that of E. cloacae pqqE gene. Confocal laser scanning microscope revealed strong colonization of fluorescently labeled Fs-11 with sunflower roots. Sunflower inoculation with Fs-11 and its rifampicin resistant derivative in sterile sand and natural soil showed that Fs-11 colonized sunflower roots up to 30 days after transplanting in both sterile sand as well as natural soil. Moreover, Fs-11 inoculation resulted in increased plant height, fresh weight, dry weight and total phosphorus contents as compared to un-inoculated plants. The data showed that Enterobacter sp. Fs-11 is an efficient phosphate solubilizing and plant growth promoting rhizobacterium and has great potential to be used as bio-inoculant for sunflower under phosphorus deficient conditions.

Characterization and screening of bacteria from rhizosphere of maize grown in Indonesian and Pakistani soils.

Thirty rhizobacteria isolated from maize grown in Pakistani and Indonesian soils were evaluated for their morphological characteristics, nitrogen fixation, P-solubilization, indole acetic acid (IAA) and siderophores production. Nitrogenase activity was detected in nineteen isolates ranging from 21.8-3624 n moles C2H4 produced/h/mg protein. Most of the isolates produced IAA, ten were capable of siderophore production while four were P-solubilizers. Ultrastructural studies of Pseudomonas sp. F14 indicated characteristic rhizospheric colonization within 48 h that was observed to change considerably with the passage of time from few bacteria to micro colonies. Random amplified polymorphic DNA (RAPD) analysis of 30 bacterial strains using 30 oligonucleotide primers resulted in considerable level of genetic diversity, with genetic distance ranging from 2-16%. Indonesian isolates were found to be more diverse as compared to Pakistani isolates. The characterization and screening of rhizobacteria of maize rhizosphere has helped in selection of isolates F7, LS-1, 3.1.1.C, F2, F3 and F13 as superior strains for use as bioinoculant. Moreover isolate F14 identified, as Pseudomonas fulgida by partial 16S rRNA sequence analysis is a novel strain regarding its tremendous potentials for inoculum production to enhance the yield of maize.