Mitigation of water scarcity with sustained growth of Rice by plant growth promoting bacteria.

Climate change augments the risk to food security by inducing drought stress and a drastic decline in global rice production. Plant growth-promoting bacteria (PGPB) have been known to improve plant growth under drought stress. Here in the present study, we isolated, identified, and well-characterized eight drought-tolerant bacteria from the rice rhizosphere that are tolerant to 20% PEG-8000. These strains exhibited multiple plant growth-promoting traits, i.e., 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, exopolysaccharide production, phosphate (P)-solubilizing activity (51-356 µg ml-1), indole-3 acetic acid (IAA) production (14.3-46.2 µg ml-1), and production of organic acids (72-178 µg ml-1). Inoculation of bacterial consortium (Bacillus subtilis NM-2, Brucella haematophilum NM-4, and Bacillus cereus NM-6) significantly improved seedling growth and vigor index (1009.2-1100) as compared to non-inoculated stressed plants (630-957). Through rhizoscanning, efficiency of the consortium was validated by improved root parameters such as root length (17%), diameter, and surface area (18%) of all tested genotypes as compared with respective non-inoculated stressed treatments. Furthermore, the response of consortium inoculation on three rice genotypes was positively correlated with improved plant growth and drought stress ameliorating traits by the accumulation of osmoprotectant, i.e., proline (85.8%-122%), relative water content (51%), membrane stability index (64%), and production of antioxidant enzymes to reduce oxidative damage by reactive oxygen species. A decrease in temperature and improved chlorophyll content of inoculated plants were found using infrared thermal imaging and soil plant analyzer development (SPAD), respectively. The key supporting role of inoculation toward stress responses was validated using robust techniques like infrared thermal imaging and an infrared gas analyzer. Furthermore, principal component analysis depicts the contribution of inoculation on stress responses and yield of tested rice genotypes under water stress. The integration of drought-tolerant rice genotype (NIBGE-DT02) and potential bacterial strains, i.e., NM-2, NM-4, and NM-6, can serve as an effective bioinoculant to cope with water scarcity under current alarming issues related to food security in fluctuating climate.

Draft Genome Sequence of Streptomyces sp. Strain R1, Isolated from Water Canal Sediments, Possessing Antimicrobial and Plant Growth Promoting Capabilities.

We present the genome sequence of Streptomyces sp. strain R1, isolated from water canal sediments and possessing genes responsible for antimicrobial metabolites and plant growth promotion. The genome assembly contains 7,936,694 bp with 72.24% of guanine-cytosine content. This genome will provide basic knowledge of the genes and pathways involved in the above mechanisms.

Designing Synergistic Biostimulants Formulation Containing Autochthonous Phosphate-Solubilizing Bacteria for Sustainable Wheat Production.

Applying phosphate-solubilizing bacteria (PSB) as biofertilizers has enormous potential for sustainable agriculture. Despite this, there is still a lack of information regarding the expression of key genes related to phosphate-solubilization (PS) and efficient formulation strategies. In this study, we investigated rock PS by Ochrobactrum sp. SSR (DSM 109610) by relating it to bacterial gene expression and searching for an efficient formulation. The quantitative PCR (qPCR) primers were designed for PS marker genes glucose dehydrogenase (gcd), pyrroloquinoline quinone biosynthesis protein C (pqqC), and phosphatase (pho). The SSR-inoculated soil supplemented with rock phosphate (RP) showed a 6-fold higher expression of pqqC and pho compared to inoculated soil without RP. Additionally, an increase in plant phosphorous (P) (2%), available soil P (4.7%), and alkaline phosphatase (6%) activity was observed in PSB-inoculated plants supplemented with RP. The root architecture improved by SSR, with higher root length, diameter, and volume. Ochrobactrum sp. SSR was further used to design bioformulations with two well-characterized PS, Enterobacter spp. DSM 109592 and DSM 109593, using the four organic amendments, biochar, compost, filter mud (FM), and humic acid. All four carrier materials maintained adequate survival and inoculum shelf life of the bacterium, as indicated by the field emission scanning electron microscopy analysis. The FM-based bioformulation was most efficacious and enhanced not only wheat grain yield (4-9%) but also seed P (9%). Moreover, FM-based bioformulation enhanced soil available P (8.5-11%) and phosphatase activity (4-5%). Positive correlations were observed between the PSB solubilization in the presence of different insoluble P sources, and soil available P, soil phosphatase activity, seed P content, and grain yield of the field grown inoculated wheat variety Faisalabad-2008, when di-ammonium phosphate fertilizer application was reduced by 20%. This study reports for the first time the marker gene expression of an inoculated PSB strain and provides a valuable groundwork to design field scale formulations that can maintain inoculum dynamics and increase its shelf life. This may constitute a step-change in the sustainable cultivation of wheat under the P-deficient soil conditions.

Integrated Analysis of Osmotic Stress and Infrared Thermal Imaging for the Selection of Resilient Rice Under Water Scarcity.

The climate change scenario has increased the severity and frequency of drought stress, which limits the growth and yield of rice worldwide. There is a dire need to select drought-tolerant rice varieties to sustain crop production under water scarcity. Therefore, the present study effectively combined morpho-physiological and biochemical approaches with the technology of infrared thermal imaging (IRTI) for a reliable selection of drought-tolerant genotypes. Initially, we studied 28 rice genotypes including 26 advance lines and three varieties for water stress tolerance under net house conditions. Three genotypes NIBGE-DT-02, KSK-133, and NIBGE-DT-11 were selected based on the Standard Evaluation System (SES) scoring for drought tolerance. NIBGE-DT-02 showed tolerance to polyethylene glycol (20%) induced osmotic stress indicated by a minimum reduction in seedling length, biomass, chlorophyll content, and increased leaf proline content as compared to susceptible varieties under a hydroponic system. NIBGE-DT-02 was further evaluated for water withholding at varying growth stages, i.e., 30 and 60 days after transplantation (DAT) in pots under net house conditions. NIBGE-DT-02 showed a significantly lower reduction (35.9%) in yield as compared to a susceptible variety (78.06%) under water stress at 60 DAT with concomitant induction of antioxidant enzymes such as peroxidase, catalase, and polyphenol oxidase. A significant increase (45.9%) in proline content, a low increase (7.5%) in plant temperature, along with a low reduction in relative water content (RWC) (5.5%), and membrane stability index (MSI) (9%) were observed under water stress at 60 DAT as compared to the well-watered control. Pearson correlation analysis showed the strong correlation of shoot length with MSI and root length with RWC in rice genotypes at the later growth stage. Furthermore, Regression analysis indicated a negative correlation between plant temperature of NIBGE-DT-02 and proline, RWC, MSI, and peroxidase enzyme under variable water stress conditions. All these responses collectively validated the adaptive response of selected genotypes under water stress during different growth stages. Tolerant genotypes can be used in breeding programs aimed at improving drought tolerance and can expand rice cultivation. Furthermore, this study provides a foundation for future research directed to utilize IRTI as a fast and non-destructive approach for the selection of potent rice genotypes better adapted to water scarcity from wide germplasm collection.

Integrated analysis of potential microbial consortia, soil nutritional status, and agro-climatic datasets to modulate P nutrient uptake and yield effectiveness of wheat under climate change resilience.

Climate change has a devastating effect on wheat production; therefore, crop production might decline by 2030. Phosphorus (P) nutrient deficiency is another main limiting factor of reduced yield. Hence, there is a dire need to judiciously consider wheat yield, so that human requirements and nutrition balance can be sustained efficiently. Despite the great significance of biostimulants in sustainable agriculture, there is still a lack of integrated technology encompassing the successful competitiveness of inoculated phosphate-solubilizing bacteria (PSB) in agricultural systems in the context of climatic conditions/meteorological factors and soil nutritional status. Therefore, the present study reveals the modulation of an integrated P nutrient management approach to develop potential PSB consortia for recommended wheat varieties by considering the respective soil health and agro-climatic conditions. The designed consortia were found to maintain adequate viability for up to 9 months, verified through field emission scanning electron microscopy and viable count. Furthermore, a significant increase in grain yield (5%-8%) and seed P (4%) content was observed in consortia-inoculated wheat plants with 20% reduced Diammonium phosphate (DAP) application under net house conditions. Fluorescence in situ hybridization analysis of roots and amplification of the gcd gene of Ochrobactrum sp. SSR indicated the survival and rhizosphere competency of the inoculated PSB. Categorical principal component analysis (CAT-PCA) showed a positive correlation of inoculated field-grown wheat varieties in native soils to grain yield, soil P content, and precipitation for sites belonging to irrigated plains and seed P content, soil organic matter, and number of tillers for sites belonging to Northern dry mountains. However, the impact of inoculation at sites belonging to the Indus delta was found significantly correlated to soil potassium (K) content, electrical conductivity (EC), and temperature. Additionally, a significant increase in grain yield (15%) and seed P (14%) content was observed in inoculated wheat plants. Thus, the present study demonstrates for the first time the need to integrate soil biological health and agro-climatic conditions for consistent performance of augmented PSB and enhanced P nutrient uptake to curtail soil pollution caused by the extensive use of agrochemicals. This study provides innovative insights and identifies key questions for future research on PSB to promote its successful implementation in agriculture.

Differential Root Exudation and Architecture for Improved Growth of Wheat Mediated by Phosphate Solubilizing Bacteria.

Phosphorous (P) deficiency is a major challenge faced by global agriculture. Phosphate-solubilizing bacteria (PSB) provide a sustainable approach to supply available phosphates to plants with improved crop productivity through synergistic interaction with plant roots. The present study demonstrates an insight into this synergistic P-solubilizing mechanism of PSB isolated from rhizosphere soils of major wheat-growing agro-ecological zones of Pakistan. Seven isolates were the efficient P solubilizers based on in vitro P-solubilizing activity (233-365 µg ml-1) with a concomitant decrease in pH (up to 3.5) by the production of organic acids, predominantly acetic acid (∼182 µg ml-1) and gluconic acid (∼117 µg ml-1). Amplification and phylogenetic analysis of gcd, pqqE, and phy genes of Enterobacter sp. ZW32, Ochrobactrum sp. SSR, and Pantoea sp. S1 showed the potential of these PSB to release orthophosphate from recalcitrant forms of phosphorus. Principal component analysis indicates the inoculation response of PSB consortia on the differential composition of root exudation (amino acids, sugars, and organic acids) with subsequently modified root architecture of three wheat varieties grown hydroponically. Rhizoscanning showed a significant increase in root parameters, i.e., root tips, diameter, and surface area of PSB-inoculated plants as compared to uninoculated controls. Efficiency of PSB consortia was validated by significant increase in plant P and oxidative stress management under P-deficient conditions. Reactive oxygen species (ROS)-induced oxidative damages mainly indicated by elevated levels of malondialdehyde (MDA) and H2O2 contents were significantly reduced in inoculated plants by the production of antioxidant enzymes, i.e., superoxide dismutase, catalase, and peroxidase. Furthermore, the inoculation response of these PSB on respective wheat varieties grown in native soils under greenhouse conditions was positively correlated with improved plant growth and soil P contents. Additionally, grain yield (8%) and seed P (14%) were significantly increased in inoculated wheat plants with 20% reduced application of diammonium phosphate (DAP) fertilizer under net house conditions. Thus, PSB capable of such synergistic strategies can confer P biofortification in wheat by modulating root morphophysiology and root exudation and can alleviate oxidative stress under P deficit conditions.

The wheat growth-promoting traits of Ochrobactrum and Pantoea species, responsible for solubilization of different P sources, are ensured by genes encoding enzymes of multiple P-releasing pathways.

Production and release of organic acids and phosphatase enzymes by microbes are important for inorganic and organic phosphorus cycling in soil. The presence of microorganisms with corresponding traits in the plant rhizosphere lead to improved plant P uptake and ultimately growth promotion. We studied the potential of two rhizosphere-competent strains, Pantoea sp. MR1 and Ochrobactrum sp. SSR, for solubilization of different organic and inorganic P sources in vitro. In a pot experiment we further revealed the impact of the two strains on wheat seedling performance in soil amended with either phytate, rock phosphate or K2HPO4 as solely P source. To directly link P-solubilizing activity to the strain-specific genetic potential, we designed novel primers for glucose dehydrogenase (gcd), phosphatase (pho) and phytase (phy) genes, which are related to the organic and inorganic P solubilization potential. Quantitative tracing of these functional genes in the inoculated soils of the conducted pot experiment further allowed to compare strain abundances in the soil in dependency on the present P source. We observed strain- and P source-dependent patterns of the P solubilization in vitro as well as in the pot experiment, whereby P release, particularly from phytate, was linked to the strain abundance. We further revealed that the activity of microbial phosphatases is determined by the interplay between functional gene abundance, available soil P, and substrate availability. Moreover, positive impacts of microbial seed inoculation on wheat root architecture and aboveground growth parameters were observed. Our results suggest that screening for rhizosphere-competent strains with gcd, pho and phy genes may help to identify new microbial taxa that are able to solubilize and mineralize inorganic as well as organic bound P. Subsequently, the targeted use of corresponding strains may improve P availability in agricultural soils and consequently reduce fertilizer application.

Tailored Bioactive Compost from Agri-Waste Improves the Growth and Yield of Chili Pepper and Tomato.

An extensive use of chemical fertilizers has posed a serious impact on food and environmental quality and sustainability. As the organic and biofertilizers can satisfactorily fulfill the crop's nutritional requirement, the plants require less chemical fertilizer application; hence, the food is low in chemical residues and environment is less polluted. The agriculture crop residues, being a rich source of nutrients, can be used to feed the soil and crops after composting and is a practicable approach to sustainable waste management and organic agriculture instead of open-field burning of crop residues. This study demonstrates a feasible strategy to convert the wheat and rice plant residues into composted organic fertilizer and subsequent enrichment with plant-beneficial bacteria. The bioactive compost was then tested in a series of in vitro and in vivo experiments for validating its role in growing organic vegetables. The compost was enriched with a blend of micronutrients, such as zinc, magnesium, and iron, and a multi-trait bacterial consortium AAP (Azospirillum, Arthrobacter, and Pseudomonas spp.). The bacterial consortium AAP showed survival up to 180 days post-inoculation while maintaining their PGP traits. Field emission scanning electron microscopic analysis and fluorescence in situ hybridization (FISH) of bioactive compost further elaborated the morphology and confirmed the PGPR survival and distribution. Plant inoculation of this bioactive compost showed significant improvement in the growth and yield of chilies and tomato without any additional chemical fertilizer yielding a high value to cost ratio. An increase of ≈35% in chlorophyll contents, ≈25% in biomass, and ≈75% in yield was observed in chilies and tomatoes. The increase in N was 18.7 and 25%, while in P contents were 18.5 and 19% in chilies and tomatoes, respectively. The application of bioactive compost significantly stimulated the bacterial population as well as the phosphatase and dehydrogenase activities of soil. These results suggest that bioactive compost can serve as a source of bioorganic fertilizer to get maximum benefits regarding vegetable yield, soil quality, and fertilizer saving with the anticipated application for other food crops. It is a possible win-win situation for environmental sustainability and food security.

Precise CRISPR-Cas9 Mediated Genome Editing in Super Basmati Rice for Resistance Against Bacterial Blight by Targeting the Major Susceptibility Gene.

Basmati rice is famous around the world for its flavor, aroma, and long grain. Its demand is increasing worldwide, especially in Asia. However, its production is threatened by various problems faced in the fields, resulting in major crop losses. One of the major problems is bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xoo hijacks the host machinery by activating the susceptibility genes (OsSWEET family genes), using its endogenous transcription activator like effectors (TALEs). TALEs have effector binding elements (EBEs) in the promoter region of the OsSWEET genes. Out of six well-known TALEs found to have EBEs in Clade III SWEET genes, four are present in OsSWEET14 gene's promoter region. Thus, targeting the promoter of OsSWEET14 is very important for creating broad-spectrum resistance. To engineer resistance against bacterial blight, we established CRISPR-Cas9 mediated genome editing in Super Basmati rice by targeting 4 EBEs present in the promoter of OsSWEET14. We were able to obtain four different Super Basmati lines (SB-E1, SB-E2, SB-E3, and SB-E4) having edited EBEs of three TALEs (AvrXa7, PthXo3, and TalF). The edited lines were then evaluated in triplicate for resistance against bacterial blight by choosing one of the locally isolated virulent Xoo strains with AvrXa7 and infecting Super Basmati. The lines with deletions in EBE of AvrXa7 showed resistance against the Xoo strain. Thus, it was confirmed that edited EBEs provide resistance against their respective TALEs present in Xoo strains. In this study up to 9% editing efficiency was obtained. Our findings showed that CRISPR-Cas9 can be harnessed to generate resistance against bacterial blight in indigenous varieties, against locally prevalent Xoo strains.

Illumina sequencing of 16S rRNA tag shows disparity in rhizobial and non-rhizobial diversity associated with root nodules of mung bean (Vigna radiata L.) growing in different habitats in Pakistan.

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.

Isolation and Characterization of Plant Growth Promoting Antagonistic Bacteria from Cotton and Sugarcane Plants for Suppression of Phytopathogenic Fusarium Species.

BACKGROUND: Plant Growth Promoting Rhizobacteria (PGPR) may be utilized to augment plant growth and suppress the plant pathogens. Objective: The present study was conducted to isolate and characterize the antagonistic bacteria indigenous to cotton and sugarcane rhizosphere in Pakistan, and to evaluate their ability to suppress phytopathogenic Fusarium spp. Out of 63 isolates 37 different morphotypes were studied for their antagonistic activity against Fusarium monoliformae, Fusarium oxysporum and Fusarium solani. Among these 31 strains showed the percentage suppression ranging from 40 to 66% against Fusarium spp. OBJECTIVES: The antagonistic bacteria having antifungal activity were studied for different morphological and physiological characteristics using Gram staining and light microscopy. Most of them were Gram negative and tentatively identified as Pseudomonas spp. The selected strains were screened in vitro for plant growth regulation and antifungal traits. MATERIAL AND METHODS: Our study included 1000 premature CAD patients that classified into two groups with history of MI (n = 461) and without of MI (n = 539). The polymorphism variants in 10% of samples were determined by PCR-RFLP technique and genotyping of the polymorphism in all subjects was conducted by High Resolution Melting method. Given the two conditions of patients residing in Tehran and also faced with their first episode of MI, 640 out of 1000 study samples that had been previously followed-up were assessed in a retrospective cohort phase regarding long-term major adverse cardiac events (MACE). RESULTS: Four bacterial strains were able to produce the chitinase enzyme while four other bacterial strains showed protease production. Ten strains were positive for HCN production. Out of 37, eight strains showed phosphate solubilization ranging from 13 to 24 µg/ml. eighteen strains produced indole acetic acid ranging from 5 to 19 µg/ml. CONCLUSIONS: This study identified specific traits in the isolated rhizobacteria which make them good candidates as PGPR and might contribute to enhance growth of crop plants. This information is of general interest and also helpful for devising strategies to manage diseases caused by Fusarium in cotton and sugarcane.

Glucose dehydrogenase gene containing phosphobacteria for biofortification of Phosphorus with growth promotion of rice.

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.

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.

Phosphate solubilizing bacteria with glucose dehydrogenase gene for phosphorus uptake and beneficial effects on wheat.

The aim of this study was to isolate, characterize and use phosphate solubilizing bacteria to enhance the bioavailability of insoluble Ca-phosphate for wheat plants. For this purpose, 15 phosphorus solubilizing bacteria (PSB) were isolated from wheat rhizospheric soils of Peshawar and southern Punjab region, Pakistan. These isolates were identified using light microscopy and 16S rRNA gene. Among the isolated bacteria, two strains (Pseudomonas sp. MS16 and Enterobacter sp. MS32) were the efficient P solubilizers based on their P solubilization activity determined qualitatively (solubilization index 3.2-5.8) as well as quantitatively (136-280 µg mL-1). These two strains produced indole-3-acetic acid (25.6-28.1 µg mL-1), gibberellic acid (2.5-11.8), solubilized zinc compounds (SI 2.8-3.3) and showed nitrogenase and 1-Aminocyclopropane-1-carboxylic acid deaminase activity in vitro. Phosphate solubilization activity of Pseudomonas sp. MS16 was further validated by amplification, sequencing and phylogenetic analysis of glucose dehydrogenase (gcd) gene (LT908484) responsible for P solubilization. Response Surface Methodology for large-scale production was used to find optimal conditions (Temperature 22.5°C, pH 7) for P solubilization. Glucose was found to support higher P solubilization in vitro. In an in vitro experiment, PSB treated wheat seedlings improved germination and seedling vigor (11% increases) as compared to un-inoculated control. Rhizoscanning of these seedlings showed an increase in various root growth parameters. Wheat inoculation with selected strain MS16 showed pronounced effect on grain yield in pot (38.5% increase) and field (17-18% increase) experiments compared to non-inoculated control. Root colonization by PSB through Florescent in situ Hybridization and Confocal Laser Scanning Microscopy confirmed their rhizosphere competence in soil. BOX-PCR confirmed the re-isolated colonies of Pseudomonas sp. MS16. The results indicated that gluconic acid producing Pseudomonas sp. MS16 from un-explored soils may be cost effective and environment friendly candidate to improve plant growth and phosphorous uptake by wheat plants.

Retrieved 16S rRNA and nifH sequences reveal co-dominance of Bradyrhizobium and Ensifer (Sinorhizobium) strains in field-collected root nodules of the promiscuous host Vigna radiata (L.) R. Wilczek.

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.

Interactive effect of trivalent iron on activated sludge digestion and biofilm structure in attached growth reactor of waste tire rubber.

Waste tire rubber (WTR) has been introduced as an alternative, novel media for biofilm development in several experimental systems including attached growth bioreactors. In this context, four laboratory-scale static batch bioreactors containing WTR as a support material for biofilm development were run under anoxic condition for 90 days using waste activated sludge as an inoculum under the influence of different concentrations (2.5, 6.5, 8.5 mg/l) of trivalent ferric iron (Fe3+). The data revealed that activated sludge with a Fe3+ concentration of 8.5 mg/l supported the maximum bacterial biomass [4.73E + 10 CFU/ml cm2]; besides, it removed 38% more Chemical oxygen demand compared to Fe3+ free condition from the reactor. Biochemical testing and 16S rDNA phylogenetic analysis of WTR-derived biofilm communities further suggested the role of varying concentrations of Fe3+ on the density and diversity of members of Enterobacteria(ceae), ammonium (AOB) and nitrite oxidizing bacteria. Furthermore, Fluorescent in situ hybridization with phylogenetic oligonucleotide probes and confocal laser scanning microscopy of WTR biofilms indicated a significant increase in density of eubacteria (3.00E + 01 to.05E + 02 cells/cm2) and beta proteobacteria (8.10E + 01 to 1.42E + 02 cells/cm2), respectively, with an increase in Fe3+ concentration in the reactors, whereas, the cell density of gamma proteobacteria in biofilms decreased.

Biocontrol of Bacterial Leaf Blight of Rice and Profiling of Secondary Metabolites Produced by Rhizospheric Pseudomonas aeruginosa BRp3.

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.

Plant Growth Promotion and Suppression of Bacterial Leaf Blight in Rice by Inoculated Bacteria.

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.

Association of plant growth-promoting Serratia spp. with the root nodules of chickpea.

Serratia species-affiliated DNA sequences have recently been discovered in the root nodules of two chickpea cultivars; however, little is known about their potential influence on chickpea plant growth. All Serratia-affiliated sequences (1136) could be grouped into two clusters at 98% DNA similarity. The major cluster, represented by 96% of sequences, was closely associated with Serratia marcescens sequences from GenBank. In the current study, we isolated two Serratia strains, 5D and RTL100, from root nodules of a field-grown Desi cultivar from Faisalabad and Thal areas, respectively. In vitro, strain 5D showed significantly higher phosphate (P) solubilization and lactic acid production than RTL100, whereas a comparable concentration of phytohormone was produced by both isolates. The application of Serratia strain 5D as an inoculum resulted in 25.55% and 30.85% increases in the grain yield of crops grown on fertile soil in irrigated areas and nutrient-deficient soil in rainfed areas, respectively, compared to the non-inoculated control. Results of plant inoculations indicated that Serratia sp. 5D and RTL100 can serve as effective microbial inoculants, particularly in nutrient-deficient soils in rainfed areas, where chickpea is the only major crop grown during the entire year.

Biological control of potato black scurf by rhizosphere associated bacteria

The present work was carried out to study the potential of plant rhizosphere associated bacteria for the biocontrol of potato black scurf disease caused by Rhizoctonia solani Khun AG-3. A total of twenty-eight bacteria isolated from diseased and healthy potato plants grown in the soil of Naran and Faisalabad, Pakistan were evaluated for their antagonistic potential. Nine bacterial strains were found to be antagonistic in vitro, reduced the fungal growth and caused the lysis of sclerotia of R. solani in dual culture assay as well as in extracellular metabolite efficacy test. The selected antagonistic strains were further tested for the production and efficacy of volatile and diffusible antibiotics, lytic enzymes and siderophores against R. solani. Selected antagonistic bacteria were also characterized for growth promoting attributes i.e., phosphate solubilization, nitrogen fixation and indole acetic acid production. Biocontrol efficacy and percent yield increase by these antagonists was estimated in greenhouse experiment. Statistical analysis showed that two Pseudomonas spp. StT2 and StS3 were the most effective with 65.1 and 73.9 percent biocontrol efficacy, as well as 87.3 and 98.3 percent yield increase, respectively. Potential antagonistic bacterial strain StS3 showed maximum homology to Pseudomonas sp. as determined by 16S rRNA gene sequencing. These results suggest that bacterial isolates StS3 and StT2 have excellent potential to be used as effective biocontrol agents promoting plant growth with reduced disease incidence.