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.

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.

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.

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.

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.

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.