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.

Biochar-induced priming effects in soil via modifying the status of soil organic matter and microflora: A review.

Biochar (BC) application has the potential to be integrated into a carbon-trading framework owing to its multiple environmental and economic benefits. Despite the increasing research attention over the past ten years, the mechanisms of BC-induced priming effects on soil organic carbon mineralization and their influencing factors have not been systematically considered. This review aims to document the recent progress in BC research by focusing on (1) how BC-induced priming effects change the soil environment, (2) the factors governing the mechanisms underlying BC amendment effects on soils, and (3) how BC amendments alter soil microbial communities and nutrient dynamics. Here, we carried out a detailed examination of the origins of different biochar, its pyrolysis conditions, and potential interactions with various factors that affect BC characteristics and mechanisms of C mineralization in primed soil. These findings clearly addressed the strong linkage between BC properties and abiotic factors that leads to change the soil microclimate, priming effects, and carbon stabilization. This review offers an overview of a fragmented body of evidence and the current state of understanding to support the application of BC in different soil environments with the aim of sustaining or improving the agricultural crop production.

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.

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.

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.

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.