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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 15 N 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 15 N 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 15 N 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflictof interest., (Copyright © 2022 Naqqash, Malik, Imran, Hameed, Shahid, Hanif, Majeed, Iqbal, Qaisrani and van Elsas.)

During and after the green revolution in the last century, agrochemicals especially nitrogen (N) were extensively used. However, it resulted in a remarkable increase in crop yield but drastically reduced soil fertility; increased the production cost, food prices, and carbon footprints; and depleted the fossil reserves with huge penalties to the environment and ecological sustainability. The groundwater, rivers, and oceans are loaded with N excess which is an environmental catastrophe. Nitrogen emissions (e.g., ammonia, nitrogen oxide, nitrous oxide) play an important role in global climate change and contribute to particulate matter and acid rain causing respiratory problems, cancers, and damage to forests and buildings. Therefore, the nitrogen-polluted planet Earth needs concerted global efforts to avoid the disaster. Improved agricultural N management focuses on the synchronization of crop N demand and N supply along with improving the N-use efficiency of the crops. However, there is very little focus on the natural sources of N available for plants in the form of diazotrophic bacteria present inside or on the root surface and the rhizosphere. These diazotrophs are the mini-nitrogen factories that convert available (78%) atmospheric N 2 to ammonia through a process known as "biological nitrogen fixation" which is then taken up by the plants for its metabolic functioning. Diazotrophs also stimulate root architecture by producing plant hormones and hence improve the plant's overall ability to uptake nutrients and water. In recent years, nanotechnology has revolutionized the whole agri-industry by introducing nano-fertilizers and coated/slow-releasing fertilizers. With this in mind, we tried to explore the following questions: To what extent can the crop N requirements be met by diazotroph inoculation? Can N input to agriculture be managed in a way leading to environmental benefits and farmers saving money? Can nanotechnology help in technological advancement of diazotroph application? The review suggests that an integrated technology based on slow-releasing nano-fertilizer combined with diazotrophs should be adopted to decrease nitrogen inputs to the agricultural system. This integrated technology would minimize N pollution and N losses to much extent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Imran, Hakim, Tariq, Nawaz, Laraib, Gulzar, Hanif, Siddique, Hayat, Fraz and Ahmad.)

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.

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 β-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.