Employment of Halotolerant Plant Growth Promoting Bacteria to Mitigate Soil Salinity in Wheat and Chickpea

Abstract

Soil salinity is one of the primary yield-limiting factors, in a variety of crops. Numerous rhizobacteria have shown an encouraging effect in improving the growth of plants and help them to cope with various stresses under various environmental challenges. This study was designed to isolate and identify halotolerant plant growth promoting bacteria (PGPB) from saline environment. These PGPB were further applied to the soil to mitigate salinity stress in wheat and chickpea. In the first part of this study, twenty bacterial strains were isolated and identified from the rhizospheric soil of Justicia adhatoda, Chenopodium murale, and Cenchrus ciliaris, growing in Khewra salt mine. It is the world's second-largest salt mine. Six of these bacterial strains were discovered to be extremely salt tolerant, as they could grow in Luria Bertani (LB) medium, supplemented with 10% NaCl. On the basis of their morphological, biochemical, and partial 16S rRNA gene sequencing, isolated bacteria were characterized and identified as Bacillus megaterium, Bacillus tequilensis, Bacillus xiamenensis, Pseudomonas putida, Pseudomonas aeruginosa, and Staphylococcus pasteuri. B. megaterium, B. tequilensis, and P. putida were chosen for further examination based on the best plant growth-promoting activities and extracellular enzyme secretions. Selected PGPB demonstrated varying degrees of antibiotic tolerance. Among three selected bacterial strains, B. tequilensis exhibited tolerance against maximum number of antibiotics. All of the chosen bacterial strains were capable of producing phytohormones such as indole-3-acetic acid (IAA), gibberellic acid (GA3), and abscisic acid (ABA). Under salt stress conditions. These bacterial strains increased root length, shoot length, and leaf area of wheat seedlings by increasing macronutrient uptake (nitrogen, phosphorus and potassium). In the second part of study, selected plant growth promoting rhizobacteria (PGPR) including Plant growth promoting attributes of B. megaterium, B. tequilensis, and P. putida were investigated further. These strains exhibited potential to secrete 1- aminocyclopropane-1-carboxylic acid (ACC) deaminase and exopolysaccharides (EPS). These bacterial strains improved the physiology, biochemistry, and antioxidant enzyme activities of the wheat plant under salt stress. Plants inoculated with PGPR exhibited higher relative water content, higher photosynthetic pigments, lower levels of hydrogen peroxide DRSML QAU (H2O2) and malondialdehyde (MDA), and improved enzymatic activity for the scavenging of reactive oxygen species (ROS). The qPCR revealed increased expression of salt overly sensitive genes (SOS1 and SOS4), indicating their possible role in stress tolerance. These genes can be overexpressed in wheat plants to make them more resistant to salinity stress. Based on these findings, it is possible to conclude that priming seeds with the aforementioned PGPRs can reduce the negative effects of salinity on wheat plants. In the third part of the study, salinity tolerance mechanism of one best performing halotolerant bacterium (B. tequilensis) was investigated. Scanning electron microscopy (SEM) revealed the highest floc yield and biofilm formation ability of B. tequilensis at 100 mM NaCl concentration. Fourier Transformed Infrared Spectroscopy (FTIR) depicted the presence of amino, hydroxyl and carboxyl groups in the exopolysaccharides (EPS) of B. tequilensis, indicating the presence of carbohydrates and proteins. Using PCR, plant growth promoting bacterial genes viz., 1-aminocyclopropane-1-carboxylate deaminase (acdS) and pyrroloquinoline quinone (pqqE) were successfully amplified from the genome of B. tequilensis. To further confirm its plant growth promoting ability, B. tequilensis was inoculated in the soil and chickpea plants were grown. Chickpea seedlings displayed increased chlorophyll content, relative water contents, higher soluble sugars, and proline contents, while their electrolytic leakage was decreased. The activity of several antioxidant enzymes, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), was increased. Salt stress also resulted in higher production of malondialdehyde and hydrogen peroxide (H2O2). In the fourth part of the study, B. tequilensis (strain MPP8) was inoculated to chickpea plants at 100 mM NaCl concentration and the expression of CaRab genes was investigated, using qRT-PCR. These genes have been reported to be involved in intracellular trafficking and play key role in salinity stress. In this study, the expression of CaRabA2 gene was found to be higher than all other genes. A strong positive correlation (R2 = 0.6615) of CaRabA2 gene expression with Na+ buildup in leaves was observed. Moreover, a variety of leaf traits like stomata assay, gas exchange assay, and the concentrations of sodium (Na+ ) and potassium ions (K+ ) in the leaves were also studied. Under salinity stress, net CO2 absorption, intracellular CO2 concentration, stomatal conductance, net transpiration, and photosynthetic rate were observed to be decreased. DRSML QAU Saline conditions increased leaf vapor pressure deficit and leaf temperature. Inoculation of B. tequilensis improved gas exchange properties of chickpea and improved plant vigor. At 100 mM NaCl stress conditions, limited gaseous exchange was observed due to closed stomata and deformed guard cells. Findings of this study suggested the sustainable use of B. tequilensis to mitigate salinity stress of chickpea and other crops.