Designing of a multi-epitopes based vaccine against Haemophilius parainfluenzae and its validation through integrated computational approaches.

Haemophilus parainfluenzae is a Gram-negative opportunist pathogen within the mucus of the nose and mouth without significant symptoms and has an ability to cause various infections ranging from ear, eye, and sinus to pneumonia. A concerning development is the increasing resistance of H. parainfluenzae to beta-lactam antibiotics, with the potential to cause dental infections or abscesses. The principal objective of this investigation is to utilize bioinformatics and immuno-informatic methodologies in the development of a candidate multi-epitope Vaccine. The investigation focuses on identifying potential epitopes for both B cells (B lymphocytes) and T cells (helper T lymphocytes and cytotoxic T lymphocytes) based on high non-toxic and non-allergenic characteristics. The selection process involves identifying human leukocyte antigen alleles demonstrating strong associations with recognized antigenic and overlapping epitopes. Notably, the chosen alleles aim to provide coverage for 90% of the global population. Multi-epitope constructs were designed by using suitable linker sequences. To enhance the immunological potential, an adjuvant sequence was incorporated using the EAAAK linker. The final vaccine construct, comprising 344 amino acids, was achieved after the addition of adjuvants and linkers. This multi-epitope Vaccine demonstrates notable antigenicity and possesses favorable physiochemical characteristics. The three-dimensional conformation underwent modeling and refinement, validated through in-silico methods. Additionally, a protein-protein molecular docking analysis was conducted to predict effective binding poses between the multi-epitope Vaccine and the Toll-like receptor 4 protein. The Molecular Dynamics (MD) investigation of the docked TLR4-vaccine complex demonstrated consistent stability over the simulation period, primarily attributed to electrostatic energy. The docked complex displayed minimal deformation and enhanced rigidity in the motion of residues during the dynamic simulation. Furthermore, codon translational optimization and computational cloning was performed to ensure the reliability and proper expression of the multi-Epitope Vaccine. It is crucial to emphasize that despite these computational validations, experimental research in the laboratory is imperative to demonstrate the immunogenicity and protective efficacy of the developed vaccine. This would involve practical assessments to ascertain the real-world effectiveness of the multi-epitope Vaccine.

Metal-tolerant morganella morganii isolates can potentially mediate nickel stress tolerance in Arabidopsis by upregulating antioxidative enzyme activities.

Plant growth-promoting rhizobacteria (PGPRs) have been utilized to immobilize heavy metals, limiting their translocation in metal contaminated settings. However, studies on the mechanisms and interactions that elucidate how PGPRs mediate Nickel (Ni) tolerance in plants are rare. Thus, in this study we investigated how two pre-characterized heavy metal tolerant isolates of Morganella morganii (ABT9 and ABT3) improve Ni stress tolerance in Arabidopsis while enhancing its growth and yield. Arabidopsis seedlings were grown for five weeks in control/Ni contaminated (control, 1.5 mM and 2.5 mM) potted soil, in the presence or absence of PGPRs. Plant growth characteristics, quantum yield, and antioxidative enzymatic activities were analyzed to assess the influence of PGPRs on plant physiology. Oxidative stress tolerance was quantified by measuring MDA accumulation in Arabidopsis plants. As expected, Ni stress substantially reduced plant growth (shoot and root fresh weight by 53.25% and 58.77%, dry weight by 49.80% and 57.41% and length by 47.16% and 64.63% over control), chlorophyll content and quantum yield (by 40.21% and 54.37% over control). It also increased MDA content by 84.28% at higher (2.5 mM) Ni concentrations. In contrast, inoculation with M. morganii led to significant improvements in leaf chlorophyll, quantum yield, and Arabidopsis biomass production. The mitigation of adverse effects of Ni stress on biomass observed in M. morganii-inoculated plants was attributed to the enhancement of antioxidative enzyme activities compared to Ni-treated plants. This upregulation of the antioxidative defense mechanism mitigated Ni-induced oxidative stress, leading to improved performance of the photosynthetic machinery, which, in turn, enhanced chlorophyll content and quantum yield. Understanding the underlying mechanisms of these tolerance-inducing processes will help to complete the picture of PGPRs-mediated defense signaling. Thus, it suggests that M. morganii PGPRs candidate can potentially be utilized for plant growth promotion by reducing oxidative stress via upregulating antioxidant defense systems in Ni-contaminated soils and reducing Ni metal uptake.

Heavy metal-resistant rhizobacteria fosters to alleviate the cadmium toxicity in Arabidopsis by upregulating the plant physiological responses.

This study was designed to investigate the role of Morganella morganii strains in alleviating Cd stress in Arabidopsis seedlings under controlled conditions. Both M. morganii strains ABT3 (ON316873) and ABT9 (ON316874) strains isolated from salt-affected areas showed higher resistance against Cd and possess plant growth-promoting traits such as nitrogen fixation, indole-acetic acid production, ammonia production, phosphate solubilization, and, catalase, gelatinase and protease enzyme production. Plant inoculation assay showed that varying concentration of Cd (1.5 mM and 2.5 mM) significantly reduced Arabidopsis growth, quantum yield (56.70%-66.49%), and chlorophyll content (31.90%-42.70%). Cd toxicity also triggered different associations between lipid peroxidation (43.61%-69.77%) and enzymatic antioxidant mechanisms. However, when both strains were applied to the Arabidopsis seedlings, the shoot and root length and fresh and dry weights were improved in the control and Cd-stressed plants. Moreover, both strains enhanced the resistance against Cd stress by increasing antioxidant enzyme activities [catalase (19.47%-27.39%) and peroxidase (37.50%-48.07%)]that ultimately cause a substantial reduction in lipid peroxidation (27.71%-41.90%). Both strains particularly ABT3 also showed positive results in improving quantum yield (73.84%-98.64%) and chlorophyll content (41.13%-48.63%), thus increasing the growth of Arabidopsis seedlings. The study suggests that PGPR can protect plants from Cd toxicity, and Cd-tolerant rhizobacterial strains can remediate heavy metal polluted sites and improve plant growth.

In order to develop sustainable and effective agricultural techniques in areas polluted with heavy metals, it is important to have a deeper understanding of the characteristics of metal-resistant PGPR. Hence, this study focuses on the efficacy of M. morganii in promoting the growth and increasing the photosynthetic pigments of Arabidopsis seedlings under Cd toxicity.

Mineral Solubilizing Rhizobacterial Strains Mediated Biostimulation of Rhodes Grass Seedlings.

Minerals play a dynamic role in plant growth and development. However, most of these mineral nutrients are unavailable to plants due to their presence in fixed forms, which causes significant losses in crop production. An effective strategy to overcome this challenge is using mineral solubilizing bacteria, which can convert insoluble forms of minerals into soluble ones that plants can quickly assimilate, thus enhancing their availability in nutrient-depleted soils. The main objective of the present study was to isolate and characterize mineral solubilizing rhizobacteria and to assess their plant growth-promoting potential for Rhodes grass. Twenty-five rhizobacterial strains were isolated on a nutrient agar medium. They were characterized for solubilization of insoluble minerals (phosphate, potassium, zinc, and manganese), indole acetic acid production, enzymatic activities, and various morphological traits. The selected strains were also evaluated for their potential to promote the growth of Rhodes grass seedlings. Among tested strains, eight strains demonstrated strong qualitative and quantitative solubilization of insoluble phosphate. Strain MS2 reported the highest phosphate solubilization index, phosphate solubilization efficiency, available phosphorus concentration, and reduction in medium pH. Among tested strains, 75% were positive for zinc and manganese solubilization, and 37.5% were positive for potassium solubilization. Strain MS2 demonstrated the highest quantitative manganese solubilization, while strains MS7 and SM4 reported the highest solubilization of zinc and potassium through acidifying their respective media. The strain SM4 demonstrated the most increased IAA production in the presence and absence of L-tryptophan. The majority of strains were positive for various enzymes, including urease, catalase protease, and amylase activities. However, these strains were negative for coagulase activity except strains SM7 and MS7. Based on 16S rRNA gene sequencing, six strains, namely, SM2, SM4, SM5, MS1, MS2, and MS4, were identified as Bacillus cereus, while strains SM7 and MS7 were identified as Staphylococcus saprophyticus and Staphylococcus haemolyticus. These strains significantly improved growth attributes of Rhodes grass, such as root length, shoot length, and root and shoot fresh and dry biomasses compared to the uninoculated control group. The present study highlights the significance of mineral solubilizing and enzyme-producing rhizobacterial strains as potential bioinoculants to enhance Rhodes grass growth under mineral-deficient conditions sustainably.

First report on the probiotic potential of Mammaliicoccus sciuri isolated from raw goat milk.

Probiotics are considered effective microbial dietary supplements that provide beneficial effects to consumers, usually by restoring or improving gut microflora. Goat milk is one of the rich sources of probiotics as well as nutrients. Therefore, the primary aim of this research was to isolate and evaluate the potential of novel indigenous probiotic strains present in goat milk. Six different raw goat milk samples were collected from different areas of Multan, Pakistan. For bacterial characterization, samples were cultured and isolated on MRS agar plates for different morphological and biochemical tests. The probiotic potential of the six isolates, all of which were gram positive (G1, G2, G3, G4, G5, and G6) and five of which were catalase negative (all except G1), were assessed via a milk coagulation assay and antimicrobial activity, pH tolerance, phenol tolerance, and sodium chloride (NaCl) tolerance tests, which revealed that all the isolates coagulated in milk and showed protease and lipase activity, except G3. All six isolates showed tolerance against 0.2% phenol and 2-4% NaCl and were able to survive in both alkaline and acidic conditions. Only five isolates showed antimicrobial activity against indicator strain Aspergillus niger strain STA9, validating their probiotic nature. The most potent bile-tolerant and bacteriocin-producing isolate, G1, also showed γ-hemolytic activity and resistance to penicillin but showed susceptibility to other antibiotics. The lactic acid-producing (0.60% titratable acidity) G1 isolate was identified as a novel strain of Mammaliicoccus sciuri based on 16S rDNA sequencing. The above findings suggest that the potent M. sciuri GMN01 strain can serve as a potential probiotic strain. A potent probiotic strain isolated from raw goat milk could be utilized as a dietary supplement, and goat milk could become an alternative to other sources of milk, particularly cow milk. However, safety aspects of this strain require further investigation because the present safety tests are insufficient to conclude that the GMN01 isolate is safe.

Application of zinc oxide nanoparticles immobilizes the chromium uptake in rice plants by regulating the physiological, biochemical and cellular attributes.

Zinc oxide nano particles (ZnO NPs) have been employed as a novel strategy to regulate plant tolerance and alleviate heavy metal stress, but our scanty knowledge regarding the systematic role of ZnO NPs to ameliorate chromium (Cr) stress especially in rice necessitates an in-depth investigation. An experiment was performed to evaluate the effect of different concentrations of ZnO NPs (e.g., 0, 25, 50, 100 mg/L) in ameliorating the Cr toxicity and accumulation in rice seedlings in hydroponic system. Our results demonstrated that Cr (100 µM) severely inhibited the rice seedling growth, whereas exogenous treatment of ZnO NPs significantly alleviated Cr toxicity stress and promoted the plant growth. Moreover, application of ZnO NPs significantly augmented the germination energy, germination percentage, germination index, and vigor index. In addition, biomass accumulation, antioxidants (SOD, CAT, POD), nutrient acquisition (Zn, Fe) was also improved in ZnO NPs-treated plants, while the lipid peroxidation (MDA, H2O2), electrolyte leakage as well as Cr uptake and in-planta accumulation was significantly decreased. The burgeoning effects were more apparent at ZnO NPs (100 mg/L) suggesting the optimum treatment to ameliorate Cr induced oxidative stress in rice plants. Furthermore, the treatment of ZnO NPs (100 mg/L) reduced the level of endogenous abscisic acid (ABA) and stimulated the growth regulator hormones such as brassinosteroids (BRs) possibly linked with enhanced phytochelatins (PCs) levels. The ultrastructure analysis at cellular level of rice revealed that the application of 100 mg/L ZnO NPs protected the chloroplast integrity and other cell organells via improvement in plant ionomics, antioxidant activities and down regulating Cr induced oxidative stress in rice plants. Conclusively, observations of the current study will be helpful in developing stratigies to decrease Cr contamination in food chain by employing ZnO NPs and to mitigate the drastic effects of Cr in plants for the sustainable crop growth.

Inoculation With Azospirillum spp. Acts as the Liming Source for Improving Growth and Nitrogen Use Efficiency of Potato.

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 15N 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 15N 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 15N 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.

Comparison of bacterial diversity, root exudates and soil enzymatic activities in the rhizosphere of AVP1-transgenic and nontransgenic wheat (Triticum aestivum L.).

AIMS: Soil microbial communities are among the most diverse communities that might be affected due to transgenic crops. Therefore, risk assessment studies on transgenes are essentially required as any adverse effects may depend not only on the specific gene and crop involved but also on soil conditions. METHODS AND RESULTS: The present study deals with the comparison of bacterial populations, root exudates and activities of soil enzymes in nontransgenic and AVP1-transgenic wheat rhizosphere, overexpressing vacuolar H + pyrophosphatase for salinity and drought stress tolerance. Amounts of organic acids and sugars produced as root exudates and activities of dehydrogenase, phosphatase and protease enzymes in soil solution showed no significant differences in AVP1-transgenic and nontransgenic wheat rhizosphere, except for urease and phenol oxidase activities. The higher copy number of nifH gene showed the abundance of nitrogen-fixing bacteria in the rhizosphere of AVP1-transgenic wheat compared with nontransgenic wheat. nifH gene sequence analysis indicated the common diazotrophic genera Azospirillum, Bradyrhizobium, Rhizobium and Pseudomonas in AVP1-transgenic and nontransgenic wheat except for Zoogloea detected only in nontransgenic wheat. Using 454-pyrosequencing of 16S rRNA gene from soil DNA, a total of 156, 282 sequences of 18 phyla were obtained, which represented bacterial (128,006), Archeal (7928) and unclassified (21,568) sequences. Proteobacteria, Crenarchaeota and Firmicutes were the most abundant phyla in the transgenic and nontransgenic wheat rhizosphere. Further comparison of different taxonomic units at the genus level showed similar distribution in transgenic and nontransgenic wheat rhizospheres. CONCLUSION: We conclude that the AVP1 gene in transgenic wheat has no apparent adverse effects on the soil environment and different bacterial communities. However, the bacterial community depends on several other factors, not only genetic composition of the host plants. SIGNIFICANCE OF THE STUDY: The present research supports introduction and cultivation of transgenic plants in agricultural systems without any adverse effects on indigenous bacterial communities and soil ecosystems.

Efficacy of organic-based carrier material for plant beneficial rhizobacteria application in okra under normal and salt-affected soil conditions.

AIMS: Plant beneficial rhizobacteria (PBR) improve salt tolerance and plant yield in vegetable plants by producing 1-aminocyclopropane-1-carboxylate-deaminase, indole-3-acetic acid and phosphate solubilization. Organic-based carrier material is needed to ensure the PBR's uniform application, distribution, survival and functioning in a variety of fields. The PBR also use carbon present in the carrier as food and energy source. The selection of a suitable organic-based carrier material for the application of the PBR in normal and saline soils always has received less attention. The current study compared the PBR suitability of different organic-based carrier materials (biochar, biogas residues [BGRs] and coconut powder) and evaluated their effects on okra productivity under normal and saline soil conditions. METHODS AND RESULTS: In a pot experiment, the PBR strain Bacillus sp. MR-1/2 (accession number, MG548383) was applied with/or without organic-based carrier materials to okra grown in three different soils: S1 (EC 1.0 dS m-1 ), S2 (EC 3.0 dS m-1 ) and S3 (EC 5.0 dS m-1 ). The experiment was set up in a completely randomized design with five replicates in factorial arrangement. Results indicated that in soil S1, PBR + BGR increased the number of pods per plant, plant dry weight and indole compounds by 64%, 68% and 17% while reduced the electrolyte leakage (ELL), malonaldehyde (MDA) contents and stress ethylene level by 17%, 55% and 38%, respectively over the PBR application without any carrier. Similarly, in soil S2, the treatment PBR + BGR increased the number of pods by 81%, plant dry weight by 40% and indole compounds by 13% while reduced the ELL by 17%, MDA contents by 50% and stress ethylene by 30% over the PBR alone treatment. In soil S3, PBR + biochar increased the number of pods by 51%, plant dry weight by 62% and indole compounds by 20%, while reduced the ELL by 21%, MDA by 40% and indole compounds by 54% over the PBR alone treatment. CONCLUSIONS: Results concluded that in soil S1 and S2 (normal soils), BGR as carrier for PBR showed best results, while in soil S3, biochar as carrier for PBR resulted in enhanced potassium (K+ ) and calcium (Ca+2 ) uptake and increased the productivity of okra. SIGNIFICANCE AND IMPACT OF STUDY: Response of different carrier materials in supporting PBR under different soil conditions was variable. This study will help in the selection and use of best suitable carrier material for PBR application under different soil conditions. It is recommended that farmer should use BGR as carrier material for PBR application in normal soils while biochar should be used as carrier for the PBR application in saline soil.

First report of diazotrophic Brevundimonas spp. as growth enhancer and root colonizer of potato.

Rhizobacteria contain various plant-beneficial traits and their inoculation can sustainably increase crop yield and productivity. The present study describes the growth-promoting potential of Brevundimonas spp. isolated from rhizospheric soil of potato from Sahiwal, Pakistan. Four different putative strains TN37, TN39, TN40, and TN44 were isolated by enrichment on nitrogen-free malate medium and identified as Brevundimonas spp. based on their morphology, 16S rRNA gene sequence, and phylogenetic analyses. All strains contained nifH gene except TN39 and exhibited nitrogen fixation potential through acetylene reduction assay (ARA) except TN40. Among all, the Brevundimonas sp. TN37 showed maximum ARA and phosphate solubilization potential but none of them exhibited the ability to produce indole acetic acid. Root colonization studies using transmission electron microscopy and confocal laser scanning microscopy showed that Brevundimonas sp. TN37 was resident over the root surface of potato; forming sheets in the grooves in the rhizoplane. TN37, being the best among all was further evaluated in pot experiment using potato cultivar Kuroda in sterilized sand. Results showed that Brevundimonas sp. TN37 increased growth parameters and nitrogen uptake as compared to non-inoculated controls. Based on the results obtained in this study, it can be suggested that Brevundimonas spp. (especially TN37) possess the potential to improve potato growth and stimulate nitrogen uptake. This study is the first report of Brevundimonas spp. as an effective PGPR in potato.

Heterologous expression of azoreductase-encoding gene azrS of Bacillus sp. MR-1/2 for enhanced azo dye decolorization and wastewater treatment.

In Pakistan, 55% of textile exports are contributed by textile-units of Faisalabad. The effluents of these textile units, being discharged without any treatment, contain the contamination of a huge amount of synthetic azo dyes. The objective of the current research was to evaluate the contribution of an azoreductase-encoding gene (azrS) from a pre-characterized azo dye decolorizing bacterial strain Bacillus sp. MR-1/2 in a high copy number host system (pUC19-T7-Top-T) of Escherichia coli strain DH5α followed by in-silico prediction of azoreductase enzyme (AzrS) function. The recombinant cells that contained azrS had a significantly higher rate of color removal in congo red and reactive black-5 dyes when compared to wild-type MR-1/2 and E. coli DH5α after 72 h of incubation. Moreover, we were able to show that the recombinant strain significantly reduced the values of all tested parameters (pH, EC, turbidity, TSS, and COD) in actual wastewater. In support of our results, it was also predicted through bioinformatics analysis that the deduced azoreductase protein of strain MR-1/2 is linked with the dye decolorization ability of the strain through NAD(P)H-ubiquinone: oxidoreductase activity. Furthermore, we also found that the deduced protein resembled closely related proteins of protein databank in many features, yet some unique features were predicted in the enzyme activity of strain MR-1/2. It was concluded that the recombinant strain could be examined in pilot-scale experiments for textile wastewater treatment.

Growth stimulatory effect of AHL producing Serratia spp. from potato on homologous and non-homologous host plants.

Plant growth promoting rhizobacteria are known to improve plant performance by developing healthy and productive interactions with the host plants. These associations may be symbiotic or asymbiotic depending upon the genetic potential of the resident microbe and promiscuity of the host. Present study describes the potential of two Serratia spp. strains for promotion of plant growth in homologous as well as non-homologous hosts. The strains KPS-10 and KPS-14; native to potato rhizosphere belong to genus Serratia based on 16S rRNA gene sequences (accession no. LN831934 and LN831937 respectively) and contain multiple plant growth promoting properties along-with the production of quorum sensing acyl homoserine lactone (AHL) molecules. Both Serratia spp. strains showed solubilization of inorganic tri-calcium phosphate while KPS-14 also exhibited phytase activity (1.98 10-10 kcat). KPS-10 showed higher P-solubilization activity (128.5 µg/mL), IAA production (8.84 µg/mL), antifungal activity and also showed the production of two organic acids i.e., gluconic acid and lactic acid. Both strains produced three common AHLs: C6-HSL, 3oxo-C10-HSL, 3oxo-C12-HSL while some strain-specific AHLs (3OH-C5-HSL, 3OH-C6-HSL, C10-HSL specific to KPS-10 and 3OH-C6-HSL, C8-HSL, 3oxo-C9-HSL, 3OH-C9-HSL specific to KPS-14). Strains showed roots and rhizosphere colonization of potato and other non-homologous hosts up to one month. In planta AHLs-detection confirmed a likely role of AHLs during seedling growth and development where both extracted AHLs or bacteria inoculated roots showed extensive root hair. A significant increase in root/shoot lengths, root/ shoot fresh weights, root/shoot dry weights was observed by inoculation in different hosts. PGP-characteristics along with the AHLs-production signify the potential of both strains as candidate for the development of bio-inoculum for potato crop in specific and other crops in general. This inoculum will not only reduce the input of chemical fertilizer to the environment but also improve soil quality and plant growth.

Communication of plants with microbial world: Exploring the regulatory networks for PGPR mediated defense signaling.

Agricultural manipulation of potentially beneficial rhizosphere microbes is increasing rapidly due to their multi-functional plant-protective and growth related benefits. Plant growth promoting rhizobacteria (PGPR) are mostly non-pathogenic microbes which exert direct benefits on plants while there are rhizosphere bacteria which indirectly help plant by ameliorating the biotic and/or abiotic stress or induction of defense response in plant. Regulation of these direct or indirect effect takes place via highly specialized communication system induced at multiple levels of interaction i.e., inter-species, intra-species, and inter-kingdom. Studies have provided insights into the functioning of signaling molecules involved in communication and induction of defense responses. Activation of host immune responses upon bacterial infection or rhizobacteria perception requires comprehensive and precise gene expression reprogramming and communication between hosts and microbes. Majority of studies have focused on signaling of host pattern recognition receptors (PRR) and nod-like receptor (NLR) and microbial effector proteins under mining the role of other components such as mitogen activated protein kinase (MAPK), microRNA, histone deacytylases. The later ones are important regulators of gene expression reprogramming in plant immune responses, pathogen virulence and communications in plant-microbe interactions. During the past decade, inoculation of PGPR has emerged as potential strategy to induce biotic and abiotic stress tolerance in plants; hence, it is imperative to expose the basis of these interactions. This review discusses microbes and plants derived signaling molecules for their communication, regulatory and signaling networks of PGPR and their different products that are involved in inducing resistance and tolerance in plants against environmental stresses and the effect of defense signaling on root microbiome. We expect that it will lead to the development and exploitation of beneficial microbes as source of crop biofertilizers in climate changing scenario enabling more sustainable agriculture.

Green copper nanoparticles from a native Klebsiella pneumoniae strain alleviated oxidative stress impairment of wheat plants by reducing the chromium bioavailability and increasing the growth.

Chromium (Cr) concentration has been increasing substantially in the environment due to industrial and anthropogenic factors. Plants can absorb Cr and undergo unrestrained oxidation cascades, resulting in cell injury. The ameliorative role of biogenic copper nanoparticles to relieve wheat plants from Cr stress by supporting their growth is still unclear. The present work aims at the biosynthesis and characterization of copper nanoparticles (CuNPs) from a native Klebsiella pneumoniae strain, followed by assessment of wheat growth and physiological responses to CuNPs mixed in Cr-rich soil. The taxonomic rank of K. pneumoniae SN35 was established by the 16 S rRNA gene sequence analysis. The properties of biogenic CuNPs were elucidated by using UV-vis spectroscopy, FTIR, XRD, SEM, and TEM. It was found that 19.01-47.47 nm spherical shaped CuNPs were stabilized by different functional groups produced extracellularly by the strain SN35. The XRD data revealed the crystalline nature of CuNPs as a face-centered cubic structure. Different concentrations of CuNPs (0, 25, 50 and 100 mg kg-1 of soil) were added into the soil mixed with 3.5 mg kg-1 K2Cr2O7 and the pots were placed in a growth chamber for 30 days. The results revealed that the CuNPs, at 25 and 50 mg kg-1 of soil, augmented plant growth, biomass, and cellular antioxidants contents, whereas decreased the reactive oxygen species and Cr translocation from soil to roots and shoots as compared to control plants. Overall, the results revealed that the soil amendment of CuNPs could immobilize the Cr in the soil to prevent its translocation to the upper plant parts and support wheat growth by relieving cellular oxidative stress.

Achromobacter sp. FB-14 harboring ACC deaminase activity augmented rice growth by upregulating the expression of stress-responsive CIPK genes under salinity stress.

Soil salinity is one of the major plant growth and yield-limiting constraints in arid and semi-arid regions of the world. In addition to the oxidative damage, increasing salt stress is associated with elevated cellular ethylene levels due to the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) in large amounts. The objective of the current study was to elucidate the inoculation effect of an ACC deaminase (ACCD)-producing phytobeneficial strain Achromobacter sp. FB-14 on rice plants to alleviate the salinity effects by upregulation of the stress-responsive CIPK genes. The strain FB-14 was isolated by using nutrient agar medium at 855 mM NaCl concentration and it was taxonomically identified as Achromobacter sp. with more than 99% 16S rRNA gene sequence similarity with many Achromobacter species. The strain FB-14 demonstrated substantial in vitro potential for ACCD activity, synthesis of indole compounds, and phosphate solubilization up to 100 mM NaCl concentration in the culture medium. The gene corresponding to ACCD activity (acdS) was amplified and sequenced in order to confirm the inherent enzyme activity of the strain at a molecular level. The rifampicin-resistant derivative of strain FB-14 was recovered from the rice rhizosphere on antibiotic medium up to 21 days of sowing. Moreover, the strain FB-14 was inoculated on rice plants under salinity and it not only enhanced the growth of rice plants in terms of root and shoot length, and fresh and dry weight, but also upregulated the expression of stress-responsive CIPK genes (OsCIPK03, OsCIPK12, and OsCIPK15) according to the results of qRT-PCR analysis. To the best of our knowledge, this is the first report deciphering the role of plant-beneficial Achromobacter strain relieving the rice plants from salt stress by promoting the growth and enhancing the expression of stress-responsive CIPK genes.

A comparative study of bacterial diversity based on culturable and culture-independent techniques in the rhizosphere of maize (Zea mays L.).

OBJECTIVE: Maize is an important crop for fodder, food and feed industry. The present study explores the plant-microbe interactions as alternative eco-friendly sustainable strategies to enhance the crop yield. METHODOLOGY: Bacterial diversity was studied in the rhizosphere of maize by culture-dependent and culture-independent techniques by soil sampling, extraction of DNA, amplification of gene of interest, cloning of desired fragment and library construction. RESULTS: Culturable bacteria were identified as Achromobacter, Agrobacterium, Azospirillum, Bacillus, Brevibacillus, Bosea, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus, Stenotrophomonas and Xanthomonas genera. For culture-independent approach, clone library of 16S ribosomal RNA gene was assembled and 100 randomly selected clones were sequenced. Majority of the sequences were related to Firmicutes (17%), Acidobacteria (16%), Actinobacteria (17%), Alpha-Proteobacteria (7%), Delta-proteobacteria (4.2%) and Gemmatimonadetes (4.2%) However, some of the sequences (30%) were novel that showed no homologies to phyla of cultured bacteria in the database. Diversity of diazotrophic bacteria in the rhizosphere investigated by analysis of PCR-amplified nifH gene sequence that revealed abundance of sequences belonging to genera Azoarcus (25%), Aeromonas (10%), Pseudomonas (10%). The diazotrophic genera Azotobacter, Agrobacterium and Zoogloea related nifH sequences were also detected but no sequence related to Azospirillum was found showing biasness of the growth medium rather than relative abundance of diazotrophs in the rhizosphere. CONCLUSION: The study provides a foundation for future research on focussed isolation of the Azoarcus and other diazotrophs found in higher abundance in the rhizosphere.

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.

Differential Response of Potato Toward Inoculation with Taxonomically Diverse Plant Growth Promoting Rhizobacteria.

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.

Isolation and characterization of a ß-propeller gene containing phosphobacterium Bacillus subtilis strain KPS-11 for growth promotion of potato (Solanum tuberosum L.).

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 ß-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.