Genome analysis of a halophilic Virgibacillus halodenitrificans ASH15 revealed salt adaptation, plant growth promotion, and isoprenoid biosynthetic machinery

Globally, due to widespread dispersion, intraspecific diversity, and crucial ecological components of halophilic ecosystems, halophilic bacteria is considered one of the key models for ecological, adaptative, and biotechnological applications research in saline environments. With this aim, the present study was to enlighten the plant growth-promoting features and investigate the systematic genome of a halophilic bacteria, Virgibacillus halodenitrificans ASH15, through single-molecule real-time (SMRT) sequencing technology. Results showed that strain ASH15 could survive in high salinity up to 25% (w/v) NaCl concentration and express plant growth-promoting traits such as nitrogen fixation, plant growth hormones, and hydrolytic enzymes, which sustain salt stress. The results of pot experiment revealed that strain ASH15 significantly enhanced sugarcane plant growth (root shoot length and weight) under salt stress conditions. Moreover, the sequencing analysis of the strain ASH15 genome exhibited that this strain contained a circular chromosome of 3,832,903 bp with an average G+C content of 37.54%: 3721 predicted protein-coding sequences (CDSs), 24 rRNA genes, and 62 tRNA genes. Genome analysis revealed that the genes related to the synthesis and transport of compatible solutes (glycine, betaine, ectoine, hydroxyectoine, and glutamate) confirm salt stress as well as heavy metal resistance. Furthermore, functional annotation showed that the strain ASH15 encodes genes for root colonization, biofilm formation, phytohormone IAA production, nitrogen fixation, phosphate metabolism, and siderophore production, which are beneficial for plant growth promotion. Strain ASH15 also has a gene resistance to antibiotics and pathogens. In addition, analysis also revealed that the genome strain ASH15 has insertion sequences and CRISPRs, which suggest its ability to acquire new genes through horizontal gene transfer and acquire immunity to the attack of viruses. This work provides knowledge of the mechanism through which V. halodenitrificans ASH15 tolerates salt stress. Deep genome analysis, identified MVA pathway involved in biosynthesis of isoprenoids, more precisely “Squalene.” Squalene has various applications, such as an antioxidant, anti-cancer agent, anti-aging agent, hemopreventive agent, anti-bacterial agent, adjuvant for vaccines and drug carriers, and detoxifier. Our findings indicated that strain ASH15 has enormous potential in industries such as in agriculture, pharmaceuticals, cosmetics, and food

Complete genome analysis of sugarcane root associated endophytic diazotroph Pseudomonas aeruginosa DJ06 revealing versatile molecular mechanism involved in sugarcane development

Sugarcane is an important sugar and bioenergy source and a significant component of the economy in various countries in arid and semiarid. It requires more synthetic fertilizers and fungicides during growth and development. However, the excess use of synthetic fertilizers and fungicides causes environmental pollution and affects cane quality and productivity. Plant growth-promoting bacteria (PGPB) indirectly or directly promote plant growth in various ways. In this study, 22 PGPB strains were isolated from the roots of the sugarcane variety GT42. After screening of plant growth-promoting (PGP) traits, it was found that the DJ06 strain had the most potent PGP activity, which was identified as Pseudomonas aeruginosa by 16S rRNA gene sequencing. Scanning electron microscopy (SEM) and green fluorescent protein (GFP) labeling technology confirmed that the DJ06 strain successfully colonized sugarcane tissues. The complete genome sequencing of the DJ06 strain was performed using Nanopore and Illumina sequencing platforms. The results showed that the DJ06 strain genome size was 64,90,034 bp with a G+C content of 66.34%, including 5,912 protein-coding genes (CDSs) and 12 rRNA genes. A series of genes related to plant growth promotion was observed, such as nitrogen fixation, ammonia assimilation, siderophore, 1-aminocyclopropane-1-carboxylic acid (ACC), deaminase, indole-3-acetic acid (IAA) production, auxin biosynthesis, phosphate metabolism, hydrolase, biocontrol, and tolerance to abiotic stresses. In addition, the effect of the DJ06 strain was also evaluated by inoculation in two sugarcane varieties GT11 and B8. The length of the plant was increased significantly by 32.43 and 12.66% and fresh weight by 89.87 and 135.71% in sugarcane GT11 and B8 at 60 days after inoculation. The photosynthetic leaf gas exchange also increased significantly compared with the control plants. The content of indole-3-acetic acid (IAA) was enhanced and gibberellins (GA) and abscisic acid (ABA) were reduced in response to inoculation of the DJ06 strain as compared with control in two sugarcane varieties. The enzymatic activities of oxidative, nitrogen metabolism, and hydrolases were also changed dramatically in both sugarcane varieties with inoculation of the DJ06 strain. These findings provide better insights into the interactive action mechanisms of the P. aeruginosa DJ06 strain and sugarcane plant development

High-Throughput Sequencing-Based Analysis of Rhizosphere and Diazotrophic Bacterial Diversity Among Wild Progenitor and Closely Related Species of Sugarcane (Saccharum spp. Inter-Specific Hybrids)

Considering the significant role of genetic background in plant-microbe interactions and that most crop rhizospheric microbial research was focused on cultivars, understanding the diversity of root-associated microbiomes in wild progenitors and closely related crossable species may help to breed better cultivars. This study is aimed to fill a critical knowledge gap on rhizosphere and diazotroph bacterial diversity in the wild progenitors of sugarcane, the essential sugar and the second largest bioenergy crop globally. Using a high-throughput sequencing (HTS) platform, we studied the rhizosphere and diazotroph bacterial community of SaccharumofficinarumL. cv. Badila (BRS), Saccharumbarberi (S. barberi) Jesw. cv Pansahi (PRS), Saccharum robustum [S. robustum; (RRS), Saccharum spontaneum (S. spontaneum); SRS], and Saccharum sinense (S. sinense) Roxb. cv Uba (URS) by sequencing their 16S rRNA and nifH genes. HTS results revealed that a total of 6,202 bacteria-specific operational taxonomic units (OTUs) were identified, that were distributed as 107 bacterial groups. Out of that, 31 rhizobacterial families are commonly spread in all five species. With respect to nifH gene, S. barberi and S. spontaneum recorded the highest and lowest number of OTUs, respectively. These results were validated by quantitative PCR analysis of both genes. A total of 1,099 OTUs were identified for diazotrophs with a core microbiome of 9 families distributed among all the sugarcane species. The core microbiomes were spread across 20 genera. The increased microbial diversity in the rhizosphere was mainly due to soil physiochemical properties. Most of the genera of rhizobacteria and diazotrophs showed a positive correlation, and few genera negatively correlated with the soil properties. The results showed that sizeable rhizospheric diversity exists across progenitors and close relatives. Still, incidentally, the rhizosphere microbial abundance of progenitors of modern sugarcane was at the lower end of the spectrum, indicating the prospect of Saccharum species introgression breeding may further improve nutrient use and disease and stress tolerance of commercial sugarcane. The considerable variation for rhizosphere microbiome seen in Saccharum species also provides a knowledge base and an experimental system for studying the evolution of rhizobacteria-host plant association during crop domestication

Multifarious Plant Growth-Promoting Rhizobacterium Enterobacter sp. CM94-Mediated Systemic Tolerance and Growth Promotion ofChickpea (Cicer arietinum L.) under Salinity Stress

Background: Chickpea is one of the most important leguminous crops and its productivity is significantly affected by salinity stress. The use of ecofriendly, salt-tolerant, plant growth-promoting rhizobacteria (PGPR) as a bioinoculant can be very effective in mitigating salinity stress in crop plants. In the present study, we explored, characterized, and evaluated a potential PGPR isolate for improving chickpea growth under salt stress. Methods: A potential PGPR was isolated from rhizospheric soils of chickpea plants grown in the salt-affected area of eastern Uttar Pradesh, India. The isolate was screened for salt tolerance and characterized for its metabolic potential and different plant growth-promoting attributes. Further, the potential of the isolate to promote chickpea growth under different salt concentrations was determined by a greenhouse experiment. Results: A rhizobacteria isolate, CM94, which could tolerate a NaCl concentration of up to 8% was selected for this study. Based on the BIOLOG carbon source utilization, isolate CM94 was metabolically versatile and able to produce multiple plant growth-promoting attributes, such as indole acetic acid, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, siderophore, hydrogen cyanide (HCN), and ammonia as well as solubilized phosphate. A polyphasic approach involving the analysis of fatty acid methyl ester (FAME) and 16S rRNA gene sequencing confirmed the identity of the isolate as Enterobacter sp. The results of greenhouse experiments revealed that isolate CM94 inoculation significantly enhanced the shoot length, root length, and fresh and dry weight of chickpea plants, under variable salinity stress. In addition, inoculation improved the chlorophyll, proline, sugar, and protein content in the tissues of the plant, while lowering lipid peroxidation. Furthermore, isolate CM94 reduced oxidative stress by enhancing the enzymatic activities of superoxide dismutase, catalase, and peroxidase compared to in the respective uninoculated plants. Conclusions: Overall, the results suggested that using Enterobacter sp. CM94 could significantly mitigate salinity stress and enhance chickpea growth under saline conditions. Such studies will be helpful in identifying efficient microorganisms to alleviate salinity stress, which in turn will help, to devise ecofriendly microbial technologies

De novo assembly, functional annotation and comparative alignment of whole genome of a halo-tolerant Exiguobacterium profundum PHM11 with related genomes

Advances in the next-generation sequencing (NGS) technologies have invigorated the exploration of microbial genomes in retrieving the hidden traits. In this study, high-throughput next generation whole genome sequencing of a halotolerant E. profundum PHM11 was performed on Illumina HiSeq paired end sequencing plateform and assembled through de novo Linux based approaches using Velvet (V 1.2.10.) algorithm package. Quality filtering in de novo sequencing produced 72,947,390 reads with total size of 7335195362 bp (7335.1 Mb). High quality reads having minimum and maximum contig lengths of 202 and 958471 (~0.95 Mb) bp were further considered for assembly. Final PHM11 genome has a size of ~2.92 Mb comprising 70 contigs, 47.93% G+C content with 761858 (26.08%), 757313 (25.92%), 699924 (23.96%), and 700172 (23.97%) percentages of adenine, thymine, cytosine and guanine nucleotides. Throughout micro-satellite mining of genome showed a total of 3005 SSRs, covering 0.1 of whole PHM11 genome, with relative abundance; 1029, relative density; 10951, and percentages of penta repeats; 65.75, hexa repeats; 28.75, mono repeats; 3.89, and tetra-repeats; 1.59, respectively. Gene networks related to the arrangement of key genes and presence of lysogenic phage DNA were reflected through generating the chromosome map of PHM11 genome. Functional annotations of genome reflected the different protein families, and hidden inherent metabolic pathways providing unusual features. A total of 3033 protein coding genes and 33 non-protein coding genes were identified; out of these only 2316 could be characterized and 737 were reported as hypothetical. Random genes of different metabolic pathways were amplified from its genome and authenticated through their sequencing. Genome-rearrangements in PHM11 could be deciphered through aligning its genome with thirteen other genomes of different Exiguobacterium species

Combination of UV-Fenton oxidation process with biological technique for treatment of polycyclic aromatic hydrocarbons using <em>Pseudomonas pseudoalcaligenes</em> NRSS3 isolated from petroleum contaminated site

460-469Polycyclic aromatic hydrocarbons (PAHs), often from petroleum oil spill, by-product of petroleum refining, incomplete combustion of fossil fuel, leakage in pipeline and underground storage, apart from the effluents of pesticide, dye, pigment, and drug industries, are considered carcinogenic and mutagenic. As the abundance of PAHs in the environment cause adverse effects on humans and ecosystem, the PAHs contamination needs to be monitored and such polluted sites require remediation. Conventional methods available for remediation of PAHs are adsorption, advance oxidation process, electrochemical remediation, solvent extraction, use of synthetic surfactants and photocatalytic remediation. These methods including the alternative Fenton oxidation technology are not only expensive but also produce secondary pollutants. In this study, we evaluated the performance of UV-Fenton-PBBR (Packed bed bioreactor) hybrid system for the treatment of polycyclic aromatic hydrocarbons (naphthalene and fluorene). Pseudomonas pseudoalcaligenes NRSS3 isolated from petroleum contaminated site and immobilized on Sterculia alata was used as packing media in the PBBR. The naphthalene and fluorene were taken as model polycyclic aromatic hydrocarbon (PAHs) with initial concentration of 400 mg/L. The optimum conditions for UV-Fenton oxidation were (pH: 3, Fe2+: 2.5 g/L, H2O2: 1000 mg/L) for naphthalene and (pH: 3, Fe2+: 3.0 g/L, H2O2: 1200 mg/L) for fluorene. The overall maximum removal efficiency of the combined system was found to be 96 and 94.7% for naphthalene and fluorene, respectively. GC-MS analysis confirmed the formation of catechol, 1-napthol, salicylic acid and phthalic anhydride as metabolites during degradation process. Biodegradation kinetics of naphthalene and fluorene were studied using Monod model and kinetics constants were found to be µmax: 0.3057 per day; Ks: 112.87 mg/L for naphthalene and µmax: 0.2921 per day; Ks: 114.75 mg/L for fluorene

Silicon and soil microorganisms improve rhizospheric soil health with bacterial community, plant growth, performance and yield

The interaction of silicon and soil microorganisms stimulates crop enhancement to ensure sustainable agriculture. Silicon may potentially increase nutrient availability in rhizosphere with improved plants’ growth, development as it does not produce phytotoxicity. The rhizospheric microbiome accommodates a variety of microbial species that live in a small area of soil directly associated with the hidden half plants’ system. Plant growth-promoting rhizobacteria (PGPR) play a major role in plant development in response to adverse climatic conditions. PGPRs may enhance the growth, quality, productivity in variety of crops, and mitigate abiotic stresses by reprogramming stress-induced physiological variations in plants via different mechanisms, such as synthesis of indole-3-acetic acid, 1-aminocyclopropane-1-carboxylate deaminase, exopolysaccharides, volatile organic compounds, atmospheric nitrogen fixation, and phosphate solubilization. Our article eye upon interactions of silicon and plant microbes which seems to be an opportunity for sustainable agriculture for series of crops and cropping systems in years to come, essential to safeguard the food security for masses

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Not AvailableCold shock proteins (CSPs) are greatly conserved family of structurally related DNA binding proteins which are produced during temperature drift. A 213 bp long cspA gene was cloned and sequenced from Pseudomonas koreensis P2 in the present study. The expression analysis of the cspA showed 2.5 folds increase in the mRNA level at 15 C while the expression was almost on par at 30 C and 5 C indicating its role in moderately low temperature. In silico analyses of the gene showed that the gene codes for 7.69 kDa protein which was phylogenetically very similar to CspA present in Pseudomonads. Amino acid composition of the CspA from P. koreensis was different from that of mesophilic Pseudomonas and tiny/small amino varied significantly between CspA of cold adaptive and mesophilic species. The CspA from P. koreensis P2 contained RNP motifs involved in binding of DNA and RNA. Phylogenetic analyses revealed that the CspA protein of P. koreensis P2 was more close to CspA of distant subgroups of Pseudomonas like P. fluorescens and P. putida subgroup indicating a possible intra-specific gene transfer.The authors gratefully acknowledge the financial assistance under network project ‘Application of Microorganisms Agriculture and Allied Sectors (AMAAS)’ and ‘‘CRP Genomics’’ from Indian Council of Agricultural Research (ICAR), India. i

Halotolerant Exiguobacterium profundum PHM11 Tolerate Salinity by Accumulating L-Proline and Fine-Tuning Gene Expression Profiles of Related Metabolic Pathways

Salinity stress is one of the serious factors, limiting production of major agricultural crops; especially, in sodic soils. A number of approaches are being applied to mitigate the salt-induced adverse effects in agricultural crops through implying different halotolerant microbes. In this aspect, a halotolerant, Exiguobacterium profundum PHM11 was evaluated under eight different salinity regimes; 100, 250, 500, 1000, 1500, 2000, 2500, and 3000 mM to know its inherent salt tolerance limits and salt-induced consequences affecting its natural metabolism. Based on the stoichiometric growth kinetics; 100 and 1500 mM concentrations were selected as optimal and minimal performance limits for PHM11. To know, how salt stress affects the expression profiles of regulatory genes of its key metabolic pathways, and total production of important metabolites; biomass, carotenoids, beta-carotene production, IAA and proline contents, and expression profiles of key genes affecting the protein folding, structural adaptations, transportation across the cell membrane, stress tolerance, carotenoids, IAA and mannitol production in PHM11 were studied under 100 and 1500 mM salinity. E. profundum PHM11 showed maximum and minimum growth, biomass and metabolite production at 100 and 1500 mM salinity respectively. Salt-induced fine-tuning of expression profiles of key genes of stress pathways was determined in halotolerant bacterium PHM11

Insights into the Bacterial and Nitric Oxide-Induced Salt Tolerance in Sugarcane and Their Growth-Promoting Abilities

Soil salinity causes severe environmental stress that affects agriculture production and food security throughout the world. Salt-tolerant plant-growth-promoting rhizobacteria (PGPR) and nitric oxide (NO), a distinctive signaling molecule, can synergistically assist in the alleviation of abiotic stresses and plant growth promotion, but the mechanism by which this happens is still not well known. In the present study, in a potential salt-tolerant rhizobacteria strain, ASN-1, growth up to 15% NaCl concentration was achieved with sugarcane rhizosphere soil. Based on 16S-rRNA gene sequencing analysis, the strain ASN-1 was identified as a Bacillus xiamenensis. Strain ASN-1 exhibits multiple plant-growth-promoting attributes, such as the production of indole-3-acetic acid, 1-aminocyclopropane-1-carboxylate deaminase, siderophores, HCN, ammonia, and exopolysaccharides as well as solubilized phosphate solubilization. Biofilm formation showed that NO enhanced the biofilm and root colonization capacity of the PGPR strain ASN-1 with host plants, evidenced by scanning electron microscopy. The greenhouse study showed that, among the different treatments, the combined application of PGPR and sodium nitroprusside (SNP) as an NO donor significantly (p ≤ 0.05) enhanced sugarcane plant growth by maintaining the relative water content, electrolyte leakage, gas exchange parameters, osmolytes, and Na+/K+ ratio. Furthermore, PGPR and SNP fertilization reduced the salinity-induced oxidative stress in plants by modulating the antioxidant enzyme activities and stress-related gene expression. Thus, it is believed that the acquisition of advanced information about the synergistic effect of salt-tolerant PGPR and NO fertilization will reduce the use of harmful chemicals and aid in eco-friendly sustainable agricultural production under salt stress conditions