Deciphering the antiviral nature of endophytic Bacillus spp. against groundnut bud necrosis virus in cowpea and tomato.

Tomato, the important vegetable crop, is severely affected by Orthotospovirus arachinecrosis which impacts heavy economic losses. The application of insecticide to manage viral diseases is not an environmentally safe approach. In view of these issues, we investigated the antiviral efficacy of 21 bacterial endophytes against GBNV in local lesion host (Cowpea-VBN3). Based on the reduction in lesion number and virus titer as estimated through both DAC ELISA and qPCR in cowpea, the bacterial endophytes viz., Bacillus licheniformis Soya1, Bacillus tequilensis NBL6, and Bacillus velezensis VB7 were selected and further tested in tomato. The study revealed the well-defined antiviral efficacy of these endophytes against GBNV. The percentage of disease incidence ranged from 16 to 24% in endophyte-treated tomato plants compared with untreated plants (88%). In addition, symptom severity was reduced, and the application of endophytes also in promotion of the growth compared with untreated control. DAC ELISA revealed that the tomato plants treated with bacterial endophytes challenged with GBNV showed reduction in the virus titer (0.26-0.39 @ OD 405 nm) at different days of interval after inoculation (0, 5, and 10 days) compared with untreated control (3.475 @ OD 405 nm). Additionally, reduction in the viral copy number in bacterial endophyte-treated plants was evident by real-time PCR. Furthermore, tomato plants bacterized with endophytes depicted significant correlation and reduction in viral load and disease incidence as revealed by the principal-component biplot analysis. Thus, the application of bacterial endophytes has a potential role in reducing the disease incidence, severity, and titer value of GBNV, which will be the promising management approach in future to mitigate the virus infection in tomato plants.

Metagenomic profiling of tomato rhizosphere delineates the diverse nature of uncultured microbes as influenced by Bacillus velezensis VB7 and Trichoderma koningiopsis TK towards the suppression of root-knot nematode under field conditions.

The plant-parasitic Root Knot Nematodes (Meloidogyne spp.,) play a pivotal role to devastate vegetable crops across the globe. Considering the significance of plant-microbe interaction in the suppression of Root Knot Nematode, we investigated the diversity of microbiome associated with bioagents-treated and nematode-infected rhizosphere soil samples through metagenomics approach. The wide variety of organisms spread across different ecosystems showed the highest average abundance within each taxonomic level. In the rhizosphere, Proteobacteria, Firmicutes, and Actinobacteria were the dominant bacterial taxa, while Ascomycota, Basidiomycota, and Mucoromycota were prevalent among the fungal taxa. Regardless of the specific treatments, bacterial genera like Bacillus, Sphingomonas, and Pseudomonas were consistently found in high abundance. Shannon diversity index vividly ensured that, bacterial communities were maximum in B. velezensis VB7-treated soil (1.4-2.4), followed by Root Knot Nematode-associated soils (1.3-2.2), whereas richness was higher with Trichoderma konigiopsis TK drenched soils (1.3-2.0). The predominant occurrence of fungal genera such as Aspergillus Epicoccum, Choanephora, Alternaria and Thanatephorus habituate rhizosphere soils. Shannon index expressed the abundant richness of fungal species in treated samples (1.04-0.90). Further, refraction and species diversity curve also depicted a significant increase with maximum diversity of fungal species in B. velezensis VB7-treated soil than T. koningiopsis and nematode-infested soil. In field trial, bioagents-treated tomato plant (60% reduction of Meloidogyne incognita infection) had reduced gall index along with enhanced plant growth and increased fruit yield in comparison with the untreated plant. Hence, B. velezensis VB7 and T. koingiopsis can be well explored as an antinemic bioagents against RKN. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03851-1.

Molecular Docking of Nimbolide Extracted from Leaves of Azadirachta indica with Protein Targets to Confirm the Antifungal, Antibacterial and Insecticidal Activity.

Nimbolide, a tetranortriterpenoid (limonoid) compound isolated from the leaves of Azadirachta indica, was screened both in vitro and in silico for its antimicrobial activity against Fusarium oxysporum f. sp. cubense, Macrophomina phaseolina, Pythium aphanidermatum, Xanthomonas oryzae pv. oryzae, and insecticidal activity against Plutella xylostella. Nimbolide exhibited a concentration-dependent, broad spectrum of antimicrobial and insecticidal activity. P. aphanidermatum (82.77%) was more highly inhibited than F. oxysporum f. sp. cubense (64.46%) and M. phaseolina (43.33%). The bacterium X. oryzae pv. oryzae forms an inhibition zone of about 20.20 mm, and P. xylostella showed about 66.66% mortality against nimbolide. The affinity of nimbolide for different protein targets in bacteria, fungi, and insects was validated by in silico approaches. The 3D structure of chosen protein molecules was built by homology modelling in the SWISS-MODEL server, and molecular docking was performed with the SwissDock server. Docking of homology-modelled protein structures shows most of the chosen target proteins have a higher affinity for the furan ring of nimbolide. Additionally, the stability of the best-docked protein-ligand complex was confirmed using molecular dynamic simulation. Thus, the present in vitro and in silico studies confirm the bioactivity of nimbolide and provide a strong basis for the formulation of nimbolide-based biological pesticides. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01104-6.

Triamcinolone Acetonide Produced by Bacillus velezensis YEBBR6 Exerts Antagonistic Activity Against Fusarium oxysporum f. sp. Cubense: A Computational Analysis.

Fusarium oxysporum f. sp. cubense is one of the most severe and threatening pathogens of bananas, causing "Panama wilt" worldwide. Confrontation assay of Foc antagonistic bacterial endophyte, Bacillus velezensis YEBBR6, with the Foc and GC-MS profiling of excised agar from the zone of inhibition, led to the unveiling of secondary metabolites produced by the endophyte. To refine the probable antifungal compounds among the numerous biomolecules formed during their di-trophic interaction with the pathogen, fungal protein targets were modeled, and docking studies (AutoDock Vina module of the PyRx 0.8 server) were done with all the compounds. Triamcinolone acetonide exhibited the most excellent affinity for the protein targets among the compounds studied. It had a maximum binding affinity of 11.2 kcal/mol for XRN2 (5' → 3'). Further, the protein-ligand complex formation kinetics was done through Molecular Dynamic Simulation studies. Graphs for the RMSD, RMSF, Rg, potential energy, and SASA were generated, and the values during the simulation period suggested the stability of the biomolecule as a complex with the protein. This indicated Triamcinolone acetonide's potential ability to act as a functional disrupter of the target protein and likely an antifungal molecule. Further, the biomolecule was tested for its activity against Foc by screening in the wet lab through the poisoned plate technique, and it was found to be fully inhibitory to the growth of the pathogen at 1000 ppm.

Nematicidal Property of Clindamycin and 5-hydroxy-2-methyl Furfural (HMF) from the Banana Endophyte Bacillus velezensis (YEBBR6) Against Banana Burrowing Nematode Radopholus similis.

Radopholus similis is a burrowing nematode which causes banana toppling disease and is of major economic threat for the banana production. Bacterial endophyte Bacillus velezensis (YEBBR6) produce biomolecules like 5-hydroxy-2-methyl furfural (HMF) and clindamycin in during interaction with Fusarium oxysporum f.sp. cubense. Molecular modelling and docking studies were performed on Radopholus similis protein targets such as calreticulin, cathepsin S-like cysteine proteinase, ß-1,4 -endoglucanase, reticulocalbin, venom allergen-like protein and serine carboxypeptidase to understand the mode of action of HMF and clindamycin against Radopholus similis. Structurally validated protein targets of R. similis were docked with biomolecules through AutoDock Vina module in PyRx 0.8 software to predict the binding energy of ligand and target protein. Among the chosen six targets, docking analysis revealed that clindamycin had the maximum binding affinity for ß-1,4-endoglucanase (- 7.2 kcal/mol), reticulocalbin (- 7.5 kcal/mol) and serine carboxypeptidase (- 6.9 kcal/mol) in comparison with HMF and the nematicide, carbofuran 3G. Besides, clindamycin also had the maximum binding energy for the target sites calreticulin and venom allergen-like protein compared to the small molecule HMF. Novel molecule, clindamycin produced by B. velezensis served as a potential inhibitor of the target sites associated in interrupting the functions of ß-1,4-endoglucanase, reticulocalbin, serine carboxypeptidase, calreticulin, cathepsin S-like cysteine proteinase, and venom allergen-like proteins. Besides, increased binding affinity of clindamycin with the protein target sites facilitated to explore it as a novel nematicidal molecule for the management of banana burrowing nematode R. similis. Thus, present investigation confirmed that, the small molecules clindamycin can be explored for nematicidal activity. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01011-2.

Mining the Genome of Bacillus velezensis VB7 (CP047587) for MAMP Genes and Non-Ribosomal Peptide Synthetase Gene Clusters Conferring Antiviral and Antifungal Activity.

Chemical pesticides have an immense role in curbing the infection of plant viruses and soil-borne pathogens of high valued crops. However, the usage of chemical pesticides also contributes to the development of resistance among pathogens. Hence, attempts were made in this study to identify a suitable bacterial antagonist for managing viral and fungal pathogens infecting crop plants. Based on our earlier investigations, we identified Bacillus amyloliquefaciens VB7 as a potential antagonist for managing Sclerotinia sclerotiorum infecting carnation, tobacco streak virus infecting cotton and groundnut bud necrosis infecting tomato. Considering the multifaceted action of B. amyloliquefaciens VB7, attempts were made for whole-genome sequencing to assess the antiviral activity against tomato spotted wilt virus infecting chrysanthemum and antifungal action against Fusarium oxysporum f. sp. cubense (Foc). Genome annotation of the isolate B. amyloliquefaciens VB7 was confirmed as B. velezensis VB7 with accession number CP047587. Genome analysis revealed the presence of 9,231,928 reads with an average read length of 149 bp. Assembled genome had 1 contig, with a total length of 3,021,183 bp and an average G+C content of 46.79%. The protein-coding sequences (CDS) in the genome was 3090, transfer RNA (tRNA) genes were 85 with 29 ribosomal RNA (rRNA) genes and 21 repeat regions. The genome of B. velezensis VB7 had 506 hypothetical proteins and 2584 proteins with functional assignments. VB7 genome had the presence of flagellin protein FlaA with 987 nucleotides and translation elongation factor TU (Ef-Tu) with 1191 nucleotides. The identified ORFs were 3911 with 47.22% GC content. Non ribosomal pepide synthetase cluster (NRPS) gene clusters in the genome of VB7, coded for the anti-microbial peptides surfactin, butirosin A/butirosin B, fengycin, difficidin, bacillibactin, bacilysin, and mersacidin the Ripp lanthipeptide. Antiviral action of VB7 was confirmed by suppression of local lesion formation of TSWV in the local lesion host cowpea (Co-7). Moreover, combined application of B. velezensis VB7 with phyto-antiviral principles M. Jalapa and H. cupanioides increased shoot length, shoot diameter, number of flower buds per plant, flower diameter, and fresh weight of chrysanthemum. Further, screening for antifungal action of VB7 expressed antifungal action against Foc in vitro by producing VOC/NVOC compounds, including hexadecanoic acid, linoelaidic acid, octadecanoic acid, clindamycin, formic acid, succinamide, furanone, 4H-pyran, nonanol and oleic acid, contributing to the total suppression of Foc apart from the presence of NRPS gene clusters. Thus, our study confirmed the scope for exploring B. velezensis VB7 on a commercial scale to manage tomato spotted wilt virus, groundnut bud necrosis virus, tobacco streak virus, S. sclerotiorum, and Foc causing panama wilt of banana.

Optimization of adsorption process parameters by response surface methodology for hexavalent chromium removal from aqueous solutions using Annona reticulata Linn peel microparticles.

Fruit peel microparticles of Annona reticulata Linn were used as biosorbent for the sequestration of hexavalent chromium (CR(VI)). Characterization of the biosorbent was done using scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDXS), Fourier transfer infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GCMS), carbon, hydrogen, nitrogen and sulphur (CHNS) elemental analysis, mercury intrusion porosimetry and point of zero charge. Influential parameters were optimized using response surface methodology (RSM) with a total of 17 experimental runs based on the Box-Behnken design and found to be pH 1.0, temperature 25 °C and 100 mg/L initial chromium concentration. pH and concentration were found to be more influential than temperature. The analysis of variance indicated that a second-order polynomial regression equation was the most suitable for fitting the experimental data. The experimental runs showed a good correlation with the predicted responses (R2 = 0.9956). The biosorption process fitted well with the Langmuir isotherm with an adsorption capacity of 108. 32 mg/g out of the other isotherms such as Freundlich and Dubinin-Radushkevich that were analyzed. Non linear pseudo first order, pseudo second order, and intraparticle diffusion kinetics were applied to describe the interaction between the biosorbent and Cr(VI). Desorption and regeneration performances showed that fruit peels of Annona reticulata Linn can be an environmental friendly option for hexavalent chromium removal from aqueous solutions.

Hexavalent chromium removal from aqueous solutions by a novel powder prepared from Colocasia esculenta leaves.

In this study, batch removal of hexavalent chromium from aqueous solutions by powdered Colocasia esculenta leaves was investigated. Batch experiments were conducted to study the effects of adsorption of Cr(VI) at different pH values, initial concentrations, agitation speeds, temperatures, and contact times. The biosorbent was characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and Fourier transform infrared spectrometer analysis. The biosorptive capacity of the adsorbent was dependent on the pH of the chromium solution in which maximum removal was observed at pH 2. The adsorption equilibrium data were evaluated for various adsorption isotherm models, kinetic models, and thermodynamics. The equilibrium data fitted well with Freundlich and Halsey models. The adsorption capacity calculated was 47.62 mg/g at pH 2. The adsorption kinetic data were best described by pseudo-second-order kinetic model. Thus, Colocasia esculenta leaves can be considered as one of the efficient and cheap biosorbents for hexavalent chromium removal from aqueous solutions.

Analysis of secondary structural and physicochemical changes in protein-protein complexes.

Conformation switching in protein-protein complexes is considered important for the molecular recognition process. Overall analysis of 123 protein-protein complexes in a benchmark data-set showed that 6.8% of residues switched over their secondary structure conformation upon complex formation. Amino acid residue-wise preference for conformation change has been analyzed in binding and non-binding site residues separately. In this analysis, residues such as Ser, Leu, Glu, and Lys had higher frequency of secondary structural conformation change. The change of helix to coil and sheet to coil conformation and vice versa has been observed frequently, whereas the conformation change of helix to extended sheet occurred rarely in the studied complexes. Influence of conformation change toward the N and C terminal on either side of the binding site residues has been analyzed. Further, analysis on φ and ψ angle variation, conservation, stability, and solvent accessibility have been performed on binding site residues. Knowledge obtained from the present study could be effectively employed in the protein-protein modeling and docking studies.

Impact of protein binding cavity volume (PCV) and ligand volume (LV) in rigid and flexible docking of protein-ligand complexes.

The importance of protein binding cavity volume (PCV) and ligand volume (LV) in rigid and flexible docking has been studied in 48 protein-ligand complexes belonging to eight protein families. In continuation of our earlier study on protein flexibility in relationship to PCV and LV, this study analyzes the importance of PCV and LV in the scoring and ranking of ligands in docking experiments. Crystal structures of protein-ligand complexes with varied PCV were chosen for docking ligands of varied volume in each protein family. Docking and scoring accuracy have been evaluated by self and cross docking of ligands to the given protein conformation. Effect of PCV and LV in rigid and flexible docking has been studied both in apo and holo proteins. Rigid docking has performed well when appropriate protein conformation is used. Selecting the proteins with appropriate PCV based on the LV information is suggested for better results in ensemble docking.

Synthesis, characterization, and antimicrobial activity of nano-hydroxyapatite-zinc for bone tissue engineering applications.

The bone implants used in tissue repair are susceptible to infections caused by staphylococci, specifically Staphylococcus aureus. Hence, the development of better biological materials that provide antimicrobial activity in bone tissue engineering is required. The nanoparticles of hydroxyapatite (nHAp) and nHAp dopped with Zn (nHAp-Zn) were prepared by the wet chemical method and the ion exchange method, respectively. They were characterized using SEM, AFM, FTIR and XRD. The antibacterial activity of nHAp and nHAp-Zn was determined with Gram-negative and Gram-positive bacterial strains. The results indicated that nHAp alone was acting as an inert matrix and when substituted with Zn, it showed better antibacterial activity. The nHAp-Zn was found to be non-toxic to osteoprogenitor cells. Thus, due to the antimicrobial property of nHAp-Zn nanoparticles, we suggest that they would have potential applications towards bone tissue engineering.

Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes.

BACKGROUND: Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. METHODS: We have developed an energy based approach for identifying the binding site residues in protein-protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. RESULTS: We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. CONCLUSIONS: The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

Enhanced osteoblast adhesion on polymeric nano-scaffolds for bone tissue engineering.

Bone tissue engineering is an interdisciplinary field which is emerged for the development of viable substitutes that restore and maintain the function of human bone tissues. The success of bone tissue engineering depends on designing of the scaffolds. The polymer-based composite scaffolds containing micro- and nano-structures could provide a platform influencing osteoblastic cell adhesion, spreading, proliferation, and differentiation. Osteoblasts may adhere strongly to the nano-structures than micro-structures in the scaffolds due to the large surface area, better osteo-integrative property and mechanical reliability etc. In this review we are focusing the factors such as pore size, surface topography and roughness, protein adsorption and wettability of nano-structures and their interaction with cell surface integrins molecules. A better understanding of the interactions of nano-structures with osteoblastic cells will have potential applications in the regeneration of bone.

Chitosan and its derivatives for gene delivery.

Gene delivery can particularly be used for the treatment of diseases by the insertion of genetic materials (DNA and RNA) into mammalian cells either to express new proteins or to prevent the expression of existing proteins. Chitosan, a natural polymer is nontoxic, biocompatible, and biodegradable and it is used as a support material for gene delivery. However, practical use of chitosan has been mainly limited to its unmodified forms, and thus modified chitosans can be used for the wide range of biomedical applications including the interaction and intracellular delivery of genetic materials. In this context, this review paper provides the recent development on chitosan derivatives available for gene delivery.

Synthesis and characterization of nanoscale-hydroxyapatite-copper for antimicrobial activity towards bone tissue engineering applications.

The bacterial infection is one of the major problems associated with implant and reconstructive surgery of bone. Hence, the aim of this study was to develop biomaterials having antibacterial activity for bone tissue engineering. The hydroxyapatite nanoparticles (nHAp) improve the mechanical properties and incorporate nanotopographic features that mimic the nanostructure of natural bone. We report here for the first time the synthesis and characterization of nHAp and nHAp soaked with copper (nHAp-Cu) using SEM, AFM, FTIR and XRD. The antibacterial activity of nHAp and nHAp-Cu was determined using Gram-positive and Gram-negative bacterial strains. To have accelerated antibacterial activity, polyethylene glycol 400 (PEG 400), a synthetic biodegradable polymer was also added along with nHAp-Cu. The nHAp-Cu/PEG 400 had increased antibacterial activity towards Gram-positive than Gram-negative bacterial strains. The cytotoxicity of nHAp-Cu/PEG 400 was determined using MTT assay with rat primary osteoprogenitor cells and these biomaterials were found to be non-toxic. Hence, based on these results we suggest that the biomaterials containing nHAp-Cu/PEG 400 can be used as antibacterial materials in bone implant and bone regenerative medicine.

Variation of protein binding cavity volume and ligand volume in protein-ligand complexes.

We have systematically analyzed the variation of protein binding cavity volume of 200 protein-ligand complexes belonging to eight protein families. Wide variation in protein binding cavity volume for the same protein is observed on binding different ligands. Analysis of individual protein families shows high correlation between atom-atom interactions in binding site and ligand volume. This study implies the significance of protein flexibility in docking small molecule inhibitors on the basis of protein binding cavity volume with respect to ligand volume.

Arbitrarily primed PCR- a rapid and simple method for typing of leptospiral serovars.

PURPOSE: To investigate the use of arbitrarily primed polymerase chain reaction (AP-PCR) for typing of leptospiral serovars. METHODS: AP-PCR was adopted for identification of laboratory strains of leptospires and leptospiral cultures at serovar level. A primer of 12 bp was used for amplifying DNA of 13 laboratory strains of leptospires as well as culture pellets of leptospires. RESULTS: Each serovar produced distinct DNA fingerprint which was characteristic for each serovar. These patterns were used for typing of 81 serum culture samples obtained from human leptospiral cases. Of these samples, 39 could be typed based on AP-PCR fingerprints belonging to serovars autumnalis, pomona, canicola, javanica, icterohaemorrhagiae, patoc and pyrogenes. These results were confirmed by RAPD fingerprinting of the DNA samples of the respective leptospiral serovars after culturing -*them in EMJH media. One of the important findings of this work was that straight culture sample could be used for AP-PCR assay, without purification of DNA. By having more number of AP-PCR reference fingerprints, more serovars could be typed. CONCLUSIONS: AP-PCR technique provides great potential for simple and rapid identification of leptospires at serovar level, which could be useful in molecular epidemiological studies of leptospirosis.