Cell-free Filtrates Research Articles

Extracellular biosynthesis and characterization of silver and zinc oxide nanoparticles using Pseudomonas aeruginosa and Escherichia coli

Abstract Background: The development of dependable and environmentally friendly nanomaterial resources is an essential aspect of recent nanotechnology research and application. Microorganisms have recently been investigated as a potential biofactor for the synthesis of many nanoparticles (NPs). Objectives: The aim of this article is to evaluate the extracellular biosynthesis capabilities of metallic NPs such as silver (Ag) and zinc oxide (ZnO) by screening common bacteria. Materials and Methods: Silver nitrate and zinc nitrate were used as a source of Ag and ZnO NPs, by means of Escherichia coli and Pseudomonas aeruginosa as potential candidates for the rapid synthesis of NPs. Ag and ZnO NPs were synthesized using a reduction of aqueous solutions with cell-free filtrates of bacteria. Characterization of synthesized NPs was conducted by UV–Vis spectroscopy. Results: Transmission electron microscopy (TEM) was used to examine the particle size of (Ag, ZnO) NPs. The maximum absorbance was around (425, 450 nm) for Ag and (300, 380 nm) for ZnO by E. coli and P. aeruginosa, respectively. We found that particle size of Ag NP obtained from P. aeruginosa was smaller than that of NP obtained from E. coli. The range of particle size was 24–32 nm with an average of 29.5 nm for Ag P. aeruginosa and was 25–37 nm with an average of 32 nm for Ag E. coli. The particle size range of ZnO of P. aeruginosa was 23–28 nm with an average of 25 nm and was 25–34 nm with an average of 29.7 nm for E. coli. Conclusion: E. coli and P. aeruginosa are good candidates for biosynthesis of NPs. As a result, obliged characteristics and green synthesis and the potential issue for various environmental and health-related applications may exist.

Dual extracellular mycofabrication of cobalt and zinc nano metal oxides mediated by mycelial-cell free filtrate of Aspergillus sojae: Characterization and assessment of antibacterial activity

Foodborne pathogenic bacteria cause a wide range of infectious diseases that pose a serious threat to human health worldwide with significant social and economic consequences. Nano metal oxides have attracted paramount attention owing to their remarkable antimicrobial potency. Microbial synthesis of metal oxide nanoparticles has gained prominence due to cost-effectiveness, biocompatibility, and environmental friendliness. This study reports extracellular mycosynthesis of two nano metal oxides mediated by Aspergillus sojae mycelial cell-free filtrate (MCFF). A. sojae MCFF was reacted with cobalt chloride hexahydrate and zinc nitrate hexahydrate for the preparation of Co3O4 NPs and ZnO NPs, respectively. After reacting with each metal salt, A. sojae MCFF changed from transparent to greyish-brown and white-milky suspensions, indicating the mycosynthesis of Co3O4 NPs and ZnO NPs, respectively. Physicochemical and morphological properties of the two nano metal oxides were determined using various characterization techniques, involving XRD, FTIR, EDX, DLS, zeta potential analysis, XPS, and electron microscopy. The two nano metal oxides exhibited antibacterial potency against three foodborne pathogens. Co3O4 NPs had zones of inhibition (ZOI) of 19.33 ± 0.58, 20.67 ± 0.58, and 22.67 ± 0.58 mm against Escherichia coli (ATCC 43889), Salmonella enterica Serovar Typhimurium (ATCC 43174), and Staphylococcus aureus (ATCC 12600), respectively. Whereas ZnO NPs had ZOI of 15.67 ± 0.58, 15.67 ± 0.58, and 14.33 ± 0.58 mm against the three foodborne pathogens, respectively. The intact morphological structures of the bacterial cells collapsed following exposure to A. sojae-mediated Co3O4 NPs and ZnO NPs, with some cells having irregular, twisted, and rough surfaces. The current study concluded that A. sojae mycelia acted as potential biological entity for the biosynthesis of Co3O4 NPs and ZnO NPs. A. sojae-derived Co3O4 NPs and ZnO NPs can be potential candidates for use as antimicrobial nano metal oxides in food and pharmaceutical industries.

Characterization of a Bacillus velezensis strain as a potential biocontrol agent against soft rot of eggplant fruits

Postharvest soft rot of eggplant fruits caused by Pectobacterium carotovorum is a bacterial disease with a high disease incidence and produces substantial economic losses. This study aimed to control postharvest soft rot of eggplant fruits by Bacillus velezensis and investigate the possible control mechanisms based on the effects of B. velezensis on P. carotovorum subsp. carotovorum (Pcc) and eggplant fruits, respectively. B. velezensis effectively controlled postharvest soft rot of eggplant fruits and directly inhibited Pcc growth in vitro. The volatile metabolites produced by B. velezensis showed no inhibition on Pcc. Whereas the cell-free filtrate of B. velezensis significantly inhibited the growth of Pcc in vitro and in vivo. Notably, methanol-soluble precipitates obtained from cell-free filtrate showed significant inhibition on Pcc, and the primary inhibitory substances were identified as surfactin isoforms. Besides, iturin and fengycin isoforms with much lower relative abundance were also detected in the methanol-soluble precipitates. Furthermore, B. velezensis enhanced the activities of reactive oxygen species (ROS) scavenging enzymes in eggplant fruits that alleviated ROS and oxidative damage; thereby, B. velezensis enhanced the fruits' disease resistance.

Inhibition of foodborne bacterial pathogens using novel mycosynthesized cobalt nano ferrites and CoFe2O4 nanoparticles anchored on functionalized multi-walled carbon nanotube bionanocomposites

Cobalt nanoferrites and their nanocomposites have attracted significant attention because of their numerous applications. This study reports a novel mycosynthesis of cobalt ferrite nanoparticles (CoFe2O4 NPs) and cobalt ferrite/functionalized multi-walled carbon nanotube (CoFe2O4/fMWCNT) bionanocomposites using the mycelial cell-free filtrate (MCFF) of Aspergillus sojae (KACC 45023). The reaction of A. sojae MCFF with ferric nitrate nonahydrate and cobalt chloride hexahydrate resulted in changing the colorless solution of the MCFF to reddish-brown color and formation of a reddish-brown precipitate, indicating the mycosynthesis of CoFe2O4 NPs. The mycosynthesized CoFe2O4 NPs were biologically anchored onto fMWCNTs using A. sojae MCFF, yielding CoFe2O4/fMWCNT bionanocomposites. Physicochemical and morphological properties of the mycosynthesized CoFe2O4 NPs and CoFe2O4/fMWCNT bionanocomposites were characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, dynamic light scattering, zeta potential analysis, X-ray photoelectron spectroscopy, energy-dispersive X-ray spectroscopy, field-emission scanning electron microscopy (FESEM), and high-resolution transmission electron microscopy. The mycosynthesized CoFe2O4 NPs and CoFe2O4/fMWCNT bionanocomposites showed significant antibacterial potential against three foodborne pathogenic bacteria, Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus using an agar-well diffusion assay. The morphological structures of the bacterial cells examined by FESEM revealed changes in the intact bacterial cell structures into misshapen and twisted bacterial cells, as well as deposition of the mycosynthesized CoFe2O4 NPs and CoFe2O4/fMWCNT bionanocomposites on the bacterial cells. The present study reveals the high potential of fungi as valuable bionanofactories for the mycosynthesis of ferrite-based NPs and their bionanocomposites as eco-friendly biosynthesis routes.

Multi-species endophytic Bacillus for improved control of potato soilborne and tuber-borne diseases in Tunisia: from laboratory to field conditions

BackgroundDue to the soilborne and tuber-borne nature of their causative agents, Fusarium wilt, Rhizoctonia root canker and black dot diseases are still leading to serious problems in potato production in Tunisia and worldwide. Among endophytic bacteria, the genus Bacillus is one of the most exploited microbial groups known as potent biocontrol agents against several potato diseases. In the present study, six strains belonging to five Bacillus species were screened for their abilities to inhibit root and wilt potato pathogens in vitro, in vivo and under natural conditions over three cropping seasons and to promote plant growth.ResultsBased on the dual-culture assays, the whole-cell suspensions of SV39 and SV104 (Bacillus tequilensis), SV41 (B. subtilis), SV44 (B. methylotrophicus) and SV65 (B. amyloliquefaciens subsp. plantarum) strains exhibited potent antifungal activity against important potato soilborne phytopathogens with ~ 65 to 70% inhibition rates. Significant inhibition rates were also induced by the cell-free culture filtrates, the butanolic and the chloroformic extracts depending on the target pathogens, the concentration used and the Bacillus strain tested. In pot experiment, a decrease in Rhizoctonia root canker severity, ranging from 43 to 65% compared to the inoculated and untreated control, was induced by all Bacillus spp. strain-based treatments, while SV39-, S42- and SV65-based treatments were the most effective in suppressing by 50–53 and 65–52%, black dot severity and the relative vascular discoloration extent induced by F. oxysporum f. sp. tuberosi, respectively, relative to positive control. This biocontrol potential was associated with an enhancement of potato growth parameters. Field studies indicated that soil treatment with the most of the Bacillus spp. strains had significantly controlled all the target fungal soilborne diseases and improved at the least two growth and/or production parameters depending on the strain used and the cropping seasons. SV39-, SV41-, SV44- and SV104-based treatments resulted in a significant increase in tuber yield in one cropping season.ConclusionThese Bacillus spp. strains could be used in combinations and/or introduced with other existing practices in order to provide supplemental control of target diseases and yield promotion under organic or conventional potato production systems.

Production of Copper Nanoparticles by Aspergillus niger and Evaluation of their Biomedical Applications

Nanotechnology is a well advanced area and different types of nanoparticles are now produced. The most revolutionary topic of the twenty-first century is nanotechnology. Recently, metallic nanoparticles (NPs) have gained a lot of attention. In this study a straightforward, safe, and economical method is used for synthesizing copper nanoparticles (CuNPs) by using cell free filtrate of Aspergillus niger. Fungi secretes enormous amount of enzymes which act as reducing as well stabilizing agents. Synthesized CuNPs were characterized by different techniques. Ultraviolet (UV)-visible spectrophotometer showed a characteristic peak at 380nm, Dynamic Light Scattering (DLS) analysis indicated that CuNPs have average size of 570nm with 0.480 PDI. SEM (Scanning Electron Microscopy) revealed CuNPs are of cubic shape. As per FTIR (Fourier Transform Infrared Analysis) two functional groups i.e., -O-H- and C=C- on the surface of CuNPs. Genetic analysis of Aspergillus niger revealed the presence of nitrate reductase enzyme that may involve in reduction of metal into NPs. Furthermore, CuNPs showed significant antibacterial action against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Klebsiella pneumoniae with 17mm, 20mm, 17mm and 20mm respectively. CuNPs also showed antioxidant potential with 59.41µg/ml.
 Fungi secretes valuable enzymes with inherent ability to reduce metal ions into NPs. This reduced the cost of NPs synthesis and also synthesized NPs exhibit greater biomedical applications.

Biological control potential of a soil fungus Trichoderma asperellum K1-02 against Neoscytalidium dimidiatum causing stem canker of dragon fruit

Stem canker on dragon fruit caused by the fungus Neoscytalidium dimidiatum negatively impact dragon fruit cultivation and production. The use of Trichoderma species, the biological control agent against stem canker disease is an alternative way instead of use of synthetic fungicides. This study aimed to test the antifungal activities of soil fungi Trichoderma against N. dimidiatum. Ten isolates of Trichoderma species were tested against N. dimidiatum via dual culture assay, and the results showed that percentages of inhibition ranged from 72.75 to 84.45%. Trichoderma species K1-02 was found to be the most effective strain against N. dimidiatum (84.45%). Trichoderma K1-02 was identified based on a morphology and molecular study of internal transcribed spacer (ITS), the second largest subunit of RNA polymerase II (rbp2), and the translation elongation factor 1-alpha (tef1-α) as Trichoderma asperellum K1-02. The pre-colonized plate method demonstrated that T. asperellum K1-02 covered the tested plates at a rate of 60%, which was significantly higher than N. dimidiatum (40%), and T. asperellum K1-02 aggressively covered the mycelia of N. dimidiatum as observed via stereomicroscopy. An enzyme assay was conducted to test the activities of cell wall-degrading enzymes (CWDEs) in cell-free culture filtrate (CF) released by T. asperellum K1-02, and the results showed that the activities of chitinase and β-1,3-glucanase were significantly higher than those of the control (potato dextrose broth alone). Scanning electron microscopy of N. dimidiatum mycelia incubated with cell free CF of T. asperellum K1-02 showed abnormal morphology. Furthermore, an in vivo test was conducted on dragon fruit cladodes to test the ability of Trichoderma with respect to the reduction of canker lesions. The results revealed that the application of 106 spore/mL T. asperellum K1-02 spore suspension suppressed disease development via the reduction of canker areas (0.09 cm2) on dragon fruit cladodes at a significantly higher level than those inoculated by pathogens alone (0.49 cm2). Thus, T. asperellum K1-02 demonstrated antifungal abilities against N. dimidiatum and has the potential to control stem canker on dragon fruit.

The Bactericidal Activity of a Novel Aneurinibacillus aneurinilyticus Isolate Effectively Controls Foodborne Pathogens Campylobacter jejuni and Listeria monocytogenes

Human zoonotic infection with Campylobacter is a major cause of gastroenteritis in the United States and worldwide. Listeria monocytogenes causes a potentially fatal infection in humans and is often attributed to contaminated food. Genetic typing has demonstrated that Campylobacter infection is often associated with the consumption of contaminated poultry products, with Campylobacter often colonizing the poultry gastrointestinal tract, while listeriosis is commonly associated with the consumption of contaminated ready-to-eat (RTE) foods. In this study, a strain of endospore-forming bacterium (strain NH) that is bactericidal towards the human food pathogens Campylobacter jejuni and Listeria monocytogenes was identified and characterized. Transwell experiments demonstrated that the bactericidal effect on both C. jejuni and L. monocytogenes is due to secretions from the spore former. These foodborne pathogens consistently exhibited 7 log reductions in growth when exposed to the NH cell-free culture filtrate. Sequencing of the 16s rRNA gene V4 variable region and analysis of the full-length 16s rRNA gene sequence from the WGS indicated that strain NH belongs to the species Aneurinibacillus aneurinilyticus. A microplate bioassay demonstrated that a bactericidal substance that is sensitive to protease could be collected from cell-free filtrates by salting out with ammonium sulfate. Gel filtration chromatography indicated a native molecular weight for the bactericidal protein of ca. 50 kDa, consistent with a class III bacteriocin. The active protein bound strongly to a cation-exchange resin and with an isoelectric point of ten, suggesting a positively charged protein. Both cation-exchange chromatography and isoelectric focusing indicated the enrichment of an 11 kDa protein on SDS-PAGE. This protein was identified through mass spectroscopy as the flgM protein, an anti-sigma factor. Analysis of whole genome sequencing (WGS) of the strain NH genome indicated the presence of a number of non-conservative amino acid substitutions in the flgM-gene-derived amino acid sequence of strain NH and A. aneurinilyticus compared to other members of the Aneurinibacillus genus. Further investigation is needed to determine whether these substitutions are correlated with the bactericidal activity. The identified strain may be useful as a feed additive for the pre-harvest control of Campylobacter jejuni in poultry.

Biological Control Mechanisms of Bacillus cabrialesii subsp. tritici TSO2T against Fusarium languescens, the Causal Agent of Wilt in Jalapeño Peppers

Jalapeño peppers (Capsicum annuum var. Jalapeño) represent one of the most important crops in Mexico. However, many plant diseases, such as wilt caused by strains of the genus Fusarium, reduce its yield. A sustainable alternative to control diseases is the use of biological control agents (BCAs), for example, beneficial microorganisms such as strains of the genus Bacillus. This study aims to analyze the potential use of B. cabrialesii subsp. tritici TSO2T as a BCA and elucidate its potential modes of action against Fusarium strains causing wilt in Jalapeño peppers. For this, symptomatic samples were collected in a commercial field in the Yaqui Valley, Mexico. Six Fusarium isolates were morphologically and molecularly characterized. After pathogenicity tests, F. languescens CE2 was found to be pathogenic. In screening assays for biocontrol bacteria, strain TSO2T, which was isolated from soil in a wheat commercial field under an organic production system and preserved in the Culture Collection of Native Soil and Endophytic Microorganisms (COLMENA), had the best biocontrol effect against CE2, and its cell-free filtrate reduced mycelial growth by 30.95%. Genome mining (antiSMASH) of strain TSO2T allows us to identify gene clusters associated with biocontrol, such as fengycin, surfactin, bacillibactin, bacilysin, bacillaene, subtilosin A, and sporulation killing factor, which codify to antimicrobial metabolites and are associated with swarming motility of the studied beneficial strain. These results demonstrate the effectiveness of B. cabrialesii subsp. tritici TSO2T as a potential BCA for the control of Fusarium wilt, through competition and a complex of antifungal metabolites, which still need to be validated through metabolomic analysis. This study highlights the importance of the use of integrative genomic and bioactivity-guided methodologies in understanding biocontrol pathosystems.

Harnessing antifungal metabolites from macro basidiomycetes against wilt inciting <i>Fusarium</i> spp.

Plant diseases especially wilt disease caused by Fusarium spp. pose a major threat to the cultivation of vegetables. In the present study, experiments were undertaken to explore the potential antifungal metabolites produced by macro basidiomycetes viz., Lentinus edodes, Ganoderma lucidum and Schizophyllum commune against Fusarium oxysporum and F. solani causing wilt disease of cucumber and capsicum. Among these, the ethyl acetate fraction of Cell-Free Culture Filtrate (CFC) of L. edodes exhibited maximum per cent inhibition of the mycelial growth of F. oxysporum and F. solani (61.11 and 57.77 %, respectively) at a concentration of 2000 ppm. Characterization of antifungal metabolites of Cell Free Condensate (CFC) of ethyl acetate fraction of L. edodes observed as prominent bands in Thin Layer Chromatography (TLC) indicated with an RF value of 0.25 and 0.69. Further GC-MS characterization of TLC-eluted compounds from L. edodes indicated the presence of 14 different compounds including 2H-pyran-2-one 6-pentyl-, possessing antifungal activity. The Fouriertransform Infrared Spectroscopy (FTIR) spectrum revealed the functional groups such as alcohol (O-H), amides (C-O), aliphatic polyes (CH2), triazenes (N=N), silicon compounds (Si-O-Si), amines (C-N) and phosphorus (P=S). The comparison of metabolite distribution patterns by Principal Component Analysis (PCA) obtained from L. edodes (PC 1) showed a positive correlation between the compounds. This study infers that L. edodes possess antifungal activity against F. oxysporum and F. solani that can be explored for formulation and application of these antifungal compounds in plant protection.

Effect of Metarhizium anisopliae (MetA1) on growth enhancement and antioxidative defense mechanism against Rhizoctonia root rot in okra

Rhizoctonia solani is an important necrotrophic pathogenic fungus that causes okra root disease and results in severe yield reduction. Many biocontrol agents are being studied with the intent of improving plant growth and defense systems and reducing crop loss by preventing fungal infections. Recently, a member of the Hypocrealean family, Metarhizium anisopliae, has been reported for insect pathogenicity, endophytism, plant growth promotion, and antifungal potentialities. This research investigated the role of M. anisopliae (MetA1) in growth promotion and root disease suppression in okra. The antagonism against R. solani and the plant growth promotion traits of MetA1 were tested in vitro. The effects of endophytic MetA1 on promoting plant growth and disease suppression were assessed in planta. Dual culture and cell-free culture filtrate assays showed antagonistic activity against R. solani by MetA1. Some plant growth promotion traits, such as phosphate solubilization and catalase activity were also exhibited by MetA1. Seed primed with MetA1 increased the shoot, root, leaves, chlorophyll content, and biomass content compared to control okra plants. The plants challenged with R. solani showed the highest hydrogen peroxide (H2O2) and lipid peroxidation (MDA) contents in the leaves of okra. Whereas MetA1 applied plants showed a reduction of H2O2 and MDA by 5.21 and 14.96%, respectively, under pathogen-inoculated conditions by increasing antioxidant enzyme activities, including catalase (CAT), peroxidase (POD), glutathione S-transferase (GST), and ascorbate peroxidase (APX), by 30.11, 10.19, 5.62, and 5.06%, respectively. Moreover, MetA1 increased soluble sugars, carbohydrates, proline, and secondary metabolites, viz., phenol and flavonoid contents in okra resulting in a better osmotic adjustment of diseases infecting plants. MetA1 reduced disease incidence by 58.33% at 15 DAI compared to the R. solani inoculated plant. The results revealed that MetA1 improved plant growth, elevated the plant defense system, and suppressed root diseases caused by R. solani. Thus, MetA1 was found to be an effective candidate for the biological control program.

The Extracellular Lipopeptides and Volatile Organic Compounds of Bacillus subtilis DHA41 Display Broad-Spectrum Antifungal Activity against Soil-Borne Phytopathogenic Fungi.

Fusarium oxysporum f. sp. niveum (Fon) is a devastating soil-borne fungus causing Fusarium wilt in watermelon. The present study investigated the biochemical mechanism underlying the antifungal activity exhibited by the antagonistic bacterial strain DHA41, particularly against Fon. Molecular characterization based on the 16S rRNA gene confirmed that DHA41 is a strain of Bacillus subtilis, capable of synthesizing antifungal lipopeptides, such as iturins and fengycins, which was further confirmed by detecting corresponding lipopeptide biosynthesis genes, namely ItuB, ItuD, and FenD. The cell-free culture filtrate and extracellular lipopeptide extract of B. subtilis DHA41 demonstrated significant inhibitory effects on the mycelial growth of Fon, Didymella bryoniae, Sclerotinia sclerotiorum, Fusarium graminearum, and Rhizoctonia solani. The lipopeptide extract showed emulsification activity and inhibited Fon mycelial growth by 86.4% at 100 µg/mL. Transmission electron microscope observations confirmed that the lipopeptide extract disrupted Fon cellular integrity. Furthermore, B. subtilis DHA41 emitted volatile organic compounds (VOCs) that exhibited antifungal activity against Fon, D. bryoniae, S. sclerotiorum, and F. graminearum. These findings provide evidence that B. subtilis DHA41 possesses broad-spectrum antifungal activity against different fungi pathogens, including Fon, through the production of extracellular lipopeptides and VOCs.

Isolation of bacteria with plant growth-promoting properties from microalgae-bacterial flocs produced in high-rate oxidation ponds

ABSTRACT Exploring plant growth-promoting (PGP) bacterial activity of microbial components aggregated by wastewater treatment can reduce dependence on fossil fuel-derived fertilisers. This study describes the isolation and identification of bacteria from microalgae-bacteria flocs (MaB-flocs) generated in high-rate algal oxidation ponds (HRAOP) of an integrated algal pond system (IAPS) remediating municipal wastewater. Amplified 16S rRNA gene sequence analysis determined the molecular identity of the individual strains. Genetic relatedness to known PGP rhizobacteria in the NCBI GenBank database was by metagenomics. Isolated strains were screened for the production of indoles (measured as indole-3-acetic acid; IAA) and an ability to mineralise NH 4 + , PO 4 3 − , and K + . Of the twelve bacterial strains isolated from HRAOP MaB-flocs, four produced indoles, nine mineralised NH 4 + , seven solubilised P, and one K. Potential of isolated strains for PGP activity according to one-way ANOVA on ranks was: ECCN 7b > ECCN 4b > ECCN 6b > ECCN 3b = ECCN 10b > ECCN 1b = ECCN 5b > ECCN 8b > ECCN 2b > ECCN 12b > ECCN 9b = ECCN 11b. Further study revealed that cell-free filtrate from indole-producing cultures of Aeromonas strain ECCN 4b, Enterobacter strain ECCN 7b, and Arthrobacter strain ECCN 6b promoted mung bean adventitious root formation suggestive of the presence of auxin-like biological activity.

An Eco-Friendly Approach Utilizing Green Synthesized Titanium Dioxide Nanoparticles for Leather Conservation against a Fungal Strain, Penicillium expansum AL1, Involved in the Biodeterioration of a Historical Manuscript.

The main hypothesis of the present research is investigating the efficacy of titanium oxide nanoparticles (TiO2-NPs) to prevent the growth of fungal strains when applied on leather under an experimental study. Therefore, fifteen fungal strains were isolated from a deteriorated historical manuscript (papers and leathers) and identified by traditional methods and ITS sequence analysis, including Aspergillus chevalieri (one isolate), A. nidulans (two strains), A. flavus (four strains), A. cristatus (one strain), A. niger (one strain), Paecilomyces fulvus (two strains), Penicillium expansum (two strains), and P. citrinum (two strains). The enzymes cellulase, amylase, pectinase, and gelatinase, which play a crucial role in biodegradation, were highly active in these fungal strains. TiO2-NPs were formed using the cell-free filtrate of the probiotic bacterial strain, Lactobacillus plantarum, and characterized. Data showed that the TiO2-NPs were successfully formed with a spherical shape and anatase phase with sizes of 2-8 nm. Moreover, the EDX analysis revealed that the Ti and O ions occupied the main component with weight percentages of 41.66 and 31.76%, respectively. The in vitro cytotoxicity of TiO2-NPs toward two normal cell lines, WI38 and HFB4, showed a low toxicity effect against normal cells (IC50 = 114.1 ± 8.1µg mL-1 for Wi38, and 237.5 ± 3.5µg mL-1 for HFB4). Therefore, concentrations of 100 μg mL-1 were used to load on prepared leather samples before inoculation with fungal strain P. expansum AL1. The experimental study revealed that the loaded TiO2-NPs have the efficacy to inhibit fungal growth with percentages of 73.2 ± 2.5%, 84.2 ± 1.8%, and 88.8 ± 0.6% after 7, 14, and 21 days, respectively. Also, the analyses including SEM, FTIR-ART, color change, and mechanical properties for leather inoculated with fungal strain AL1 in the absence of NPs showed high damage aspects compared to those inoculated with fungal strains in the presence of TiO2-NPs.

Assessment of nematicidal and plant growth-promoting effects of Burkholderia sp. JB-2 in root-knot nematode-infested soil.

Root-knot nematodes (RKN), Meloidogyne spp., are plant-parasitic nematodes that are responsible for considerable economic losses worldwide, because of the damage they cause to numerous plant species and the inadequate biological agents available to combat them. Therefore, developing novel and eco-friendly nematicides is necessary. In the present study, Burkholderia sp. JB-2, isolated from RKN-infested rhizosphere soil in South Korea, was evaluated to determine its nematicidal and plant growth-promoting effects under in vitro and in vivo conditions. Cell-free filtrates of the JB-2 strain showed high levels of nematicidal activity against second-stage juveniles (J2) of M. incognita, with 87.5% mortality following two days of treatment. In addition, the assessment of the activity against other six plant parasitic nematodes (M. javanica, M. hapla, M. arenaria, Ditylenchus destructor, Aphelenchoides subtenuis, and Heterodera trifolii) showed that the cell-free filtrates have a broad nematicidal spectrum. The three defense-responsive (MiMIF-2, MiDaf16-like1, and MiSkn1-like1) genes were activated, while Mi-cm-3 was downregulated when treated with cell-free filtrates of JB-2 cultures on J2. The greenhouse experiments suggested that the cell-free filtrates of the JB-2 strain efficiently controlled the nematode population in soil and egg mass formations of M. incognita in tomato (Solanum lycopersicum L., cv. Rutgers). An improvement in the host plant growth was observed, in which the shoot length and fresh weights of shoots and roots increased. The treatment with 10% of JB-2 cell-free filtrates significantly upregulated the expression levels of plant defenses (SlPR1, SlPR5, and SlPAL) and growth-promoting (ACO1, Exp18, and SlIAA1) genes compared with the corresponding parameters of the control group. Therefore, JB-2 could be a promising candidate for the sustainable management of RKN.

Photocatalytic Degradation, Anticancer, and Antibacterial Studies of Lysinibacillus sphaericus Biosynthesized Hybrid Metal/Semiconductor Nanocomposites.

The biological synthesis of nanocomposites has become cost-effective and environmentally friendly and can achieve sustainability with high efficiency. Recently, the biological synthesis of semiconductor and metal-doped semiconductor nanocomposites with enhanced photocatalytic degradation efficiency, anticancer, and antibacterial properties has attracted considerable attention. To this end, for the first time, we biosynthesized zinc oxide (ZnO) and silver/ZnO nanocomposites (Ag/ZnO NCs) as semiconductor and metal-doped semiconductor nanocomposites, respectively, using the cell-free filtrate (CFF) of the bacterium Lysinibacillus sphaericus. The biosynthesized ZnO and Ag/ZnO NCs were characterized by various techniques, such as ultraviolet-visible spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and photoluminescence spectroscopy. The photocatalytic degradation potential of these semiconductor NPs and metal-semiconductor NCs was evaluated against thiazine dye, methylene blue (MB) degradation, under simulated solar irradiation. Ag/ZnO showed 90.4 ± 0.46% photocatalytic degradation of MB, compared to 38.18 ± 0.15% by ZnO in 120 min. The cytotoxicity of ZnO and Ag/ZnO on human cervical HeLa cancer cells was determined using an MTT assay. Both nanomaterials exhibited cytotoxicity in a concentration- and time-dependent manner on HeLa cells. The antibacterial activity was also determined against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus). Compared to ZnO, Ag/ZnO NCs showed higher antibacterial activity. Hence, the biosynthesis of semiconductor nanoparticles could be a promising strategy for developing hybrid metal/semiconductor nanomaterials for different biomedical and environmental applications.

Mycosynthesized magnetic iron-oxide nanoparticles for the remediation of heavy metals – An insight into the mechanism of adsorption, process optimization using algorithmic approach and its application for the treatment of groundwater

In the present study, mycosynthesized magnetic iron-oxide (γ-Fe2O3) nanoparticles (IO-NPs) were synthesized using manglicolous fungi isolated from the sediment samples of Indian Sundarbans. Out of the 15 strains isolated from the sediment samples, cell-free filtrate of Corynespora cassiicola – CNMP1 reduced the Fe(NO3)3 solution leading to the formation of IO-NPs (100 nm). It was observed that the fungal extracellular biomaterial embedding the IO-NPs provided stability to the IO-NPs in the aqueous suspension. The mycosynthesized IO-NPs were employed to adsorb heavy metals, viz. Pb, Ni, Cu, Zn, Cd, and Cr from water. Studies on adsorption isotherms delineated the energetically heterogeneous nature of IO-NPs. Batch adsorption studies showed that 1 g/L of IO-NPs at 313 K temperature and pH 6 is able to remove more than 90% of Pb, Ni, Cu, and Zn ions from the 10 mg/L solution. However, more than 90 % removal efficacy was obtained for Cr with a pH of 2, keeping the other adsorption parameters the same. Thermodynamic studies laid bare the spontaneity and endothermic nature of the adsorption process for each studied heavy metal. A multi-metric approach involving response surface methodology (RSM), and artificial neural network (ANN) analysis was used to validate the adsorption of heavy metals by IO-NPs, and it was observed that 1 g/L of IO-NPs at 313 K temperature and pH 6.1 can adsorb 92.86% of Pb, 94.93% of Ni, 95.92% of Cu, 98.11% of Zn, 98.91% of Cd and 82.12 % of Cr from water with an initial metallic concentration of 11.96 mg/L. Further, with the optimized IO-NP dose and temperature, the IO-NPs were able to remove 94 % of Cr from Cr-rich groundwater (12.68 mg/L) collected from a region around chromite ore processing residues (COPR) dumping. The magnetic nature of the IO-NPs eased their separation from the treated water.

Two Bacterial Bioagents Boost Onion Response to Stromatinia cepivora and Promote Growth and Yield via Enhancing the Antioxidant Defense System and Auxin Production

White rot, caused by Stromatinia cepivora (Anamorph: Sclerotium cepivorum Berk), is a serious soil-borne disease of the onion that restricts its cultivation and production worldwide. Herein, we isolated and characterized a plant growth-promoting rhizobacterium Stenotrophomonas maltophilia from healthy onion roots and an endophytic bacterium Serratia liquefaciens from healthy bean leaves. Both isolates showed strong fungistatic activity against S. cepivora using the dual culture and culture filtrate methods. This effect might be due to the presence of several volatile compounds, especially menthol in both culture filtrates as shown with a GC-MS analysis. Additionally, the root drench application of cell-free culture filtrates of S. maltophilia and S. liquefaciens significantly reduced the incidence and severity of white rot disease on treated onion plants, which was associated with the activation of both enzymatic (POX and PPO) and non-enzymatic (phenolics and flavonoids) antioxidant defense machineries of S. cepivora-infected onion plants. Moreover, the culture filtrates of both bacterial bioagents remarkably enhanced the growth (as expressed by root length, plant height, and number of leaves) and yield parameters (as indicated by bulb circumference, fresh weight of the bulb, and bulb yield per plot) of treated onion plants under field conditions during two successive seasons (2020/2021 and 2021/2022). This might be because of a reduced disease severity and/or the accumulation of the main auxin, indole-3-acetic acid (IAA), and its precursor, the amino acid tryptophan. Our findings suggest that both bioagents might be utilized as eco-friendly alternative control measures to reduce the utilization of chemical fungicides entirely or partially for the safer production of onion in S. cepivora-infested soils.

Identification and characterization of Brevibacillus halotolerans B-4359: a potential antagonistic bacterium against red pepper anthracnose in Korea.

Our study aimed to identify potential biocontrol agents (BCAs) against major phytopathogens under in vitro conditions by screening the Freshwater Bioresources Culture Collection (FBCC), Korea. Of the identified 856 strains, only 65 exhibited antagonistic activity, among which only one representative isolation, Brevibacillus halotolerans B-4359 was selected based on its in vitro antagonistic activity and enzyme production. Cell-free culture filtrate (CF) and volatile organic compounds (VOCs) of B-4359 were shown to be effective against the mycelial growth of Colletotrichum acutatum. Notably, B-4359 was found to promote spore germination in C. acutatum instead of exhibiting a suppressive effect when the bacterial suspension was mixed with the spore suspension of C. acutatum. However, B-4359 showed an excellent biological control effect on the anthracnose of red pepper fruits. Compared to other treatments and untreated control, B-4359 played a more effective role in controlling anthracnose disease under field conditions. The strain was identified as B. halotolerans using BIOLOG and 16S rDNA sequencing analyses. The genetic mechanism underlying the biocontrol traits of B-4359 was characterized using the whole-genome sequence of B-4359, which was closely compared with related strains. The whole-genome sequence of B-4359 consisted of 5,761,776 bp with a GC content of 41.0%, including 5,118 coding sequences, 117 tRNA, and 36 rRNA genes. The genomic analysis identified 23 putative secondary metabolite biosynthetic gene clusters. Our results provide a deep understanding of B-4359 as an effective biocontrol agent against red pepper anthracnose for sustainable agriculture.

Mycosynthesis of silver nanoparticles using psychrotrophic strains of Tulasnella albida Bourdot & Galzin from the South Orkney Islands (Antarctica)

This study is the first report on mycosynthesis of silver nanoparticles (NPs) using psychrotrophic Antarctic filamentous fungi, and the first report regarding Tulasnella (Basidiomycota). In this work, the ability to synthesize silver NPs from cell free filtrates of strains of Tulasnella albida isolated from Antarctica was assessed. All fungal filtrates were capable of synthesizing silver NPs with the addition of AgNO3. UV–vis spectroscopy, TEM and SEM microscopy analyses were performed to characterize the synthesized NPs. ATR-FTIR and Micro Raman spectroscopy analyses were conducted to find functional groups responsible for the reduction of AgNO3 and to detect the presence of silver oxide on the AgNPs. Theoretical calculations of optical absorption based on core-shell Ag–Ag2O were used to characterize the experimental absorption spectra of silver NPs colloids. Spherically shaped silver NPs, typically 2–3nm in diameter, were obtained. The largest ones showed a capping shell around them, which could be associated with the formation of small silver NPs. Functional groups corresponding to amides and alcohols were detected, confirming the presence of proteins as possible intermediates in the synthesis of AgNPs. On the other hand, the Micro Raman analysis confirms the presence of silver oxide on the surface of the AgNPs. This work presents a simple procedure for the synthesis of silver NPs using a psychrotrophic organism that could be interesting for the industry.