Potential of biosynthesized silver nanoparticles using Stenotrophomonas sp. BHU-S7 (MTCC 5978) for management of soil-borne and foliar phytopathogens.
ABSTRACT: Stenotrophomonas sp. is emerging as a popular microbe of global concern with various potential ecological roles. Biosynthesis of gold and silver nanoparticles (AgNPs) using this bacterial strain has shown promising applications in life sciences. However, there is no report on efficient agricultural applications of biosynthesized AgNPs using Stenotrophomonas sp. In this regard, successful biosynthesis of AgNPs using Stenotrophomonas sp. BHU-S7 (MTCC 5978) was monitored by Uv-visible spectrum showing surface plasmon resonance (SPR) peak at 440 nm. The biosynthesized AgNPs were spherical with an average mean size of ~12 nm. The antifungal efficacy of biosynthesized AgNPs against foliar and soil-borne phytopathogens was observed. The inhibitory impact of AgNPs (2, 4, 10 μg/ml) on conidial germination was recorded under in vitro conditions. Interestingly, sclerotia of Sclerotium rolfsii exposed to AgNPs failed to germinate on PDA medium and in soil system. Moreover, AgNPs treatment successfully managed collar rot of chickpea caused by S. rolfsii under greenhouse conditions. The reduced sclerotia germination, phenolic acids induction, altered lignification and H2O2 production was observed to be the probable mechanisms providing protection to chickpea against S. rolfsii. Our data revealed that AgNPs treated plants are better equipped to cope with pathogen challenge pointing towards their robust applications in plant disease management.
Project description:G-protein alpha subunits are involved in transmission of signals for development, pathogenicity, and secondary metabolism in plant pathogenic and saprophytic fungi. We cloned two G-protein alpha subunit genes, tgaA and tgaB, from the biocontrol fungus Trichoderma virens. tgaA belongs to the fungal Galphai class, while tgaB belongs to the class defined by gna-2 of Neurospora crassa. We compared loss-of-function mutants of tgaA and tgaB with the wild type for radial growth, conidiation, germination of conidia, the ability to overgrow colonies of Rhizoctonia solani and Sclerotium rolfsii in confrontation assays, and the ability to colonize the sclerotia of these pathogens in soil. Both mutants grew as well as the wild type, sporulated normally, did not sporulate in the dark, and responded to blue light by forming a conidial ring. The tgaA mutants germinated by straight unbranched germ tubes, while tgaB mutants, like the wild type, germinated by wavy and highly branched germ tubes. In confrontation assays, both tgaA and tgaB mutants and the wild type overgrew, coiled, and lysed the mycelia of R. solani, but tgaA mutants had reduced ability to colonize S. rolfsii colonies. In the soil plate assay, both mutants parasitized the sclerotia of R. solani, but tgaA mutants were unable to parasitize the sclerotia of S. rolfsii. Thus, tgaA is involved in antagonism against S. rolfsii, but neither G protein subunit is involved in antagonism against R. solani. T. virens, which has a wide host range, thus employs a G-protein pathway in a host-specific manner.
Project description:Purpose:In this study, silver nanoparticles (AgNPs) were biosynthesized using culture supernatant of strain Shewanella sp. ARY1, characterized and their antibacterial activity was investigated against Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Methods:The strain Shewanella sp. ARY1 was isolated from river Yamuna, Delhi and used for biosynthesis of AgNPs via extracellular approach. Biosynthesized AgNPs were characterized by UV-Visible (UV-Vis) spectrophotometer, fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), energy dispersive X-ray (EDX), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Antibacterial activity of AgNPs was determined by well diffusion, broth microdilution and streaking plate assay to determine the zone of inhibition (ZOI), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), respectively. The effect of AgNPs on treated bacteria was investigated by electron microscopy analysis. Further, the biocompatibility of AgNPs was tested against mice erythrocytes (RBC) by hemolytic assay. Results:The UV-Vis spectral analysis revealed absorption maxima at 450 nm which confirmed the formation of AgNPs. The FTIR analysis suggested the involvement of various supernatant biomolecules, as reducing and capping agents in the synthesis of AgNPs. The XRD and EDX analysis confirmed the crystalline and metallic nature of AgNPs, respectively. The TEM and SEM analysis showed nanoparticles were spherical with an average size of 38 nm. The biosynthesized AgNPs inhibited the growth and formed a clear zone of inhibition (ZOI) against tested Gram-negative strains. The MIC and MBC were determined as 8-16 µg/mL and 32 µg/mL, respectively. Further, electron microscopy analysis of treated cells showed that AgNPs can damage the outer membrane, release of cytoplasmic contents, and alter the normal morphology of Gram-negative bacteria, leading to cell death. The hemolytic assay indicated that the biosynthesized AgNPs were biocompatible at low dose concentrations. Conclusion:This study demonstrates an eco-friendly process for extracellular synthesis of AgNPs using Shewanella sp. ARY1 and these AgNPs exhibited excellent antibacterial activity, which may be used to combat Gram-negative pathogens.
Project description:Silver nanoparticles (AgNPs) are known to have bacteriostatic and bactericidal effects. The present study highlights the extracellular synthesis of AgNPs and its antibacterial properties. The AgNPs were synthesized using Pseudomonas sp. THG-LS1.4 strain which had been isolated from soil. The AgNPs were characterized by field emission-transmission electron microscopy (FE-TEM), X-ray diffraction (XRD), Fourier transform-infrared (FT-IR) spectroscopy, and particle size distribution (DLS). The AgNPs displayed maximum absorbance at 412?nm and were irregular in shape ranging from 10 to 40?nm. The XRD spectroscopy results demonstrated the crystalline nature of nanoparticles. The AgNPs showed antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Candida tropicalis, Vibrio parahaemolyticus, Escherichia coli and Pseudomonas aeruginosa. Furthermore, the AgNPs were also evaluated for their increased antibacterial activities with various antibiotics against Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica. Additionally, AgNPs showd biofilm inhibition activity. The biosynthesized AgNPs were found to be a potent agent against tested pathogens. More importantly, we highlight the applications of AgNPs as an antimicrobial agent.
Project description:In this study, we characterized sporadically occurring sclerotium rot caused by Sclerotium rolfsii in Chinese chive (Allium tuberosum Roth.) in farm fields in Sacheon, Korea. The initial symptom of the disease was water-soaked, which progressed to rotting, wilting, blighting, and eventually death. Further, mycelial mats spread over the lesions near the soil line, and sclerotia formed on the scaly stem and leaves. The sclerotia were globoid, 1~3 mm, and white to brown. The optimum temperature for growth and sclerotia formation on potato dextrose agar (PDA) was 30℃. The diameter of the hypae ranged from 4 to 8 µm. Clamp connection was observed on PDA medium after 5 days of incubation. Based on the mycological characteristics, internal transcribed spacer sequence analysis, and pathogenicity test, the causal agent was identified as Sclerotium rolfsii Saccardo. This is the first report of sclerotium rot in Chinese chive caused by S. rolfsii in Korea.
Project description:Using gamma-ray-induced mutagenesis, we have developed a mutant (named G2) of Trichoderma virens that produced two- to three-fold excesses of secondary metabolites, including viridin, viridiol, and some yet-to-be identified compounds. Consequently, this mutant had improved antibiosis against the oomycete test pathogen Pythium aphanidermatum. A transcriptome analysis of the mutant vis-à-vis the wild-type strain showed upregulation of several secondary-metabolism-related genes. In addition, many genes predicted to be involved in mycoparasitism and plant interactions were also upregulated. We used tamarind seeds as a mass multiplication medium in solid-state fermentation and, using talcum powder as a carrier, developed a novel seed dressing formulation. A comparative evaluation of the wild type and the mutant in greenhouse under high disease pressure (using the test pathogen Sclerotium rolfsii) revealed superiority of the mutant over wild type in protecting chickpea (Cicer arietinum) seeds and seedlings from infection. We then undertook extensive field evaluation (replicated micro-plot trials, on-farm demonstration trials, and large-scale trials in farmers' fields) of our mutant-based formulation (named TrichoBARC) for management of collar rot (S. rolfsii) in chickpea and lentil (Lens culinaris) over multiple locations in India. In certain experiments, other available formulations were included for comparison. This formulation consistently, over multiple locations and years, improved seed germination, reduced seedling mortality, and improved plant growth and yield. We also noticed growth promotion, improved pod bearing, and early flowering (7-10 days) in TrichoBARC-treated chickpea and lentil plants under field conditions. In toxicological studies in animal models, this formulation exhibited no toxicity to mammals, birds, or fish.
Project description:An endophytic fungal strain isolated from the leaves of Gymnema sylvestre was identified as Pestalotiopsis microspora VJ1/VS1 based on nucleotide sequencing of internal transcribed spacer region (ITS 1-5.8S-ITS 2) of 18S rRNA gene (NCBI accession number KX213894). In this study, an efficient and ecofriendly approach has been reported for the synthesis of silver nanoparticles (AgNPs) using aqueous culture filtrate of P. microspora. Ultraviolet-visible analysis confirmed the synthesis of AgNPs by showing characteristic absorption peak at 435 nm. Fourier transform infrared spectroscopy analysis revealed the presence of phenolic compounds and proteins in the fungal filtrate, which are plausibly involved in the biosynthesis and capping of AgNPs. Transmission electron microscopy (TEM) showed that the AgNPs were spherical in shape of 2-10 nm in size. Selected area electron diffraction and X-ray diffraction studies determined the crystalline nature of AgNPs with face-centered cubic (FCC) lattice phase. Dynamic light scattering analysis showed that the biosynthesized AgNPs possess high negative zeta potential value of -35.7 mV. Biosynthesized AgNPs were proved to be potential antioxidants by showing effective radical scavenging activity against 2,2'-diphenyl-1-picrylhydrazyl and H2O2 radicals with IC50 values of 76.95±2.96 and 94.95±2.18 µg/mL, respectively. The biosynthesized AgNPs exhibited significant cytotoxic effects against B16F10 (mouse melanoma, IC50 =26.43±3.41 µg/mL), SKOV3 (human ovarian carcinoma, IC50 =16.24±2.48 µg/mL), A549 (human lung adenocarcinoma, IC50 =39.83±3.74 µg/mL), and PC3 (human prostate carcinoma, IC50 =27.71±2.89 µg/mL) cells. The biosynthesized AgNPs were found to be biocompatible toward normal cells (Chinese hamster ovary cell line, IC50 =438.53±4.2 µg/mL). Cytological observations on most susceptible SKOV3 cells revealed concentration-dependent apoptotic changes that include cell membrane blebbing, cell shrinkage, pyknotic nuclei, karyorrhexis followed by destructive fragmentation of nuclei. The results together in this study strongly provided a base for the development of potential and versatile biomedical applications of biosynthesized AgNPs in the near future.
Project description:Continuous formation and utilization of nanoparticles (NPs) have resulted into significant discharge of nanosized particles into the environment. NPs find applications in numerous products and agriculture sector, and gaining importance in recent years. In the present study, silver nanoparticles (AgNPs) were biosynthesized from silver nitrate (AgNO3) by green synthesis approach using Aloe vera extract. Mustard (Brassica sp.) seedlings were grown hydroponically and toxicity of both AgNP and AgNO3 (as ionic Ag+) was assessed at various concentrations (1 and 3 mM) by analyzing shoot and root length, fresh mass, protein content, photosynthetic pigments and performance, cell viability, oxidative damage, DNA degradation and enzyme activities. The results revealed that both AgNPs and AgNO3 declined growth of Brassica seedlings due to enhanced accumulation of AgNPs and AgNO3 that subsequently caused severe inhibition in photosynthesis. Further, the results showed that both AgNPs and AgNO3 induced oxidative stress as indicated by histochemical staining of superoxide radical and hydrogen peroxide that was manifested in terms of DNA degradation and cell death. Activities of antioxidants, i.e., ascorbate peroxidase (APX) and catalase (CAT) were inhibited by AgNPs and AgNO3. Interestingly, damaging impact of AgNPs was lesser than AgNO3 on Brassica seedlings which was due to lesser accumulation of AgNPs and better activities of APX and CAT, which resulted in lesser oxidative stress, DNA degradation and cell death. The results of the present study showed differential impact of AgNPs and AgNO3 on Brassica seedlings, their mode of action, and reasons for their differential impact. The results of the present study could be implied in toxicological research for designing strategies to reduce adverse impact of AgNPs and AgNO3 on crop plants.
Project description:Sclerotium rot was found on Cymbidium orchids at Seosan-si, Chungcheongnam-do, Korea, in July, 2010. Symptoms occurred on low leaves, which turned yellowish, after which the entire plant wilted. Severely infected plants were blighted and eventually died. White mycelial mats and sclerotia appeared on pseudobulbs. Based on the mycological characteristics and pathogenicity, the causal fungus was identified as Sclerotium rolfsii. This is the first report of new Sclerotium rot on Cymbidium spp. caused by S. rolfsii in Korea.
Project description:Dry root rot (DRR) caused by the fungus Rhizoctonia bataticola (Taub.) Butler, is an emerging disease in chickpea. The disease is often mistaken with other root rots like Fusarium wilt, collar rot and black root rot in chickpea. Therefore, its timely and specific detection is important. Current detection protocols are either based on mycological methods or on protocols involving DNA amplification by polymerase chain reaction (PCR). Here we report the rapid and specific detection of R. bataticola using loop-mediated isothermal amplification (LAMP) assay targeting fungal specific 5.8S rDNA sequence for visual detection of R. bataticola. The reaction was optimized at 63?°C for 75?min using minimum 10?fg of DNA. After adding SYBR Green I in LAMP products, the amplification was found to be highly specific in all the 94 isolates of R. bataticola collected from diverse geographical regions as well as DRR infected plants and sick soil. No reaction was found in other pathogenic fungi infecting chickpea (Fusarium oxysporum f. sp. ciceris, Rhizoctonia solani, Sclerotium rolfsii and Fusarium solani) and pigeonpea (Fusarium udum and Phytophthora cajani). The standardised LAMP assay with its simplicity, rapidity and specificity is very useful for the visual detection of this emerging disease in chickpea.
Project description:The biological approach to synthesize metal nanoparticles is an important aspect of current nanotechnology research. Silver nanoparticles have been well-known for their inhibitory and antimicrobial effects. The ever-increasing antibiotic resistance in pathogenic and opportunistic microorganisms is a major threat to the health care industry. In the present investigation, silver nanoparticles have been successfully biosynthesized by Streptomyces sp JAR1. Biosynthesized silver nanoparticles were characterized by means of several analytical techniques including a UV-Visible spectrophotometer, Fourier transform infrared spectroscopy, X-ray diffraction pattern analysis, and atomic force microscopy. An evaluation of the antimicrobial activity of silver nanoparticles (AgNPs) was carried out against clinically important pathogenic microorganisms. The metal nanoparticles were also evaluated for their combined effects with antibiotics against the clinical pathogens. The antibacterial activities of the antibiotics increased in the presence of the biologically synthesized AgNPs against the clinically important pathogens. The highest enhancing effect was observed for erythromycin against the test pathogens.