Intracellular metabolomics and microRNAomics unveil new insight into the regulatory network for potential biocontrol mechanism of stress-tolerant Tricho-fusants interacting with phytopathogen Sclerotium rolfsii Sacc.

The present study employed microRNA (miRNA) sequencing and metabolome profiling of Trichoderma parental strains and fusants during normal growth and interaction with the phytopathogen Sclerotium rolfsii Sacc. In-vitro antagonism indicated that abiotic stress-tolerant Tricho-fusant FU21 was examined as a potent biocontroller with mycoparasitic action after 10 days. During interaction with the test pathogen, the most abundant uprising intracellular metabolite was recognized as l-proline, which corresponds to held-down l-alanine, associated with arginine and proline metabolism, biosynthesis of secondary metabolites, and nitrogen metabolism linked to predicted genes controlled by miRNAs viz., cel-miR-8210-3p, hsa-miR-3613-5p, and mml-miR-7174-3p. The miRNAs- mml-miR-320c and mmu-miR-6980-5p were found to be associated with phenylpropanoid biosynthesis, transcription factors, and signal transduction pathways, respectively, and were ascertained downregulated in potent FU21_IB compared with FU21_CB. The amino benzoate degradation and T cell receptor signaling pathways were regulated by miRNAs cel-miR-8210 and tca-miR-3824 as stress tolerance mechanisms of FU21. The intracellular metabolites l-proline, maleic acid, d-fructose, Myo-inositol, arabinitol, d-xylose, mannitol, and butane were significantly elevated as potential biocontrol and stress-tolerant constituents associated with miRNA regulatory pathways in potent FU21_IB. A network analysis between regulatory miRNA-predicted genes and intracellular metabolomics acknowledged possible biocontrol pathways/mechanisms in potent FU21_IB to restrain phytopathogen.

Characterization and bioefficacy of green nanosilver particles derived from fungicide-tolerant Tricho-fusant for efficient biocontrol of stem rot (Sclerotium rolfsii Sacc.) in groundnut (Arachis hypogaea L.).

An efficient and eco-friendly bioefficacy of potent Tricho-fusant (Fu21) and its green nanosilver formulation against stem rot (Sclerotium rolfsii) in groundnut was established. Fu21 demonstrated higher in-vitro growth inhibition of pathogen with better fungicide tolerance than the parental strains. The green nanosilver particles were synthesized from the extracellular metabolites of Fu21 and characterized for shape (spherical, 59.34 nm in scanning electron microscope), purity (3.00 KeV, energy dispersive X-ray analysis), size (54.3 nm in particle size analyzer), and stability (53.7 mv, zeta). The field efficacy study exhibited that the seedling emergence was high in seeds treated with green nanosilver (minimum inhibitory concentration-[MIC] 20 µg Ag/ml), and a low disease severity index of stem rot during the crop growth was followed by the live antagonist (Fu21) in addition to seed treatment with a fungicide mix under pathogen infestation. The seed quality analysis of harvested pods revealed a high oil content with balanced fatty acid composition (3.10 oleic/linoleic acid ratio) in green nanosilver treatment under pathogen infestation. The residual analysis suggested that green nanosilver applied at the MIC level as seed treatment yielded similar effects as the control for silver residue in the harvested groundnut seeds. The green nanosilver at MIC has a high pod-yield under S. rolfsii infestation, demonstrating green chemistry and sustainability of the nanoproduct.

Possible Association Between Nickel and Multiple Osteomas of the Mandible in a Gir Bullock.

A 10-year-old Gir bullock was presented with four contiguous hard nodular submucosal masses attached to the right rostral mandible. Overgrown masses were resected surgically and submitted for microstructural, elemental, and molecular spectroscopic analyses. An osteoma was diagnosed histopathologically. Elemental analysis by energy dispersive X-ray fluorescence spectroscopy revealed the presence of Ca, P, Sr, S, Zn, Cu, Ni, and Fe. Levels of the trace elements Fe, Zn, and Cu in the mandible mass were 2.39, 1.86, and 1.25 times higher, respectively, than those of normal bone. Nickel was detected in the mandible mass, but not in the normal bone. Molecular Fourier transform infrared spectroscopy confirmed the presence of inorganic ν2 CO32-, ν3 PO43-, and OH- in addition to organic collagen amide B, amide I, amide II, and amide III chemical functional groups. Multiple osteomas of the mandible in humans are a feature of Gardner syndrome and have not been recognized in animals so far. This could be the first report of multiple osteomas of the mandible in a Gir bullock associated with nickel-induced epigenetic mutation.

Microscopic and spectroscopic characterization of an extraskeletal intranasal osteoma in a Gir cow.

This is probably the first report characterizing an extraskeletal intranasal osteoma in a Gir cow through scanning electron microscopy and various spectroscopic techniques. Nasal obstruction in a 10-year-old Gir cow is investigated in this study. Skull radiograph demonstrated 174.12 mm × 81.97 mm sized well-circumscribed radiodense mass within the left nasal passage. The intranasal mass was excised completely through a rhinotomy incision. Grossly, intranasal mass was nonhyperemic, rock-hard, and calcified, 174.12 mm × 81.97 mm in size, and 650 g of weight. Excised intranasal mass was investigated through histopathologic, scanning electron microscopic (SEM), energy-dispersive X-ray (EDX) spectroscopic, X-ray fluorescence (XRF) spectroscopic, microwave plasma-atomic energy spectroscopic (MPAES), and Fourier-transform infrared (FTIR) spectroscopic techniques. A native bone of age-matched Gir cow, collected from a cadaver, was taken as a control. Microscopically, structures similar to cortical bone randomly coexisted with trabecular bone were observed. The EDX analysis of the intranasal mass indicated mean Ca/P weight ratio of 1.88, close to Ca/P weight ratio of the control. The XRF analysis revealed the presence of Ca, P, Sr, S, Zn, Cu, Fe, and Ni in the intranasal mass. Additionally, Mn was noted by MPAES analysis. Hence, the XRF and MPAES analyses confirmed a similar elemental composition of the intranasal mass and control. FTIR spectroscopic study confirmed the presence of inorganic ν1, ν3 PO4 3- , OH- in addition to organic collagen amide A, amide B, amide I, amide II, and amide III chemical functional groups in the intranasal mass. These findings of the intranasal mass were consistent with an osteoma having similar elemental and molecular compositions with the native bone.

Correction to: Identification of novel QTLs for late leaf spot resistance and validation of a major rust QTL in peanut (Arachis hypogaea L.).

[This corrects the article DOI: 10.1007/s13205-020-02446-4.].

Identification of novel QTLs for late leaf spot resistance and validation of a major rust QTL in peanut (Arachis hypogaea L.).

Co-occurrence of two devastating foliar-fungal diseases of peanut, viz., late leaf spot (LLS), and rust may cause heavy yield loss besides adversely affecting the quality of kernel and fodder. This study reports the mapping of seven novel stress-related candidate EST-SSRs in a region having major QTLs for LLS and rust diseases using an F2 mapping population (GJG17 × GPBD4) consisting of 328 individuals. The parental polymorphism using 1311 SSRs revealed 84 SSRs (6.4%) as polymorphic and of these 70 SSRs could be mapped on 14 linkage groups (LG). QTL analysis has identified a common QTL (LLSQTL1/RustQTL) for LLS and rust diseases in the map interval of 1.41 cM on A03 chromosome, explaining 47.45% and 70.52% phenotypic variations, respectively. Another major QTL for LLS (LLSQTL1), explaining a 29.06% phenotypic variation was also found on LG_A03. A major rust QTL has been validated which was found harboring R-gene and resistance-related genes having a role in inducing hypersensitive response (HR). Further, 23 linked SSRs including seven novel EST-SSRs were also validated in 177 diverse Indian groundnut genotypes. Twelve genotypes resistant to both LLS and rust were found carrying the common (rust and LLS) QTL region, LLS QTL region, and surrounding regions. These identified and validated candidate EST-SSR markers would be of great use for the peanut breeding groups working for the improvement of foliar-fungal disease resistance.

The impact of bacterial size on their survival in the presence of cationic particles of nano-silver.

BACKGROUND: Microbial surface area is one of the battlegrounds for invading microbes and host defense. Hence, infectious diseases caused by drug resistant microbes with large surface area are more difficult to treat than small size microbes. Nanobiology offers opportunities to re-explore the biological properties of conventional drugs at molecular level to combat these microbes. The purpose of the present study was to examine size depended susceptibility of Gram-positive bacteria towards nano-silver particles. METHODS: This study investigated the growth, surface charge, and morphology of emerging B. megaterium MTCC 7192 and re-emerging S. aureus MTCC 3160 cells in order to observe the susceptibility of these bacteria towards cationic nano-silver particles. Nano-silver particles were applied into wells formed on the Nutrient agar plates containing 108 CFU/mL of the bacteria. Surface potential of normal and treated cells was measured by Microtrac and the effects of nano-silver particles on bacterial cells were assessed by Scanning Electron Microscopy (SEM). RESULTS: In this work, synthesized nano-silver particles were found to be more effective against B. megaterium MTCC 7192 than S. aureus MTCC 3160. For B. megaterium MTCC 7192, a 0.30 fold increase in inhibition zone was observed after the addition of nano-silver particles in the wells. From our studies, it is reasonable to state that alternation of zeta potential may affect the cell morphology, which was further confirmed by SEM. CONCLUSION: The present study concluded that nano-silver particles appears to interact with a larger surface area more effectively.

Preparation, characterization, and xenotransplantation of the caprine acellular dermal matrix.

BACKGROUND: Caprine skin is a promising biomaterial for tissue-engineering applications. However, tissue processing is required before its xenogenic use. AIMS: Therefore, the purpose of this study was to evaluate the structural integrity and biocompatibility of the caprine skin after de-epithelialization, using sodium chloride (NaCl) and trypsin solutions, followed by de-cellularization using sodium dodecyl sulfate (SDS) solution. MATERIALS & METHODS: The caprine skin was de-epithelialized using NaCl (2-4 mol/L) and trypsin (0.25%-0.5%) followed by the treatment of SDS (1%-4%) solution over a period of time. Acellularity of the prepared matrix was confirmed histologically and characterized by appropriate staining, scanning electron microscopy (SEM), DNA quantification, and Fourier-transform infrared (FTIR) spectroscopy. The caprine acellular dermal matrix (CADM) was used for the repair of spontaneously occurring abdominal hernia in ten buffaloes. The biocompatibility of the CADM was evaluated using clinical, hematological, biochemical, and anti-oxidant parameters. RESULTS: Histologically, the skin treated with 0.25% trypsin in 4 mol/L NaCl for 8 hours resulted in complete de-epithelialization. Further treatment with 2% SDS for 48 hours demonstrated complete acellularity and orderly arranged collagen fibers. The SEM confirmed a preservation of collagen arrangement within CADM. The DNA content was significantly (P < .05) lower in CADM (46.20 ± 7.94 ng/mg) as compared to fresh skin (662.56 ± 156.11 ng/mg) indicating effective acellularity. The FTIR spectra showed characteristic collagen peaks of amide A, amide B, amide I, amide II, and amide III in CADM. All the 10 animals recovered uneventfully and remained sound. Hematological, biochemical, and anti-oxidants findings were unremarkable. CONCLUSION: Results indicated the acceptance and biocompatibility of the xenogenic caprine acellular dermal matrix for abdominal hernia repair in buffaloes without complications.

Bacterial membrane destabilization with cationic particles of nano-silver to combat efflux-mediated antibiotic resistance in Gram-negative bacteria.

AIMS: With the purpose of exploring combinatorial options that could enhance the bactericide efficacy of linezolid against Gram-negative bacteria, we assessed the extent of combination of nano-silver and linezolid. MAIN METHODS: In this study, we selected Escherichia coli MTCC 443 as a model to study the combinatorial effect of nano-silver and linezolid to combat efflux-mediated resistance in Gram-negative bacteria. The acting mechanism of nano-silver on E. coli MTCC 443 was investigated by evaluating interaction of nano-silver with bacterial membrane as well as bacterial surface charge, morphology, intracellular leakages and biological activities of membrane bound respiratory chain dehydrogenase and deoxyribonucleic acids (DNA) of the cells following treatment with nano-silver. KEY FINDINGS: The alternation of zeta potential due to the interaction of nano-silver towards bacterial membrane proteins was correlated with enhancement of membrane permeability, which allows the penetration of linezolid into the cells. In addition, the binding affinity of nano-silver towards bacterial membrane depressed biological activities of membrane bound respiratory chain dehydrogenases and DNA integrity. SIGNIFICANCE: Our findings suggested that nano-silver could not only obstruct the activities of efflux pumps, but also altered membrane integrity at the same time and thus increased the cytoplasmic concentration of the linezolid to the effective level.

Bactericidal assessment of nano-silver on emerging and re-emerging human pathogens.

With the threat of the growing number of bacteria resistant to antibiotics, the re-emergence of previously deadly infections and the emergence of new infections, there is an urgent need for novel therapeutic agent. Silver in the nano form, which is being used increasingly as antibacterial agents, may extend its antibacterial application to emerging and re-emerging multidrug-resistant pathogens, the main cause of nosocomial diseases worldwide. In the present study, a completely bottom up method to prepare green nano-silver was used. To explore the action of nano-silver on emerging Bacillus megaterium MTCC 7192 and re-emerging Pseudomonas aeruginosa MTCC 741 pathogenic bacteria, the study includes an analysis of the bacterial membrane damage through Scanning Electron Microscope (SEM) as well as alternation of zeta potential and intracellular leakages. In this work, we observed genuine bactericidal property of nano-silver as compare to broad spectrum antibiotics against emerging and re-emerging mode. After being exposed to nano-silver, the membrane becomes scattered from their original ordered arrangement based on SEM observation. Moreover, our results also suggested that alternation of zeta potential enhanced membrane permeability, and beyond a critical point, it leads to cell death. The leakages of intracellular constituents were confirmed by Gas Chromatography-Mass Spectrometry (GC-MS). In conclusion, the combine results suggested that at a specific dose, nano-silver may destroy the structure of bacterial membrane and depress its activity, which causes bacteria to die eventually.

Interruption in membrane permeability of drug-resistant Staphylococcus aureus with cationic particles of nano­silver.

The increasing drug-resistance pathogens among gram-positive bacterial species are becoming a major health concern nowadays. Over the past few years, the bactericidal efficacy of nano­silver against some drug-resistant gram-positive bacteria has been established, however further investigation is needed to determine whether nano­silver could be an option for the treatment of drug-resistant gram-positive microbial infections. The purpose of the present study was to determine the bactericidal efficacy of nano­silver with its membrane destroying property using drug-resistant Staphylococcus aureus MTCC 3160. In the present study, bactericidal assessment of nano­silver with different antibiotics was determined by agar well diffusion method. Interaction of nano­silver towards bacterial membrane was carried to understand the probable bactericidal actions of nano­silver, which was further confirmed by respiratory chain dehydrogenase, zeta potential, Scanning Electron Microscopy (SEM) and Gas Chromatography-Mass Spectrometry (GC-MS). The effect of nano­silver on bacterial Deoxyribonucleic Acids (DNA) was evaluated by agarose gel electrophoresis. Bactericidal assessment of nano­silver showed a very strong bactericidal action compare to antibiotics. The binding affinity of nano­silver towards bacterial membrane induced loss of catalytic activity for respiratory chain dehydrogenases. Zeta potential, SEM and GC-MS analysis also revealed extensive damage to the bacterial cell membrane. Moreover, the analysis of agarose gel electrophoresis revealed that nano­silver can enhance the decomposability of bacterial DNA, which was directly attached to the bacterial cell membrane. The present findings suggested that nano­silver directly interact with the bacterial cell surface without the need to penetrate; and this distinctive property raises the hope that nano­silver will remain an important bactericide in bacteria than antibiotics.

Antipathy of Trichoderma against Sclerotium rolfsii Sacc.: Evaluation of Cell Wall-Degrading Enzymatic Activities and Molecular Diversity Analysis of Antagonists.

The fungus Trichoderma is a teleomorph of the Hypocrea genus and associated with biological control of plant diseases. The microscopic, biochemical, and molecular characterization of Trichoderma was carried out and evaluated for in vitro antagonistic activity against the fungal pathogen Sclerotium rolfsii causing stem rot disease in groundnut. In total, 11 isolates of Trichoderma were examined for antagonism at 6 and 12 days after inoculation (DAI). Out of 11, T. virens NBAII Tvs12 evidenced the highest (87.91%) growth inhibition of the test pathogen followed by T. koningii MTCC 796 (67.03%), T. viride NBAII Tv23 (63.74%), and T. harzianum NBAII Th1 (60.44%). Strong mycoparasitism was observed in the best antagonist Tvs12 strain during 6-12 DAI. The specific activity of cell wall-degrading enzymes - chitinase and ß-1,3-glucanase - was positively correlated with growth inhibition of the test pathogen. In total, 18 simple sequence repeat (SSR) polymorphisms were reported to amplify 202 alleles across 11 Trichoderma isolates. The average polymorphism information content for SSR markers was found to be 0.80. The best antagonist Tvs 12 was identified with 7 unique SSR alleles amplified by 5 SSR markers. Clustering patterns of 11 Trichoderma strains showed the best antagonist T. virens NBAII Tvs 12 outgrouped with a minimum 3% similarity from the rest of Trichoderma.