Genome-Wide Characterization of Effector Protein-Encoding Genes in Sclerospora graminicola and Its Validation in Response to Pearl Millet Downy Mildew Disease Stress.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is the essential food crop for over ninety million people living in drier parts of India and South Africa. Pearl millet crop production is harshly hindered by numerous biotic stresses. Sclerospora graminicola causes downy mildew disease in pearl millet. Effectors are the proteins secreted by several fungi and bacteria that manipulate the host cell structure and function. This current study aims to identify genes encoding effector proteins from the S. graminicola genome and validate them through molecular techniques. In silico analyses were employed for candidate effector prediction. A total of 845 secretory transmembrane proteins were predicted, out of which 35 proteins carrying LxLFLAK (Leucine-any amino acid-Phenylalanine-Leucine-Alanine-Lysine) motif were crinkler, 52 RxLR (Arginine, any amino acid, Leucine, Arginine), and 17 RxLR-dEER putative effector proteins. Gene validation analysis of 17 RxLR-dEER effector protein-producing genes was carried out, of which 5genes were amplified on the gel. These novel gene sequences were submitted to NCBI. This study is the first report on the identification and characterization of effector genes in Sclerospora graminicola. This dataset will aid in the integration of effector classes that act independently, paving the way to investigate how pearl millet responds to effector protein interactions. These results will assist in identifying functional effector proteins involving the omic approach using newer bioinformatics tools to protect pearl millet plants against downy mildew stress. Considered together, the identified effector protein-encoding functional genes can be utilized in screening oomycetes downy mildew diseases in other crops across the globe.

Chitosan and chitosan-derived nanoparticles modulate enhanced immune response in tomato against bacterial wilt disease.

The present study evaluated the priming efficacy of chitosan and chitosan-derived nanoparticles (CNPs) against bacterial wilt of tomato. In the current study, seed-treated CNPs plus pathogen-inoculated tomato seedlings recorded significant protection of 62 % against pathogen-induced wilt disease and subsequently better growth. The induced resistance was witnessed by a prominent increase in lignin, callose and H2O2 deposition, followed by superoxide radical accumulation in leaves. Additionally, chitosan and CNPs-treated tomato plants recorded a remarkable increase in the upregulation of phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO), catalase (CAT) and ß-1, 3 glucanase (GLU) in comparison with untreated plants. The chitosan and CNPs-induced antioxidant enzymes were positively correlated with the stimulation of corresponding gene expression in CNPs treated plants related to pathogen-inoculated ones. The results of this study describe that how the application of chitosan and CNPs elicit defense responses at the cellular, biochemical and gene expression in tomato plants against bacterial wilt disease, thereby improve growth and yield.

Ameliorated Antibacterial and Antioxidant Properties by Trichoderma harzianum Mediated Green Synthesis of Silver Nanoparticles.

Biosynthesis of silver nanoparticles using beneficial Trichoderma harzianum is a simple, eco-friendly and cost-effective route. Secondary metabolites secreted by T. harzianum act as capping and reducing agents that can offer constancy and can contribute to biological activity. The present study aimed to synthesize silver nanoparticles using T. harzianum cell filtrate and investigate different bioactive metabolites based on LC-MS/MS analysis. The synthesized silver nanoparticles (AgNPs) from T. harzianum were characterized by ultraviolet-visible spectrophotometry, Fourier transform infrared spectrometry (FT-IR), energy-dispersive spectroscopy (EDS), dynamic light scattering (DLS), X-ray powder diffraction (XRD) and scanning electron microscopy (SEM). The surface plasmon resonance of synthesized particles formed a peak centered near 438 nm. The DLS study determined the average size of AgNPs to be 21.49 nm. The average size of AgNPs was measured to be 72 nm by SEM. The cubic crystal structure from XRD analysis confirmed the synthesized particles as silver nanoparticles. The AgNPs exhibited remarkable antioxidant properties, as determined by DPPH and ferric reducing antioxidant power (FRAP) assay. The AgNPs also exhibited broad-spectrum antibacterial activity against two Gram-positive bacteria (S. aureus and B. subtilis) and two Gram-negative bacteria (E. coli and R. solanacearum). The minimum inhibitory concentration (MIC) of AgNPs towards bacterial growth was evaluated. The antibacterial activity of AgNPs was further confirmed by fluorescence microscopy and SEM analysis.

Bioactive compounds guided diversity of endophytic fungi from Baliospermum montanum and their potential extracellular enzymes.

Baliospermum montanum (Willd.) Muell. Arg, a medicinal plant distributed throughout India from Kashmir to peninsular-Indian region is extensively used to treat jaundice, asthma, and constipation. In the current study, 203 endophytic fungi representing twenty-nine species were isolated from tissues of B. montanum. The colonization and isolation rate of endophytes were higher in stem followed by seed, root, leaf and flower. The phytochemical analysis revealed 70% endophytic isolates showed alkaloids and flavonoids, 13% were positive for phenols, saponins and terpenoids. Further, these endophytes produced remarkable extracellular enzymes such as amylase, cellulase, phosphates, protease and lipase. The most promisive three endophytic fungi were identified by ITS region and secreted metabolites were identified by gas chromatography-mass spectrometry (GC-MS/MS). The GC-MS profile detected twenty-five bioactive compounds from ethyl acetate extracts. Among endophytic fungi, Trichoderma reesei isolated from flower exhibited nine bioactive compounds namely, 2-Cyclopentenone, 2-(4-chloroanilino)-4-piperidino, Oxime-methoxy-Phenyl, Methanamine N-hydroxy-N-methyl, Strychane, Cyclotetrasiloxane, Octamethyl and 1-Acetyl-20a-hydroxy-16-methylene. The endophyte, Aspergillus brasiliensis isolated from root and Fusarium oxysporum isolated from seed produced nine and seven bioactive compounds, respectively. Overall, a significant contribution of bioactive compounds was noticed from the diverse endophytic fungi associated with B. montanum and could be explored for development of novel drug with commercial values.

GC-MS analysis of phytoconstituents from Amomum nilgiricum and molecular docking interactions of bioactive serverogenin acetate with target proteins.

Amomum nilgiricum is one of the plant species reported from Western Ghats of India, belonging to the family Zingiberaceae, with ethno-botanical values, and is well-known for their ethno medicinal applications. In the present investigation, ethyl acetate and methanol extracts of A. nilgiricum were analyzed by Fourier transform infrared spectrometer (FTIR) and gas chromatography-mass spectrometry (GC-MS) to identify the important functional groups and phytochemical constituents. The FTIR spectra revealed the occurrence of functional characteristic peaks of aromatic amines, carboxylic acids, ketones, phenols and alkyl halides group from leaf and rhizome extracts. The GC-MS analysis of ethyl acetate and methanol extracts from leaves, and methanol extract from rhizomes of A. nilgiricum detected the presence of 25 phytochemical compounds. Further, the leaf and rhizome extracts of A. nilgiricum showed remarkable antibacterial and antifungal activities at 100 mg/mL. The results of DPPH and ferric reducing antioxidant power assay recorded maximum antioxidant activity in A. nilgiricum methanolic leaf extract. While, ethyl acetate leaf extract exhibited maximum α-amylase inhibition activity, followed by methanolic leaf extract exhibiting aldose reductase inhibition. Subsequently, these 25 identified compounds were analyzed for their bioactivity through in silico molecular docking studies. Results revealed that among the phytochemical compounds identified, serverogenin acetate might have maximum antibacterial, antifungal, antiviral, antioxidant and antidiabetic properties followed by 2,4-dimethyl-1,3-dioxane and (1,3-13C2)propanedioic acid. To our best knowledge, this is the first description on the phytochemical constituents of the leaves and rhizomes of A. nilgiricum, which show pharmacological significance, as there has been no literature available yet on GC-MS and phytochemical studies of this plant species. The in silico molecular docking of serverogenin acetate was also performed to confirm its broad spectrum activities based on the binding interactions with the antibacterial, antifungal, antiviral, antioxidant and antidiabetic target proteins. The results of the present study will create a way for the invention of herbal medicines for several ailments by using A. nilgiricum plants, which may lead to the development of novel drugs.

Detection and Characterization of Antibacterial Siderophores Secreted by Endophytic Fungi from Cymbidium aloifolium.

Endophytic fungi from orchid plants are reported to secrete secondary metabolites which include bioactive antimicrobial siderophores. In this study endophytic fungi capable of secreting siderophores were isolated from Cymbidium aloifolium, a medicinal orchid plant. The isolated extracellular siderophores from orchidaceous fungi act as chelating agents forming soluble complexes with Fe3+. The 60% endophytic fungi of Cymbidium aloifolium produced hydroxamate siderophore on CAS agar. The highest siderophore percentage was 57% in Penicillium chrysogenum (CAL1), 49% in Aspergillus sydowii (CAR12), 46% in Aspergillus terreus (CAR14) by CAS liquid assay. The optimum culture parameters for siderophore production were 30 °C, pH 6.5, maltose and ammonium nitrate and the highest resulting siderophore content was 73% in P. chrysogenum. The total protein content of solvent-purified siderophore increased four-fold compared with crude filtrate. The percent Fe3+ scavenged was detected by atomic absorption spectra analysis and the highest scavenging value was 83% by P. chrysogenum. Thin layer chromatography of purified P. chrysogenum siderophore showed a wine-colored spot with Rf value of 0.54. HPLC peaks with Rts of 10.5 and 12.5 min were obtained for iron-free and iron-bound P. chrysogenum siderophore, respectively. The iron-free P. chrysogenum siderophore revealed an exact mass-to-charge ratio (m/z) of 400.46 and iron-bound P. chrysogenum siderophore revealed a m/z of 453.35. The solvent-extracted siderophores inhibited the virulent plant pathogens Ralstonia solanacearum, that causes bacterial wilt in groundnut and Xanthomonas oryzae pv. oryzae which causes bacterial blight disease in rice. Thus, bioactive siderophore-producing endophytic P. chrysogenum can be exploited in the form of formulations for development of resistance against other phytopathogens in crop plants.

Exogenous priming of chitosan induces upregulation of phytohormones and resistance against cucumber powdery mildew disease is correlated with localized biosynthesis of defense enzymes.

In recent years, exploration of biopolymer-based materials to avoid hazardous chemicals in agriculture has gained enormous importance for sustainable crop improvement. In the present study, chitosan a biopolymer derived from crab-shell was used in different concentrations as priming agent to cucumber seeds and were evaluated for its effect to enhance plant growth parameters as well as its ability to induce resistance against powdery mildew disease. Among the treatments, seeds-primed with 2.5 mg/mL exhibited early seedling germination of 90% and vigour of 2665 and also remarkably enhanced the cucumber growth parameters which might be fairly attributed to the stimulation of phytohormones content in primed plants over the controls. More importantly, under greenhouse conditions a significant induced disease protection of 66.6% against powdery mildew disease was noticed in chitosan-pretreated plants at 2.5 mg/mL. The induced resistant plants also showed a significant deposition of lignin, callose and H2O2. Notably, polyphenol oxidase, phenylalanine ammonia-lyase, peroxidase and glucanase defense-responsive enzymes were upregulated in chitosan-primed plants. Considered together, these results determine that the susceptible cucumber cultivar elicits immunity after perception of priming with chitosan to upregulate phytohormones and synthesize defense-responsive enzymes, thereby induce resistance against powdery mildew disease and strengthen the growth-promotion of cucumber plants.

Biofabrication of zinc oxide nanoparticles from Melia azedarach and its potential in controlling soybean seed-borne phytopathogenic fungi.

Present study, report the biofabrication of zinc oxide nanoparticles from aqueous leaf extract of Melia azedarach (MaZnO-NPs) through solution combustion method and their novel application in preventing the growth of seed-borne fungal pathogens of soybean (Cladosporium cladosporioides and Fusarium oxysporum). The standard blotter method was employed to isolate fungi and was identified through molecular techniques. The characterization of MaZnO-NPs was carried out by UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FT-IR), X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM) equipped with Energy Dispersive Spectroscopy (EDS) and Transmission Electron Microscopy (TEM). The physicochemical characterization confirmed the particles were of high purity and nano size (30-40 nm) with a hexagonal shape. The synthesized MaZnO-NPs inhibited the growth of C. cladosporioides and F. oxysporum in a dose dependent manner. Biomass, ergosterol, lipid peroxidation, intracellular reactive oxygen species and membrane integrity determination upon MaZnO-NPs treatment offered significant activities there by confirming the mechanism of action against the test pathogens. In conclusion, due to the effectiveness of MaZnO-NPs in controlling the growth of C. cladosporioides and F. oxysporum, the synthesized MaZnO-NPs provides insight towards their potential application in agriculture and food industries.

Green Synthesis and Characterization of Zinc Oxide Nanoparticles Using Eucalyptus globules and Their Fungicidal Ability Against Pathogenic Fungi of Apple Orchards.

Eucalyptus globules belonging to the Myrtaceae family was explored for the synthesis of zinc oxide nanoparticles and for biological applications. The aqueous extract of the synthesized zinc nanoparticles (ZnNPs) was characterized using UV-visible spectrophotometer, FTIR, SEM and TEM. The aqueous broth was observed to be an efficient reducing agent, leading to the rapid formation of ZnNPs of varied shapes with sizes ranging between 52-70 nm. In addition, antifungal activity of the biosynthesized ZnNPs was evaluated against major phytopathogens of apple orchards. At 100 ppm of ZnNPs, the fungal growth inhibition rate was found to be 76.7% for Alternaria mali, followed by 65.4 and 55.2% inhibition rate for Botryosphaeria dothidea and Diplodia seriata, respectively. The microscopic observations of the treated fungal plates revealed that ZnNPs damages the topography of the fungal hyphal layers leading to a reduced contraction of hyphae. This considerable fungicidal property of ZnNPs against phytopathogenic fungi can have a tremendous impact on exploitation of ZnNPs for fungal pest management and ensure protection in fruit crops.