Metabolomics of early blight (Alternaria solani) susceptible tomato (Solanum lycopersicum) unfolds key biomarker metabolites and involved metabolic pathways.

Tomato (Solanum lycopersicum) is among the most important commercial horticultural crops worldwide. The crop quality and production is largely hampered due to the fungal pathogen Alternaria solani causing necrotrophic foliage early blight disease. Crop plants usually respond to the biotic challenges with altered metabolic composition and physiological perturbations. We have deciphered altered metabolite composition, modulated metabolic pathways and identified metabolite biomarkers in A. solani-challenged susceptible tomato variety Kashi Aman using Liquid Chromatography-Mass Spectrometry (LC-MS) based metabolomics. Alteration in the metabolite feature composition of pathogen-challenged (m/z 9405) and non-challenged (m/z 9667) plant leaves including 8487 infection-exclusive and 8742 non-infection exclusive features was observed. Functional annotation revealed putatively annotated metabolites and pathway mapping indicated their enrichment in metabolic pathways, biosynthesis of secondary metabolites, ubiquinone and terpenoid-quinones, brassinosteroids, steroids, terpenoids, phenylpropanoids, carotenoids, oxy/sphingolipids and metabolism of biotin and porphyrin. PCA, multivariate PLS-DA and OPLS-DA analysis showed sample discrimination. Significantly up regulated 481 and down regulated 548 metabolite features were identified based on the fold change (threshold ≥ 2.0). OPLS-DA model based on variable importance in projection (VIP scores) and FC threshold (> 2.0) revealed 41 up regulated discriminant metabolite features annotated as sphingosine, fecosterol, melatonin, serotonin, glucose 6-phosphate, zeatin, dihydrozeatin and zeatin-ß-D-glucoside. Similarly, 23 down regulated discriminant metabolites included histidinol, 4-aminobutyraldehyde, propanoate, tyramine and linalool. Melatonin and serotonin in the leaves were the two indoleamines being reported for the first time in tomato in response to the early blight pathogen. Receiver operating characteristic (ROC)-based biomarker analysis identified apigenin-7-glucoside, uridine, adenosyl-homocysteine, cGMP, tyrosine, pantothenic acid, riboflavin (as up regulated) and adenosine, homocyctine and azmaline (as down regulated) biomarkers. These results could aid in the development of metabolite-quantitative trait loci (mQTL). Furthermore, stress-induced biosynthetic pathways may be the potential targets for modifications through breeding programs or genetic engineering for improving crop performance in the fields.

Untargeted Metabolomics of Alternaria solani-Challenged Wild Tomato Species Solanum cheesmaniae Revealed Key Metabolite Biomarkers and Insight into Altered Metabolic Pathways.

Untargeted metabolomics of moderately resistant wild tomato species Solanum cheesmaniae revealed an altered metabolite profile in plant leaves in response to Alternaria solani pathogen. Leaf metabolites were significantly differentiated in non-stressed versus stressed plants. The samples were discriminated not only by the presence/absence of specific metabolites as distinguished markers of infection, but also on the basis of their relative abundance as important concluding factors. Annotation of metabolite features using the Arabidopsis thaliana (KEGG) database revealed 3371 compounds with KEGG identifiers belonging to biosynthetic pathways including secondary metabolites, cofactors, steroids, brassinosteroids, terpernoids, and fatty acids. Annotation using the Solanum lycopersicum database in PLANTCYC PMN revealed significantly upregulated (541) and downregulated (485) features distributed in metabolite classes that appeared to play a crucial role in defense, infection prevention, signaling, plant growth, and plant homeostasis to survive under stress conditions. The orthogonal partial least squares discriminant analysis (OPLS-DA), comprising a significant fold change (≥2.0) with VIP score (≥1.0), showed 34 upregulated biomarker metabolites including 5-phosphoribosylamine, kaur-16-en-18-oic acid, pantothenate, and O-acetyl-L-homoserine, along with 41 downregulated biomarkers. Downregulated metabolite biomarkers were mapped with pathways specifically known for plant defense, suggesting their prominent role in pathogen resistance. These results hold promise for identifying key biomarker metabolites that contribute to disease resistive metabolic traits/biosynthetic routes. This approach can assist in mQTL development for the stress breeding program in tomato against pathogen interactions.

Naringenin-Capped Silver Nanoparticles Amalgamated Gel for the Treatment of Cutaneous Candidiasis.

The current research was aimed to synthesize a phytomolecule, naringenin (NRG)-mediated silver nanoparticles (NRG-SNPs) to study their antifungal potential against Candida albicans (C. albicans) and Candida glabrata (C. glabrata). The NRG-SNPs were synthesized by using NRG as a reducing agent. The synthesis of NRG-SNPs was confirmed by a color change and surface plasmon resonance (SPR) peak at 425 nm. Furthermore, the NRG-SNPs were analyzed for size, PDI, and zeta potential, which were found to be 35 ± 0.21 nm, 0.19 ± 0.03, and 17.73 ± 0.92 mV, respectively. In silico results demonstrated that NRG had a strong affinity towards the sterol 14α-demethylase. The docking with ceramide revealed the skin permeation efficiency of the NRG-SNPs. Next, the NRG-SNPs were loaded into the topical dermal dosage form (NRG-SNPs-TDDF) by formulating a gel using Carbopol Ultrez 10 NF. The MIC50 of NRG solution and TSC-SNPs against C. albicans was found to be 50 µg/mL and 4.8 µg/mL, respectively, significantly (P < 0.05) higher than 0.3625 µg/mL of NRG-SNPs-TDDF. Correspondingly, MIC50 results were calculated against C. glabrata and the results of NRG, TSC-SNPs, NRG-SNPs-TDDF, and miconazole nitrate were found to be 50 µg/mL, 9.6 µg/mL, 0.3625 µg/mL, and 3-µg/mL, respectively. Interestingly, MIC50 of NRG-SNPs-TDDF was significantly (P < 0.05) lower than MIC50 of miconazole nitrate against C. glabrata. The FICI (fractional inhibitory concentration index) value against both the C. albicans and C. glabrata was found to be 0.016 and 0.011, respectively, which indicated the synergistic antifungal activity of NRG-SNPs-TDDF. Thus, NRG-SNPs-TDDF warrants further in depth in vivo study under a set of stringent parameters for translating in to a clinically viable antifungal product.

Metabolomics-Driven Mining of Metabolite Resources: Applications and Prospects for Improving Vegetable Crops.

Vegetable crops possess a prominent nutri-metabolite pool that not only contributes to the crop performance in the fields, but also offers nutritional security for humans. In the pursuit of identifying, quantifying and functionally characterizing the cellular metabolome pool, biomolecule separation technologies, data acquisition platforms, chemical libraries, bioinformatics tools, databases and visualization techniques have come to play significant role. High-throughput metabolomics unravels structurally diverse nutrition-rich metabolites and their entangled interactions in vegetable plants. It has helped to link identified phytometabolites with unique phenotypic traits, nutri-functional characters, defense mechanisms and crop productivity. In this study, we explore mining diverse metabolites, localizing cellular metabolic pathways, classifying functional biomolecules and establishing linkages between metabolic fluxes and genomic regulations, using comprehensive metabolomics deciphers of the plant's performance in the environment. We discuss exemplary reports covering the implications of metabolomics, addressing metabolic changes in vegetable plants during crop domestication, stage-dependent growth, fruit development, nutri-metabolic capabilities, climatic impacts, plant-microbe-pest interactions and anthropogenic activities. Efforts leading to identify biomarker metabolites, candidate proteins and the genes responsible for plant health, defense mechanisms and nutri-rich crop produce are documented. With the insights on metabolite-QTL (mQTL) driven genetic architecture, molecular breeding in vegetable crops can be revolutionized for developing better nutritional capabilities, improved tolerance against diseases/pests and enhanced climate resilience in plants.

Salinity alleviator bacteria in rice (Oryza sativa L.), their colonization efficacy, and synergism with melatonin.

In this study, rhizospheric and endophytic bacteria were tested for the alleviation of salinity stress in rice. Endophytic isolates were taken from previous studies based on their salt stress-alleviating traits. The rhizospheric bacteria were isolated from rice and screened based on salt tolerance and plant growth-promoting traits. Molecular identification indicated the presence of class Gammaproteobacteria, Bacillota, and Actinomycetia. Two-two most potential isolates each from rhizospheric and endophytic bacteria were selected for in planta trials. Results showed that microbial inoculation significantly improved germination and seedling vigor under elevated salinity. The confocal scanning laser microscopy showed higher bacterial colonization in inoculated rice roots than in control. Based on this experiment, rhizospheric bacteria Brevibacterium frigoritolerans W19 and endophytic Bacillus safensis BTL5 were selected for pot trial along with a growth-inducing compound melatonin 20 ppm. Inoculation of these two bacteria improved the levels of chlorophyll, proline, phenylalanine ammonia-lyase, catalase, superoxide dismutase, polyphenol oxidase, root-shoot length, and dry weight under elevated salt concentration. The gene expression studies showed modulation of SOD1, CATa, NHX1, and PAL1 genes by the bacterial strains and melatonin application. The inoculation was found to have additive effects with 20 ppm melatonin. This enhancement in dry matter accumulation, compatible solute production, and oxidative stress regulation could help plants in mitigating the ill effects of high salinity. Exploring such a combination of microbes and inducer molecules could be potentially useful in developing stress-alleviating bioformulations.

SUMOylation in fungi: A potential target for intervention.

SUMOylation is a post-translational, reversible modification process which occurs in eukaryotes. Small Ubiquitin like MOdifier or (SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins to modify the target protein function. In pathogenic fungi, SUMO has been identified and preliminary studies indicate its importance either for survival and/or for virulence. In this review we provide an overview of the current state of knowledge of SUMOylation in fungi and the effects on pathogenesis. Subsequently we identify the orthologs of the SUMOylation pathway components across fungi. We also show the level of conservation of the proteins involved and identify the similarities/differences in the orthologs across fungi and the human and plant hosts to identify potential targets of intervention.

Stage-dependent concomitant microbial fortification improves soil nutrient status, plant growth, antioxidative defense system and gene expression in rice.

Stage-dependent concomitant fortification of rice (Oryza sativa L.) varieties PB1612 and CO51 with microbial inoculants Trichoderma asperellum and Pseudomonas fluorescens as seed coating, seedling root inoculation and soil application enhanced growth, activated antioxidant enzymes and modulated defence-related genes in plants. Microbial inoculants improved shoot height, tiller numbers, fresh weight and dry biomass. Co-inoculation was more impactful in enhancing plant growth and development as compared to single inoculation. Single and co-inoculation improved organic carbon (OC) and N, P and K content in the soil substantially. Mean values between control and co-inoculation varied significantly for OC in PB1612 (p0.001) and CO51 (p0.019) and phosphorus content in PB1612 (p0.044) and CO51 (p0.021). Microbial inoculation enhanced soil nutrients and increased their bioavailability for the plants. Total polyphenolics, flavonoids and protein content increased in the leaves following microbial inoculation. Enhanced non-enzymatic antioxidant parameters (ABTS, DPPH, Fe-ion reducing power and Fe-ion chelation) was found in microbe inoculated rice reflecting high free radical scavenging activity in polyphenolics-rich leaf extracts. Increased enzyme activity of superoxide dismutase (SOD), glutathione reductase (GR), phenylalanine ammonia-lyase (PAL), peroxidase (PO), glutathione peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT) showed improved ROS scavenging in rice plants having co-inoculation. Over-expression of PAL, cCuZn-SOD and CAT genes in microbial inoculated rice plants was recorded. The study concludes that plant stage-wise concomitant fortification by microbial inoculants could play multi-pronged manifestations at physiological, biochemical and molecular level in rice to positively influence growth, development and defense attributes in plants.

Microbial inoculation in rice regulates antioxidative reactions and defense related genes to mitigate drought stress.

Microbial inoculation in drought challenged rice triggered multipronged steps at enzymatic, non-enzymatic and gene expression level. These multifarious modulations in plants were related to stress tolerance mechanisms. Drought suppressed growth of rice plants but inoculation with Trichoderma, Pseudomonas and their combination minimized the impact of watering regime. Induced PAL gene expression and enzyme activity due to microbial inoculation led to increased accumulation of polyphenolics in plants. Enhanced antioxidant concentration of polyphenolics from microbe inoculated and drought challenged plants showed substantially high values of DPPH, ABTS, Fe-ion reducing power and Fe-ion chelation activity, which established the role of polyphenolic extract as free radical scavengers. Activation of superoxide dismutase that catalyzes superoxide (O2-) and leads to the accumulation of H2O2 was linked with the hypersensitive cell death response in leaves. Microbial inoculation in plants enhanced activity of peroxidase, ascorbate peroxidase, glutathione peroxidase and glutathione reductase enzymes. This has further contributed in reducing ROS burden in plants. Genes of key metabolic pathways including phenylpropanoid (PAL), superoxide dismutation (SODs), H2O2 peroxidation (APX, PO) and oxidative defense response (CAT) were over-expressed due to microbial inoculation. Enhanced expression of OSPiP linked to less-water permeability, drought-adaptation gene DHN and dehydration related stress inducible DREB gene in rice inoculated with microbial inoculants after drought challenge was also reported. The impact of Pseudomonas on gene expression was consistently remained the most prominent. These findings suggested that microbial inoculation directly caused over-expression of genes linked with defense processes in plants challenged with drought stress. Enhanced enzymatic and non-enzymatic antioxidant reactions that helped in minimizing antioxidative load, were the repercussions of enhanced gene expression in microbe inoculated plants. These mechanisms contributed strongly towards stress mitigation. The study demonstrated that microbial inoculants were successful in improving intrinsic biochemical and molecular capabilities of rice plants under stress. Results encouraged us to advocate that the practice of growing plants with microbial inoculants may find strategic place in raising crops under abiotic stressed environments.

Microbial Pyrrolnitrin: Natural Metabolite with Immense Practical Utility.

Pyrrolnitrin (PRN) is a microbial pyrrole halometabolite of immense antimicrobial significance for agricultural, pharmaceutical and industrial implications. The compound and its derivatives have been isolated from rhizospheric fluorescent or non-fluorescent pseudomonads, Serratia and Burkholderia. They are known to confer biological control against a wide range of phytopathogenic fungi, and thus offer strong plant protection prospects against soil and seed-borne phytopathogenic diseases. Although chemical synthesis of PRN has been obtained using different steps, microbial production is still the most useful option for producing this metabolite. In many of the plant-associated isolates of Serratia and Burkholderia, production of PRN is dependent on the quorum-sensing regulation that usually involves N-acylhomoserine lactone (AHL) autoinducer signals. When applied on the organisms as antimicrobial agent, the molecule impedes synthesis of key biomolecules (DNA, RNA and protein), uncouples with oxidative phosphorylation, inhibits mitotic division and hampers several biological mechanisms. With its potential broad-spectrum activities, low phototoxicity, non-toxic nature and specificity for impacts on non-target organisms, the metabolite has emerged as a lead molecule of industrial importance, which has led to developing cost-effective methods for the biosynthesis of PRN using microbial fermentation. Quantum of work narrating focused research efforts in the emergence of this potential microbial metabolite is summarized here to present a consolidated, sequential and updated insight into the chemistry, biology and applicability of this natural molecule.

Comparative analysis of silencing expression of myostatin (MSTN) and its two receptors (ACVR2A and ACVR2B) genes affecting growth traits in knock down chicken.

Myostatin (MSTN), a growth differentiation factor-8 regulates muscular development through its receptors, ACVR2A (Activin receptor type IIA) and ACVR2B (Activin receptor type IIB) by inhibiting cellular differentiation of developing somites during embryonic stage and diminishing myofibriller growth during post-embryonic period. The objective of this study was to compare the effect of knockdown of expression of myostatin, ACVR2A and ACVR2B genes on growth traits in chicken. The shRNAs for Myostatin, ACVR2A and ACVR2B genes were designed, synthesized and cloned in DEST vector. The recombinant molecules were transfected into the spermatozoa and transfected spermatozoa were inseminated artificially to the hens to obtain fertile eggs. The fertile eggs were collected, incubated in the incubator and hatched to chicks. Silencing of ACVR2B gene showed significantly higher body weight than other single, double and triple knock down of genes in transgenic birds. The carcass traits such as dressing%, leg muscle%, and breast muscle% were found with the highest magnitudes in birds with silencing of the ACVR2B gene as compared to the birds with that of other genes and control group. The lowest serum cholesterol and HDL content was found in ACVR2B silencing birds. The total RBC count was the highest in this group though the differential counts did not differ significantly among various silencing and control groups of birds. It is concluded that silencing of only one receptor of MSTN particularly, ACVR2B may augment the highest growth in chicken during juvenile stage. Our findings may be used as model for improving growth in other food animals and repairing muscular degenerative disorders in human and other animals.

Molecular characterization and expression profiling of BMP 3 gene in broiler and layer chicken.

A study was carried out to characterize and explore the expression profile of BMP 3 gene in control broiler and control layer chicken. The total open reading frame of BMP 3 (1389 bp) was cloned and sequenced. The control broiler and control layer chicken showed variation at nucleotide and amino acid level with reference gene (Gallus gallus, NCBI Acc. No. NM_001034819). When compared to reference gene, the control broiler showed four nucleotide differences (c.192A>G, c.519C>T, 903G>A and 960C>G), while, control layer showed variation at c.33G>C, 192A>G, 858G>A, 904G>A, 960C>G and 1257C>T making six differences in total. However, between control broiler and control layer lines, nucleotide differences was observed at c.33G>C, 519T>C, 858G>A, 903A>G, 904G>A and 1257C>T. The change at amino acid level between reference and control broiler was p.D320N and with control layer chicken, it was p.D302N and p.D320N. On the other hand, a single amino acid difference (p.D302N) was observed between the control broiler and control layer chicken lines. The phylogenetic study displayed a close relationship between broiler and layer lines and reference gene and also with other avian species resulting in a cluster formation. These cluster in turn displayed a distant link with the mammalian species. The expression profile of BMP 3 gene exhibited a variation at different stages of embryonic development and also at post embryonic period among the lines with control layer showing higher expression than that of broiler chicken. The protein was also detected in bone marrow tissue of broiler and layer lines by western blotting. It is concluded that the BMP 3 gene sequence differed at nucleotide and amino acid level among the lines and the gene expressed differentially at different periods of embryonic development and also at post hatch period.