In vitro Antibacterial Activity and Secondary Metabolite Profiling of Endolichenic Fungi Isolated from Genus Parmotrema.

The endolichenic fungi are an unexplored group of organisms for the production of bioactive secondary metabolites. The aim of the present study is to determine the antibacterial potential of endolichenic fungi isolated from genus Parmotrema. The study is continuation of our previous work, wherein a total of 73 endolichenic fungi were isolated from the lichenized fungi, which resulted in 47 species under 23 genera. All the isolated endolichenic fungi were screened for preliminary antibacterial activity. Five endolichenic fungi-Daldinia eschscholtzii, Nemania diffusa, Preussia sp., Trichoderma sp. and Xylaria feejeensis, were selected for further antibacterial activity by disc diffusion method. The zone of inhibition ranged from 14.3 ± 0.1 to 23.2 ± 0.1. The chemical composition of the selected endolichenic fungi was analysed through GC-MS, which yielded a total of 108 compounds from all the selected five endolichenic fungi. Diethyl phthalate, 1-hexadecanol, dibutyl phthalate, n-tetracosanol-1, 1-nonadecene, pyrrol[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methyl) and tetratetracontane were found to be common compounds among one or the other endolichenic fungi, which possibly were responsible for antibacterial activity. GC-MS data were further analysed through Principal Component Analysis which showed D. eschscholtzii to be with unique pattern of expression of metabolites. Compound confirmation test revealed coumaric acid to be responsible for antibacterial activity in D. eschscholtzii. So, the study proves that endolichenic fungi that inhabit lichenized fungal thalli could be a source of potential antibacterial compounds.

Bacillus subtilis NBRI-W9 simultaneously activates SAR and ISR against Fusarium chlamydosporum NBRI-FOL7 to increase wilt resistance in tomato.

AIMS: The study aimed to determine the pathogenicity of Fusarium species currently prevalent in tomato fields having history of chemical fungicide applications and determine the bio-efficacy of Bacillus subtilis NBRI-W9 as a potent biological control agent. METHODS AND RESULTS: Fusarium was isolated from surface-sterilized infected tomato plants collected from fields. Pathogenicity of 30 Fusarium isolates was determined by in vitro and in vivo assays. Following Koch's postulates, F. chlamydosporum (FOL7) was identified as a virulent pathogen. The biological control of FOL 7 by B. subtilis NBRI-W9 (W9) and the colonization potential of W9 were established using spontaneous rifampicin-resistant mutants. W9 showed 82% inhibition of FOL7 on a dual-culture plate and colonization levels in tomato plants of ∼5.5, ∼3.3, and ∼2.2 log10 CFU/g in root, stem, and leaf tissue, respectively. Antagonistic activity was shown by scanning electron microscopy (SEM) and cell-wall-degradative enzymes. W9 reduced FOL7 infection in net-house and field experiments by 60% and 41%, respectively. Biochemical investigation, defence enzymes, defence gene expression analysis, SEM, and field studies provide evidence of hyperparasitism and induced resistance as the mode of biological control. The study also demonstrates that the potent biocontrol agent W9, isolated from Piper, can colonize tomato plants, control fungal disease by inducing induced systemic resistance (ISR) and systemic acquired resistance (SAR) simultaneously, and increase crop yield by 21.58% under field conditions. CONCLUSIONS: This study concludes that F. chlamydosporum (NBRI-FOL7) is a potent, fungicide-resistant pathogen causing wilt in tomatoes. NBRI-W9 controlled FOL7 through mycoparasitism and simultaneously activated ISR and SAR in plants, providing an attractive tool for disease control that acts at multiple levels.

Endophytic biofungicide Bacillus subtilis (NBRI-W9) reshapes the metabolic homeostasis disrupted by the chemical fungicide, propiconazole in tomato plants to provide sustainable immunity against non-target bacterial pathogens.

Chemical and microbial fungicides (Bio/fungicide) act differentially on plant systems. The present work assessed the metabolic profile of tomato plants vis-a-vis endophytic diversity after spraying of Propiconazole (PCZ) and endophytic biofungicide Bacillus subtilis (W9). Bio/fungicides were sprayed on tomato plants and evaluated for phenotypic, biochemical, and metabolic profiles after one week. In W9 treatment, a significant increase in relative abundance of several metabolites was observed including sugars, sugar alcohols, fatty-acids, organic-acids, and amino-acids. Polysaccharides and fatty acids showed a significant positive correlation with Rhizobiales, Burkholderiales, Bacillales, and Lactobacillales, respectively (p < 0.05). The PCZ and W9 treated plant's metabolic status significantly affected their resistance to non-target, bacterial pathogen P. syringae. Compared to PCZ and control, W9 treatment reduced the ROS deposition and expression of antioxidants gene GPx, PO (~0.1-1.7fold). It enhanced the genes related to the Phenylpropanoid pathway (∼1.6-5.2 fold), PR protein (~1.2-3.4 fold), and JA biosynthesis (~1.7-4.3 fold), resulting in reduced disease incidence. The results provide novel insights into the effects of endophytic biofungicide and chemical fungicides on the plant's metabolic status, its relation to the endophytes, and role in altering the plant's immune system.

Deciphering the kinetics and pathway of lindane biodegradation by novel soil ascomycete fungi for its implication in bioremediation.

Lindane, an organochlorine pesticide, negatively affects living beings and the ecosystem. In this study, the potential of 9 Ascomycetes fungi, isolated from an hexachlorocyclohexane dumpsite soil, was tested for biodegradation of lindane. The strain Pleurostoma richardsiae (FN5) showed lindane biodegradation rate constant (K value) of 0.144 d-1 and a half-life of 4.8d. The formation of intermediate metabolites upon lindane degradation including γ-pentachlorocyclohexene, 2,4-dichlorophenol, phenol, benzene, 1,3- cyclohexadiene, and benzoic acid detected by GC-MS and the potential pathway adopted by the novel fungal strain FN5 for lindane biodegradation has been elucidated. The study of gene profiles with reference to linA and linB in strain FN5 confirmed the same protein family with the reported heterologs from other fungal strains in the NCBI database. This study for the first time provides a thorough understanding of lindane biodegradation by a novel soil-borne Ascomycota fungal strain for its possible application in field-scale bioremediation.

Mitigation of arsenic toxicity in rice by the co-inoculation of arsenate reducer yeast with multifunctional arsenite oxidizing bacteria.

The study aimed to explicate the role of microbial co-inoculants for the mitigation of arsenic (As) toxicity in rice. Arsenate (AsV) reducer yeast Debaryomyces hansenii NBRI-Sh2.11 (Sh2.11) with bacterial strains of different biotransformation potential was attempted to develop microbial co-inoculants. An experiment to test their efficacy (yeast and bacterial strains) on plant growth and As uptake was conducted under a stressed condition of 20 mg kg-1 of arsenite (AsIII). A combination of Sh2.11 with an As(III)-oxidizer, Citrobacter sp. NBRI-B5.12 (B5.12), resulted in ∼90% decrease in grain As content as compared to Sh2.11 alone (∼40%). Reduced As accumulation in rice roots under co-treated condition was validated with SEM-EDS analysis. Enhanced As expulsion in the selected combination under in vitro conditions was found to be correlated with higher As content in the soil during their interaction with plants. Selected co-inoculant mediated enhanced nutrient uptake in association with better production of indole acetic acid (IAA) and gibberellic acid (GA) in shoot, support microbial co-inoculant mediated better biomass under stressful condition. Boosted defense response in association with enhanced glutathione-S-transferase (GST) and glutathione reductase (GR), activities under in vitro and in vivo conditions were observed. These results indicated that the As(III) oxidizer-B5.12 accelerated the As detoxification property of the As(V) reducer-Sh2.11. Henceforth, the results confer that the coupled reduction-oxidation process of the co-inoculant reduces the accumulation of As in rice grain. These co-inoculants can be further developed for field trials to achieve higher biomass with alleviated As toxicity in rice.

Potential of methyltransferase containing Pseudomonas oleovorans for abatement of arsenic toxicity in rice.

Arsenic (As) has become natural health hazard for millions of people across the world due to its distribution in the food chain. Naturally, it is present in different oxidative states of inorganic [As(V) and As(III)] and organic (DMA, MMA and TMA) forms. Among different mitigation approaches, microbe mediated mitigation of As toxicity is an effective and eco-friendly approach. The present study involves the characterization of bacterial strains containing arsenite methyltransferase (Pseudomonas oleovorans, B4.10); arsenate reductase (Sphingobacterium puteale, B4.22) and arsenite oxidase (Citrobacter sp., B5.12) activity with plant growth promoting (PGP) traits. Efficient reduction of grain As content by 61 % was observed due to inoculation of methyltransferase containing B4.10 as compared to B4.22 (47 %) and B5.12 (49 %). Reduced bioaccumulation of As in root (0.339) and shoot (0.166) in presence of B4.10 was found to be inversely related with translocation factor for Mn (3.28), Fe (0.073), and Se (1.82). Bioaccumulation of these micro elements was found to be associated with the modulated expression of different mineral transporters (OsIRT2, OsFRO2, OsTOM1, OsSultr4;1, and OsZIP2) in rice shoot. Improved dehydrogenase (407 %), and ß-glucosidase (97 %) activity in presence of P. oleovorans (B4.10) as compared to arsenate reductase (198 and 50 %), and arsenite oxidase (134 and 69 %) containing bacteria was also observed. Our finding confers the potential of methyltransferase positive P. oleovorans (B4.10) for As stress amelioration. Reduced grain As uptake was found to be mediated by improved plant growth and nutrient uptake associated with enhanced soil microbial activity.

Functional Genetic Diversity and Plant Growth Promoting Potential of Polyphosphate Accumulating Bacteria in Soil.

Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.

Alleviative mechanisms of silicon solubilizing Bacillus amyloliquefaciens mediated diminution of arsenic toxicity in rice.

Silicon (Si) has gained considerable attention for its utility in improved plant health under biotic and abiotic stresses through alteration of physiological and metabolic processes. Its interaction with arsenic (As) has been the compelling area of research amidst heavy metal toxicity. However, microbe mediated Si solubilization and their role for reduced As uptake is still an unexplored domain. Foremost role of Bacillus amyloliquefaciens (NBRISN13) in impediment of arsenite (AsIII) translocation signifies our work. Reduced grain As content (52-72%) during SN13 inoculation under feldspar supplementation (Si+SN+As) highlight the novel outcome of our study. Upregulation of Lsi1, Lsi2 and Lsi3genes in Si+SN+As treated rice plants associated with restricted As translocation, frames new propositions for future research on microbemediated reduced As uptake through increased Si transport. In addition to low As accumulation, alleviation of oxidative stress markers by modulation of defense enzyme activities and differential accumulation of plant hormones was found to be associated with improved growth and yield. Thus, our findings confer the potential role of microbe mediated Si solubilization in mitigation of As stress to restore plant growth and yield.

Yeast strain Debaryomyces hansenii for amelioration of arsenic stress in rice.

Arsenic (As) is a serious threat for environment and human health. Rice, the main staple crop is more prone to As uptake. Bioremediation strategies with heavy metal tolerant rhizobacteria are well known. The main objective of the study was to characterize arsenic-resistant yeast strains, capable of mitigating arsenic stress in rice. Three yeast strains identified as Debaryomyces hansenii (NBRI-Sh2.11), Candida tropicalis (NBRI-B3.4) and Candida dubliniensis (NBRI-3.5) were found to have As reductase activity. D. hansenii with higher As tolerance has As expulsion ability as compared to other two strains. Inoculation of D. hansenii showed improved detoxification through scavenging of reactive oxygen species (ROS) by the modulation of SOD and APX activity under As stress condition in rice. Modulation of defense responsive gene (NADPH, GST, GR) along with arsR and metal cation transporter are the probable mechanism of As detoxification as evident with improved membrane (electrolyte leakage) stability. Reduced grain As (~40% reduction) due to interaction with D. hansenii (NBRI-Sh2.11) further validated it's As mitigation property in rice. To the best of our knowledge D. hansenii has been reported for the first time for arsenic stress mitigation in rice with improved growth and nutrient status of the plant.