In Vivo Studies of Inoculated Plants and In Vitro Studies Utilizing Methanolic Extracts of Endophytic Streptomyces sp. Strain DBT34 Obtained from Mirabilis jalapa L. Exhibit ROS-Scavenging and Other Bioactive Properties.

Reactive oxygen species (ROS) and other free radicals cause oxidative damage in cells under biotic and abiotic stress. Endophytic microorganisms reside in the internal tissues of plants and contribute to the mitigation of such stresses by the production of antioxidant enzymes and compounds. We hypothesized that the endophytic actinobacterium Streptomyces sp. strain DBT34, which was previously demonstrated to have plant growth-promoting (PGP) and antimicrobial properties, may also have a role in protecting plants against several stresses through the production of antioxidants. The present study was designed to characterize catalase and superoxide dismutase (SOD), two enzymes involved in the detoxification of ROS, in methanolic extracts derived from six endophytic actinobacterial isolates obtained from the traditional medicinal plant Mirabilis jalapa. The results of a preliminary screen indicated that Streptomyces sp. strain DBT34 was the best overall strain and was therefore used in subsequent detailed analyses. A methanolic extract of DBT34 exhibited significant antioxidant potential in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assays. The cytotoxicity of DBT34 against liver hepatocellular cells (HepG2) was also determined. Results indicated that methanolic extract of Streptomyces sp. strain DBT34 exhibited significant catalase and SOD-like activity with 158.21 U resulting in a 55.15% reduction in ROS. The IC50 values of a crude methanolic extract of strain DBT34 on DPPH radical scavenging and ABTS radical cation decolorization were 41.5 µg/mL and 47.8 µg/mL, respectively. Volatile compounds (VOC) were also detected in the methanolic extract of Streptomyces sp. strain DBT34 using GC-MS analysis to correlate their presence with bioactive potential. Treatments of rats with DBT34 extract and sitagliptin resulted in a significant (p ≤ 0.001) reduction in total cholesterol, LDL-cholesterol, and VLDL-cholesterol, relative to the vehicle control and a standard diabetic medicine. The pancreatic histoarchitecture of vehicle control rats exhibited a compact volume of isolated clusters of Langerhans cells surrounded by acinies with proper vaculation. An in-vivo study of Streptomyces sp. strain DBT34 on chickpea seedlings revealed an enhancement in its antioxidant potential as denoted by lower IC50 values for DPPH and ABTS radical scavenging activity under greenhouse conditions in relative comparison to control plants. Results of the study indicate that strain DBT34 provides a defense mechanism to its host through the production of antioxidant therapeutic agents that mitigate ROS in hosts subjected to biotic and abiotic stresses.

Draft Genome Sequence of Plant Growth-Promoting Endophytic Microbacterium hydrothermale BPSAC84, Isolated from the Medicinal Plant Mirabilis jalapa.

Endophytic Microbacterium hydrothermale strain BPSAC84, which has antimicrobial potential, was isolated from root tissues of Mirabilis jalapa in Mizoram, Northeast India. The draft genome consists of 3.58 Mb and 3,444 protein-coding sequences.

Elevated levels of laccase synthesis by Pleurotus pulmonarius BPSM10 and its potential as a dye decolorizing agent.

Laccases (EC 1.10.3.2) are a class of multi-copper oxidases that have industrial value. In the present study, forty-five isolates of wild mushrooms were screened for laccase production. Eight of the isolates exhibited exploitable levels of substrate oxidation capacity. Isolate BPSM10 exhibited the highest laccase activity of 103.50 U/ml. Internal Transcribed Spacer (ITS) rRNA gene sequencing was used to identify BPSM10 as Pleurotus pulmonarius. The response of BPSM10 to two nutritional media supplemented with various inducers was characterized and the results indicated that Malt Extract Broth (MEB) supplemented with Xylidine increased laccase production by 2.8× (349.5 U/ml) relative to the control (122 U/ml), while Potato Dextrose Broth (PDB) supplemented with xylidine increased laccase production by 1.9× (286 U/ml). BPSM10 has the ability to decolorize seven synthetic dyes in a liquid medium. Maximum decolorization was observed of malachite green (MG); exhibiting 68.6% decolorization at 100 mg/L. Fourier-transform infrared spectroscopy (FTIR) was employed to confirm the decolorization capacity. The present study indicates that P. pulmonarius BPSM10 has the potential to be used as a potent alternative biosorbent for the removal of synthetic dyes from aqueous solutions, especially in the detoxification of polluted water.

Determination and production of antimicrobial compounds by Aspergillus clavatonanicus strain MJ31, an endophytic fungus from Mirabilis jalapa L. using UPLC-ESI-MS/MS and TD-GC-MS analysis.

Endophytic fungi associated with medicinal plants are reported as potent producers of diverse classes of secondary metabolites. In the present study, an endophytic fungi, Aspergillus clavatonanicus strain MJ31, exhibiting significant antimicrobial activity was isolated from roots of Mirabilis jalapa L., was identified by sequencing three nuclear genes i.e. internal transcribed spacers ribosomal RNA (ITS rRNA), 28S ribosomal RNA (28S rRNA) and translation elongation factor 1- alpha (EF 1α). Ethyl acetate extract of strain MJ31displayed significant antimicrobial potential against Bacillus subtilis, followed by Micrococccus luteus and Staphylococcus aureus with minimum inhibitory concentrations (MIC) of 0.078, 0.156 and 0.312 mg/ml respectively. In addition, the strain was evaluated for its ability to synthesize bioactive compounds by the amplification of polyketide synthase (PKS) and non ribosomal peptide synthetase (NRPS) genes. Further, seven antibiotics (miconazole, ketoconazole, fluconazole, ampicillin, streptomycin, chloramphenicol, and rifampicin) were detected and quantified using UPLC-ESI-MS/MS. Additionally, thermal desorption-gas chromatography mass spectrometry (TD-GC-MS) analysis of strain MJ31 showed the presence of 28 volatile compounds. This is the first report on A. clavatonanicus as an endophyte obtained from M. jalapa. We conclude that A. clavatonanicus strain MJ31 has prolific antimicrobial potential against both plant and human pathogens and can be exploited for the discovery of new antimicrobial compounds and could be an alternate source for the production of secondary metabolites.

Distribution and antimicrobial potential of endophytic fungi associated with ethnomedicinal plant Melastoma malabathricum L.

Distributions of endophytic fungi associated with ethnomedicinal plant Melastoma malabathricum L. was studied and 91 isolates belonging to 18 genera were recovered. The isolates were distributed to sordariomycetes (62.63%), dothideomycetes (19.78%), eurotiomycetes (7.69%), zygomycetes (4.19%), agaricomycetes (1.09%), and mycelia sterilia (4.39%). Based on colony morphology and examination of spores, the isolates were classified into 18 taxa, of which Colletotrichum, Phomopsis and Phoma were dominant, their relative frequencies were 23.07%, 17.58% and 12.08% respectively. The colonization rate of endophytic fungi was determined and found to be significantly higher in leaf segments (50.76%), followed by root (41.53%) and stem tissues (27.69%). All the isolates were screened for antimicrobial activity and revealed that 26.37% endophytic fungi were active against one or more pathogens. Twenty four isolates showing significant antimicrobial activity were identified by sequencing the ITS1-5.8S-ITS2 region of rRNA gene. Results indicated that endophytic fungi associated with leaf were functionally versatile as they showed antimicrobial activity against most of the tested pathogens. The endophytic fungi Diaporthe phaseolorum var. meridionalis (KF193982) inhibited all the tested bacterial pathogens, whereas, Penicillium chermesinum (KM405640) displayed most significant antifungal activity. This seems to be the first hand report to understand the distribution and antimicrobial ability of endophytic fungi from ethno-medicinal plant M. malabathricum.

In Vitro and In Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants.

Endophytic actinomycetes have shown unique plant growth promoting as well as antagonistic activity against fungal phytopathogens. In the present study forty-two endophytic actinomycetes recovered from medicinal plants were evaluated for their antagonistic potential and plant growth-promoting abilities. Twenty-two isolates which showed the inhibitory activity against at least one pathogen were subsequently tested for their plant-growth promoting activities and were compared genotypically using DNA based fingerprinting, including enterobacterial repetitive intergenic consensus (ERIC) and BOX repetitive elements. Genetic relatedness based on both ERIC and BOX-PCR generates specific patterns corresponding to particular genotypes. Exponentially grown antagonistic isolates were used to evaluate phosphate solubilization, siderophores, HCN, ammonia, chitinase, indole-3-acetic acid production, as well as antifungal activities. Out of 22 isolates, the amount of indole-3-acetic acid (IAA) ranging between 10-32 µg/ml was produced by 20 isolates and all isolates were positive for ammonia production ranging between 5.2 to 54 mg/ml. Among 22 isolates tested, the amount of hydroxamate-type siderophores were produced by 16 isolates ranging between 5.2 to 36.4 µg/ml, while catechols-type siderophores produced by 5 isolates ranging from 3.2 to 5.4 µg/ml. Fourteen isolates showed the solubilisation of inorganic phosphorous ranging from 3.2 to 32.6 mg/100ml. Chitinase and HCN production was shown by 19 and 15 different isolates, respectively. In addition, genes of indole acetic acid (iaaM) and chitinase (chiC) were successively amplified from 20 and 19 isolates respectively. The two potential strains Streptomyces sp. (BPSAC34) and Leifsonia xyli (BPSAC24) were tested in vivo and improved a range of growth parameters in chilli (Capsicum annuum L.) under greenhouse conditions. This study is the first published report that actinomycetes can be isolated as endophytes from within these plants and were shown to have antagonistic and plant growth promoting abilities. These results clearly suggest the possibility of using endophytic actinomycetes as bioinoculant for plant growth promotion, nutrient mobilization or as biocontrol agent against fungal phytopathogens for sustainable agriculture.