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.

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.

Specific Microbial Communities Associate with the Rhizosphere of Welwitschia mirabilis, a Living Fossil.

Welwitschia mirabilis is an ancient and rare plant distributed along the western coast of Namibia and Angola. Several aspects of Welwitschia biology and ecology have been investigated, but very little is known about the microbial communities associated with this plant. This study reports on the bacterial and fungal communities inhabiting the rhizosphere of W. mirabilis and the surrounding bulk soil. Rhizosphere communities were dominated by sequences of Alphaproteobacteria and Euromycetes, while Actinobacteria, Alphaproteobacteria, and fungi of the class Dothideomycetes jointly dominated bulk soil communities. Although microbial communities within the rhizosphere and soil samples were highly variable, very few "species" (OTUs defined at a 97% identity cut-off) were shared between these two environments. There was a small 'core' rhizosphere bacterial community (formed by Nitratireductor, Steroidobacter, Pseudonocardia and three Phylobacteriaceae) that together with Rhizophagus, an arbuscular mycorrhizal fungus, and other putative plant growth-promoting microbes may interact synergistically to promote Welwitschia growth.

Effector specialization in a lineage of the Irish potato famine pathogen.

Accelerated gene evolution is a hallmark of pathogen adaptation following a host jump. Here, we describe the biochemical basis of adaptation and specialization of a plant pathogen effector after its colonization of a new host. Orthologous protease inhibitor effectors from the Irish potato famine pathogen, Phytophthora infestans, and its sister species, Phytophthora mirabilis, which is responsible for infection of Mirabilis jalapa, are adapted to protease targets unique to their respective host plants. Amino acid polymorphisms in both the inhibitors and their target proteases underpin this biochemical specialization. Our results link effector specialization to diversification and speciation of this plant pathogen.

Bioluminescence imaging of Clavibacter michiganensis subsp. michiganensis infection of tomato seeds and plants.

Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of C. michiganensis subsp. michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the C. michiganensis subsp. michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent C. michiganensis subsp. michiganensis strain using the modified Tn1409 containing a promoterless lux reporter. A total of 19 bioluminescent C. michiganensis subsp. michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent C. michiganensis subsp. michiganensis to help better elucidate the C. michiganensis subsp. michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of C. michiganensis subsp. nebraskensis with Tn1409::lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the agriculturally important C. michiganensis.