Characterization of the major antifungal extrolite from rice endophyte Lysinibacillus sphaericus against Rhizoctonia solani.

Sheath blight of rice caused by Rhizoctonia solani is regarded as one of the most widely distributed diseases of rice, and is one of the major production constraints for rice in India and most rice-growing countries of Asia. Biological control of plant diseases using antagonistic bacteria is now considered as a promising alternative to the use of hazardous chemical fungicides or bactericides. Several bacterial endophytes have been reported to support growth and improve the health of the plants and therefore, may be important as biocontrol agents. In the present study, putative antifungal metabolites were extracted from rice foliage endophyte Lysinibacillus sphaericus KJ872548 by solvent extraction methods and purified using HPTLC techniques. Separated bands were subjected to assess the in vitro antagonistic activity toward rice sheath blight pathogen Rhizoctonia solani using a dual culture method. Partially purified active fraction B2 obtained from HPTLC analysis showed the highest percentage of inhibition (76.9%). GC MS and FTIR analyses of B2 revealed the compound as1, 2-Benzenedicarboxylic acid butyl 2-Ethylhexyl ester, a strong antifungal volatile organic compound. Light microscopic analysis of the fungal mycelium from the dual culture plate of both culture filtrate and 1, 2-Benzenedicarboxylic acid butyl 2-Ethylhexyl ester disclosed strong mycolytic activity as evident by mycelial distractions and shrinkage. This is the first report on antifungal production by endophytic Lysinibacillus sphaericus against R. solani, the rice sheath blight pathogen. The findings of this study biologically prospect the endophyte L. sphaericus as an inexpensive broad spectrum bioagent for eco-friendly, economic and sustainable agriculture.

In vitro and in silico docking studies of antibacterial compounds derived from endophytic Penicillium setosum.

Occasionally, endophytic fungal species cognize as a hidden prospective source of plant secondary metabolites. In this study, a potent Penicillium setosum sp. nov. was explored for its detailed antibacterial action on Escherichia coli and Staphylococcus aureus through different in vitro and in silico assays. Fluorescence based viability assay determined increase in the number of dead cells in course of time with the continual exposure of extract during a 4 h period. Scanning electron micrographs reflect the distinguishable morphological changes in treated cells, namely shortening of size, bubbles, and blisters on the surface of E. coli, as well as open holes and deep craters on the surface of S. aureus, ultimately leading to rupture of cells. Significant intracellular changes in bacteria were remarkably noticed through different membrane permeabilization assays. The rate of Na+ and K+ leakage with respect to time, intracellular material and cytoplasmic ß-galactosidase release were measured spectroscopically. The results indisputably prove that membrane disruption of S. aureus cells occurs within 2 h and in E.coli occurs in between 2 and 4 h of exposure. Crude extract of P. setosum was fractioned using semi-preparative HPLC and the separated antibacterial active fraction showed antibacterial efficacy with the minimum inhibitory concentration of 8 µg/mL against both organisms. Active fraction contains four well-known plant metabolite belongs to the polyphenolic group (Leucodelphinidin, dihydroquercetin, kaempferol, and quercetin) and one polyketide (patulin) familiar as fungal metabolite, identified through high resolution LC-MS. Interaction mechanisms of identified compounds with nine important antimicrobial drug targets showed highest binding affinity by leucodelphinidin followed by dihydroquercetin > kaempferol > quercetin. This is the first instance of using leucodelphinidin and dihydroquercetin for detailed interaction study with multiple targets, and it was found that they showed more effective interaction than quercetin, which was earlier utilized for antibacterial studies.

Penicillium setosum, a new species from Withania somnifera (L.) Dunal.

Medicinal plants are considered as sources of novel and unexplored groups of endophytic microorganisms. A study on endophytic fungal species from the medicinal plant Withania somnifera (L.) Dunal resulted in the isolation of a Penicillium isolate (WSR 62) with antibiotic activity. Phylogenetic analysis showed that the isolate belongs to section Lanata-divaricata, and it is most closely related to P. javanicum. Subsequent detailed phylogenetic analyses using partial ß-tubulin (BenA), calmodulin (CaM) and DNA-dependent RNA polymerase II (RPB2) gene sequences of a larger number of related strains revealed the distinctiveness of the isolate in the P. javanicum-clade. The isolate grows fast on Czapek yeast autolysate agar (CYA) and malt extract agar (MEA) incubated at 25°C, 30°C and 37°C. The obverse colony colour is dominated by the conspicuous production of cleistothecia and is greyish yellow on CYA and yellowish brown on MEA. Production of cleistothecia containing prominent spinose ascospores was present on all tested agar media. Based on the phylogenetic analysis and the phenotypic characterisation, strain WSR 62 from Withania is described here as a novel species named Penicillium setosum.

Chemotaxonomic profiling of Penicillium setosum using high-resolution mass spectrometry (LC-Q-ToF-MS).

In the present study, secondary metabolites produced by an endophytic fungus Penicillium setosum were extracted using colony agar plug and culture broth extraction methods. High resolution LC-MS was used to explore the chemical nature of the secondary metabolites, as well, compare the reliability of the methods. P. setosum was chemotaxonomically distinguished from other members of section Lanata-divaricata, by its ability to produce mycotoxin, patulin and also by the presence of certain phenol-derived compounds, like quercetin, dihydroflavonols (dihydroquercetin and dihydromyricetin), kaempferol, luteolin, while some Penicillium specific compounds such as, citromycetin and andrastin D reveal its similarity towards section Lanata-Divaricata members. For the first time, the presence of dihydroquercetin is remarkably and spectrometrically confirmed from a microbial source. In addition, a few polyketides, anthroquinone compounds, hydrocarbons, and fatty acids were also detected in the culture extract. Being the first report on the production of polyphenolic compounds by an endophytic fungus of Penicillium species, the current research is crucial, and moreover the starin itself is a novel species.