Endophytic fungal isolation from Blumea axillaris: Identification and biological activity of secondary metabolites

Medicinal plants are a wealthy source of natural medicinal properties and remain as base for new drug discoveries. Endophyte from the specific medicinal plants produce the analogous metabolites as that of the host plant. The metabolites from the endophytes comprise maximum therapeutic properties and have been extensively applied in treating various diseases and disorders. This study was focused on identification of the endophytic fungi from the medicinal plant Blumea axillaris and investigates the diversity of endophytic fungi from various explants of the same plant. The explants were cultured on potato dextrose agar and 6 endophytic fungi were successfully isolated from Blumea axillaris. They were identified morphologically and confirmed with molecular analysis as Xylaria arbuscula, Paraphoma radicina, Phomopsis phaseoli, Sordaria fimicola, Aspergillus amstelodami, Diaporthe eucalyptorum. The DNA sequences were analyzed by BLAST and the phylogenetic tree was constructed with neighbor joining method. The six isolates were subjected to antagonistic activity for the selection of potential strain and the bioactive strain Xylaria arbuscula was selected for the production of secondary metabolites by optimization. The parameters like pH, temperature, incubation period, carbon and nitrogen (organic and inorganic source) were optimized for secondary metabolite production. The fungal metabolite was extracted by solvent extraction method using polar and non-polar solvents like propanol, methanol, chloroform, acetone and ethyl acetate. To investigate the bioactivities of the fungal crude extract was subjected first for its antioxidant activity using DPPH radical scavenging method, followed by antimicrobial activity of methanolic (MeOH) extract of Xylaria arbuscula, that were also analyzed by the agar well-diffusion method against the clinical pathogens Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pnuemoniae, Proteus mirabilis, Aspergillus niger and Candida albicans.

Genetic Diversity Analysis and In Silico Investigation of Post-Translational Modifications of Carboxypeptidase A1 (CpA1) in Sordaria fimicola

Post-translational modifications (PTMs) regulate different complex mechanisms of cell and affect cell growth, stress, evolution of living organisms and adaptations due to environment. The purpose of the present research is to investigate the genetic diversity and PTMs of protease (Carboxypeptidase A1) n Sordaria fimicola. They perform a variety of functions ranging from housekeeping: e.g., protein maturation, signal peptide cleavage, signal transduction, intracellular protein turnover, immune response, apoptosis, and reproduction. S. fimicola is a microscopic filamentous fungus, has been preferably used in this study because of its easy growing pattern on Potato Dextrose Agar (PDA) and a short life cycle of 7 to 12 days. The genomic DNA of six of the strains S. fimicola was used to amplify the carboxypeptidases A1 gene (CpA1), the product size was 940 bp. The multiple sequence alignment of the nucleotide sequences of six strains of S. fimicola with Neurospora crassa (as a reference strain) was studied. The numbers of polymorphic sites in six strains of S. fimicola with respect to N. crassa were six. Posttranslational modifications were depicted by using bioinformatics tools i.e., YinOYang1.2, NetPhos 3.1 and NetNES 1.1 Server to calculate O-glycosylation, phosphorylation sites, and nuclear export signals respectively. The study has predicted 19 phosphorylation sites on serine residues for protease Carboxypeptidase A1 in S1 strains of S. fimicola while 15 phosphorylation sites on serine in N7 strain and 17 serine phosphorylation modifications were predicted in N. Crassa. The results of this research will be helpful for further in vitro investigations of this industrially important enzyme under study. © 2021 Friends Science Publishers

HONGOS ENDÓFITOS EN 2 PLANTAS MEDICINALES DEL NORDESTE ARGENTINO. I: ANÁLISIS MORFOTAXONÓMICO DE SUS COMUNIDADES FOLIARES

Se evaluó la presencia de hongos endófitos en hojas de dos plantas medicinales, Acanthospermum australe y Pterocaulon alopecuroides, utilizadas entre otras cosas para tratar infecciones cutáneas. El muestreo se realizó en el entorno rural de dos ciudades del nordeste argentino. Los especimenes fueron recogidos por aborígenes de la zona conocedores de su distribución ambiental. Los hongos se aislaron mediante la técnica de Bisseger et al., 1994 y se identificaron empleando caracteristicas morfofisiológicas. Considerando las dos plantas, se encontraron 40 morfoespecies, donde los taxa más frecuentes en Acanthospermum australe fueron: Alternaria alternata, Aureobasidium pullulans, Fusarium oxysporum, F. solani, Myrothecium roridum, Phoma spp. y Sordaria fimícola, y en Pterocaulon alopecuroides fueron: Alternaria alternata, Aureobasidium pullulans, Cladosporium cladosporioides, Curvularia lunata, Fusarium proliferatum y Myrothecium roridum. Estos taxas considerados como generalistas (o de múltiples hospedadores), presentan según la literatura específica analizada, alta producción de metabolitos secundarios bioactivos con potenciales capacidades terapéuticas antimicrobianas.

ASPECTOS CULTURAIS, MORFOLÓGICOS E MORFOMÉTRICOS DE Sordaria fimicola INCIDENTE EM FOLHAS DE CAPIM-MASSAMBARÁ (Sorghum arundinaceum)

O capim-massambará (Sorghum arundinaceum) pode servir de fonte de inóculo de agentes de biocontrole como Sordaria spp. O objetivo deste trabalho foi avaliar o crescimento in vitro, a patogenicidade, caracterizar e identificar um isolado de Sordaria sp. incidente em capim-massambará. Amostras de folhas apresentando sintomas foram submetidas a isolamento em meio de cultura. A partir deste foram aplicados os seguintes testes: a) teste fisiológico – análise do crescimento micelial em meio de cultura. b) teste biológico – discos de micélio e solução de ascósporos foram inoculados em folhas; e c) identificação – caracterização morfológica das estruturas reprodutivas. O maior crescimento micelial foi observado nos meios BDA e ST. O isolado em condições artificiais não reproduziu os sintomas iniciais. O isolado apresentou peritécio gregário, semi-imerso em meio de cultura, sub-globoso, com organização celular reticulada, de dimensões de 1946,3-(1351,0)-507,9 × 1131,4-(828,2)-415,17 µm, com ostíolo de 413,85-(257,8)-123,8 µm; asca cilíndrica à clavada, unitunicada, de 195,0-(167,8)-135,6 × 21,8-(16,8)-9,1 µm; ascósporos apresentaram 29,7-(22,6)-13,7 × 14,9-(10,9)-1,6 µm. Com base nas características morfológicas e morfométricas, o isolado foi identificado como sendo S. fimicola.

The Blue-Light PhotoreceptorSfwc-1Gene Regulates the Phototropic Response and Fruiting-Body Development in the Homothallic AscomyceteSordaria fimicola

ABSTRACTSordaria fimicola, a coprophilous ascomycete, is a homothallic fungus that can undergo sexual differentiation with cellular and morphological changes followed by multicellular tissue development to complete its sexual cycle. In this study, we identified and characterized the blue-light photoreceptor gene inS. fimicola. TheS. fimicolawhite collar-1 photoreceptor (SfWC-1) contains light-oxygen-voltage-sensing (LOV), Per-Arnt-Sim (PAS), and other conserved domains and is homologous to the WC-1 blue-light photoreceptor ofNeurospora crassa. The LOV domain ofSfwc-1was deleted by homologous recombination usingAgrobacterium-mediated protoplast transformation. TheSfwc-1(Δlov)mutant showed normal vegetative growth but produced less carotenoid pigment under illumination. The mutant showed delayed and less-pronounced fruiting-body formation, was defective in phototropism of the perithecial beaks, and lacked the fruiting-body zonation pattern compared with the wild type under the illumination condition. Gene expression analyses supported the light-induced functions of theSfwc-1gene in the physiology and developmental process of perithecial formation inS. fimicola. Moreover, green fluorescent protein (GFP)-tagged SfWC-1 fluorescence signals were transiently strong upon light induction and prominently located inside the nuclei of living hyphae. Our studies focused on the putative blue-light photoreceptor in a model ascomycete and contribute to a better understanding of the photoregulatory functions and networks mediated by the evolutionarily conserved blue-light photoreceptors across diverse fungal phyla.IMPORTANCESordariasp. has been a model for study of fruiting-body differentiation in fungi. Several environmental factors, including light, affect cellular and morphological changes during multicellular tissue development. Here, we created a light-oxygen-voltage-sensing (LOV) domain-deletedSfwc-1mutant to study blue-light photoresponses inSordaria fimicola. Phototropism and rhythmic zonation of perithecia were defective in theSfwc-1(Δlov)mutant. Moreover, fruiting-body development in the mutant was reduced and also significantly delayed. Gene expression analysis and subcellular localization study further revealed the light-induced differential gene expression and cellular responses upon light stimulation inS. fimicola.