Study in vitro of the impact of endophytic bacteria isolated from Centella asiatica on the disease incidence caused by the hemibiotrophic fungus Colletotrichum higginsianum.

Thirty-one endophytic bacteria isolated from healthy leaves of Centella asiatica were screened in vitro for their ability to reduce the growth rate and disease incidence of Colletotrichum higginsianum, a causal agent of anthracnose. Isolates of Cohnella sp., Paenibacillus sp. and Pantoea sp. significantly stimulated the growth rate of C. higginsianum MUCL 44942, while isolates of Achromobacter sp., Acinetobacter sp., Microbacterium sp., Klebsiella sp. and Pseudomonas putida had no influence on this plant pathogen. By contrast, Bacillus subtilis BCA31 and Pseudomonas fluorescens BCA08 caused a marked inhibition of C. higginsianum MUCL 44942 growth by 46 and 82 %, respectively. Cell-free culture filtrates of B. subtilis BCA31 and P. fluorescens BCA08 were found to contain antifungal compounds against C. higginsianum MUCL 44942. Inoculation assays on in vitro-cultured plants of C. asiatica showed that foliar application of B. subtilis BCA31, three days before inoculation with C. higginsianum MUCL 44942, significantly reduced incidence and severity of the disease. The role of endophytic bacteria in maintaining the apparent inactivity of C. higginsianum MUCL 44942 in C. asiatica grown in the wild is discussed.

Characterization of an endophytic whorl-forming Streptomyces from Catharanthus roseus stems producing polyene macrolide antibiotic.

An endophytic whorl-forming Streptomyces sp. designated as TS3RO having antifungal activity against a large number of fungal pathogens, including Sclerotinia sclerotiorum, Rhizoctonia solani, Colletotrichum gloeosporioides, Cryphonectria parasitica, Fusarium oxysporum, Pyrenophora tritici-repentis, Epidermophyton floccosum, and Trichophyton rubrum, was isolated from surface-sterilized Catharanthus roseus stems. Preliminary identification showed that Streptomyces cinnamoneus subsp. sparsus was its closest related species. However, strain TS3RO could readily be distinguished from this species using a combination of phenotypic properties, 16S rDNA sequence similarity, and phylogenetic analyses. Thus, the whorl-forming Streptomyces sp. strain TS3RO is likely a new subspecies within the Streptomyces cinnamoneus group. Direct bioautography on a thin-layer chromatography plate with Cladosporium cucumerinum was conducted throughout the purification steps for bioassay-guided isolation of the active antifungal compounds from the crude extract. Structural elucidation of the isolated bioactive compound was obtained via LC-MS spectrometry, UV-visible spectra, and nuclear magnetic resonance data. It revealed that fungichromin, a known methylpentaene macrolide antibiotic, was the main antifungal component of TS3RO strain, as shown by thin-layer chromatography bioautography. This is the first report of an endophytic whorl-forming Streptomyces isolated from the medically important plant Catharanthus roseus.

Decolorization, cytotoxicity, and genotoxicity reduction during a combined ozonation/fungal treatment of dye-contaminated wastewater.

In view of compliance with increasingly stringent environmental legislation imposed by regional, national, and supranational (e.g., European Union) authorities, innovative environmental technologies for the treatment of dye-contaminated effluents are necessary in the color industry. In this study, effluents of an industrial dye producer were subjected to distinct treatment trains following an initial qualitative characterization. The effectiveness of ozonation and a treatment using white rot fungi (WRF) and their enzymes were compared with respect to parameters such as residual color, toxicity on human cells, and genotoxicity. A combined ozonation/WRF process was also investigated. The effluent exhibited significant toxicity that was reduced by only 10% through ozonation, whereas the fungal treatment achieved a 35% reduction. A combined treatment (ozone/WRF) caused an abatement of the toxicity by more than 70%. In addition, the initial genotoxicity of the effluent was still present after the ozone treatment, while it was completely removed through the fungal treatment.

Coupling occurs before breakdown during biotransformation of Acid Blue 62 by white rot fungi.

Only few data exist on the metabolites produced during the biotransformation of anthraquinonic dyes by white rot fungi (WRF). During the biotransformation of an anthraquinonic dye Acid Blue 62 (ABu62) using Pycnoporus sanguineus MUCL 41582 strain, it was previously demonstrated that the blue colour of the medium turned to red before complete dye decolourisation. To better understand the phenomenon, this study carried out ABu62 biotransformation with five different WRF strains (Coriolopsis polyzona MUCL 38443, Perenniporia ochroleuca MUCL 41114, Perenniporia tephropora MUCL 41562, P. sanguineus MUCL 38531 and Trametes versicolor MUCL 38412) and compared with P. sanguineus MUCL 41582 previously described. A multi-methodological approach (using capillary electrophoresis, mass spectrometry, HPLC, UV, NMR and IR spectroscopies) was developed to characterise the metabolites involved and monitor their apparition. Seven metabolites were commonly found with all strains, suggesting a common metabolic pathway for ABu62 biotransformation. During the first days, dimer and oligomers of the initial ABu62 molecule were observed: the main one absorbed in the 500nm region, explaining the red colour appearance of the medium. This main metabolite was made up of two molecules of ABu62 linked by an azo bond, minus a cyclohexyl moiety. After a longer incubation time, breakdown products were observed. Based on these products characterizations, a bioconversion pathway was proposed.

Comparative analysis of active constituents in Centella asiatica samples from Madagascar: application for ex situ conservation and clonal propagation.

A comparative quantitative analysis of the active triterpenoids in Centella asiatica samples collected in different locations in Madagascar was carried out to evaluate the natural variability in triterpenoid content and to select elite samples for further ex situ germplasm conservation and clonal propagation. The highest asiaticoside content (6.42%) was measured in samples collected in Mangoro region. In vitro propagation of C. asiatica was successfully achieved in hormone-free medium. Although lower asiaticoside content was detected in 8-week-old vitro plants, the Mangoro sample still showed the highest content in this triterpenoid constituent (1.78%). Acetoxycentellynol, a C(15)-polyacetylene, was found to be accumulated up to 18 times more in in vitro plants as compared to plant material collected in situ.