Recipient Agreement Number: R04-013 Research Program
W-2100
Localisation
Vancouver, British Columbia
Langue
English
Résumé
Western red-cedar (WRC, Thuja plicata Donn ex D. Don) wood was extracted sequentially with six solvents using two extraction methods. The extracts were prepared for subsequent bioassay and analysed by high performance liquid chromatography for known bioactive compound concentrations.
To focus identification of the extractives on those with bioactive properties, it was necessary to develop a micro-bioassay that would allow the biological activity of the unknown compounds present to be determined using minute quantities of each extracted constituent. The initial proposed technique utilised the loss of birefringence that occurs when decay fungi disrupt the crystalline cellulose structure as wood decays. Microtome sections of perishable sapwood were treated with microgram amounts of T. plicata heartwood compounds prior to exposure to decay fungi. The efficacy of the applied extract was then to be measured relative to the birefringence loss in untreated pine sapwood.
Validation of the technique required standardisation of a number of variables. Over 600 thin sections of ponderosa pine sapwood were cut and exposed to three different fungi, plus non-infected controls, under varying conditions of section thickness and orientation, media and growth conditions, viz, on grids or sterile microscope slides, with and without cover-slips, and with and without supplemental nitrogen, for six different incubation periods. Ultimately it was decided to test the extractives with two standard test brown rot fungi, Coniophora puteana and Postia placenta, using 25m radial sections which were sterilised, dipped in Abrams’s nutrient solution, and placed on a microscope slide prior to infection and incubation at 25 C, with a cover-slip placed over the inoculated section.
Analysis of the loss in birefringence was problematic and eventually abandoned due to time constraints. Towards the end of the project it was determined that the polarising filters in the microscope being used had been improperly manufactured. As a result, it was impossible to achieve 100% polarisation and we therefore could not examine loss in birefringence.
A commonly used antibiotic sensitivity test was modified to examine fungicidal efficacy of the extracts. Using 24-well tissue culture plates, four fungi were inoculated onto four media; one cellulose-based media was selected for use with Perenniporia subacida and Cephaloascus albidus isolated from a decaying second-growth WRC tree. Microlitre amounts of four known compounds dissolved in ethanol at three concentrations, plus the reference compound pentachlorophenol (PCP) and solvent controls, were pipetted onto paper disks and tested against the two fungi. Two of the extracts, ß-thujaplicin and thujic acid, plus PCP, were inhibitory to the fungi, verifying the methodology.
However, HPLC analysis of additional treated disks indicated that there was a substantial loss of chemical on the substrate over relatively short periods of time. This would indicate that the compounds were probably effective at lower concentrations than the targeted concentration. Volatilisation or decomposition when exposed to air and/or light was the likely cause of the observed mass loss of compounds. Recent tests have focused on the minimum time required for the ethanol solvent to evaporate when the extract is dispensed, so that disks may be rapidly moved onto the agar surface of the wells without any additional fungal toxicity from residual ethanol. A ventilation period of 30 minutes appears to be adequate for this. In addition, wood disks were found to be more effective than cellulose disks. In well tests, fungi grew well when wooden disks with ethanol were ventilated for 30 minutes. This test method will be used to bioassay isolated compounds.