The Morphology, Physiology and Molecular Characterisation of the Blue Stain In-service Organism Aureobasidium Pullucans

Aureobasidium pullulans is generally believed to be the main causal organism of the disfigurement of wood in service and coatings applied to wood. The disfigurement is referred to as blue stain in service. As such it represents a major problem to the paint industry and to the use of timber as a decorative product in service. Fifty two test strains and environmental isolates of blue stain fungi were collated to study the morphology, physiology and phylogeny ofA. pullulans. Sequencing of the rDNA 5.8S gene and flanking ITS regions was successful in unequivocally discriminating between the two morphologically similar blue stain fungi: A. pullulans and H. dematioides. The phylogenetic tree based on the rDNA sequence data confirmed that A. pullulans is a single species and there is no evidence of any subspecies from this region of DNA. RAPD analysis was used to investigate intraspecific genetic variability, but the technique was not successful in being able to differe,uiate accurately between these two fungi. However, different banding patterns were observed, suggesting there was a degree of genetic variability. The sequencing data highlighted many cases of misidentification of H. dematioides as A. pullulans from environmental isolations. The difficulty in correctly distinguishing morphologically between A. pullulans and H. dematioides b!'ought into question the conclusion that A. pullulans was the most commonly isolated and thus the main causal organism of blue stain in service. The actual frequency of occurrence of blue stain fungi in stained wood was investigated using PCR-DGGE analysis of environmental DNA samples. The use ofPCR-DGGE gave an insight into the actual fungi present at the time of sampling and gave a molecular snapshot of the blue stain community present on the stained timber samples. The PCR-DGGE analysis showed conclusively that H. dematioides was detected more frequently than A. pullulans. The results also show that PCR-DGGE, with a suitable primer set, is able to differentiate and partially quantify A. pullulans and H. dematioides in environmental samples. Quantitative analysis of A. pullulans was attempted from a new perspective using direct RNA analysis, in the form ofqPCR, to assess the active biomass ofA. pullulans in stained wood. The results showed the relatively constant levels ofmRNA of the two target genes over the eight week time period of the experiment and demonstrated the effectiveness of the RNA extraction procedure in harvesting extremely small amounts of viable RNA from wood. A method ofcontroIling blue stain fungi was investigated by attempting to reduce the number ofA. pullulans spores adhering to four different wood coatings, namely; a solvent based paint, a water based paint, a solvent based wood stain and a water based wood stain. These coatings were tested both before and after artificial weathering. The inhibition ofspore adhesion follows the tenet of prevention rather than cure and is particularly significant given the current environmental pressures on the coatings industry regarding the use ofbiocides to control these fungi. The main aim of the adhesion study was to examine ifzosteric acid could prevent the adhesion ofA. pullulans spores to wood coatings. Zosteric acid was shown to significantly reduce the adhesive capability ofA. pullulans spores on the four different wood coatings and weathering was shown to significantly increase the adhesion ofspores to the coatings. There is potential for utilising the reduction in spore adhesion observed with zosteric acid by its incorporation into paint formulations. Such an approach has great attraction as zosteric acid is a naturally occurring, non-toxic chemical in contrast to the current use of controlled biocides.EThOS - Electronic Theses Online ServiceGBUnited Kingdo