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Blue stain in service on wood surface coatings. Part 3: The nutritional capability of Aureobasidium pullulans compared to other fungi commonly isolated from wood surface coatings
1993 - IRG/WP 93-10035
The nutritional capability of Aureobasidium pullulans was previously examined, using agar plate tests, with regard to nutrient sources that are potentially available in fresh and weathered wood (Sharpe and Dickinson, 1992). This study compared these findings with the nutritional capability of four other fungi (Alternaria sp., Cladosporium cladosporoides, Stemphylium sp. and Trichoderma sp.) commonly isolated from wood surfaee coatings. The liquid culture techniques were used to assess the relative abilities of the fungi to utilise a range of simple sugars, wood sugar alcohols, hemicelluloses, cellulose and lignin degradation compounds. The observations were used to explain why Aureobasidium pullulans is able to occupy so successfully, often in monoculture, the wood-paint interface niche.
P R Sharpe, D J Dickinson

Blue stain in service on wood surface coatings. Part 2: The ability of Aureobasidium pullulans to penetrate wood surface coatings
1992 - IRG/WP 92-1557
The ability of Aureobasidium pullulans to penetrate seven different wood surface coatings was examined. Eight isolates of Aureobasidium pullulans were used. Following penetration, light microscopy was used to examine the effect of the fungus upon the coating, and growth at the wood-film interface. The fungus showed different growth capabilities with different types of surface coatings. Some isolates were able to penetrate alkyd based coatings, but were unable to grow in the film. In contrast, after penetration through latex based coatings several isolates were able to produce growth in the films themselves.
P R Sharpe, D J Dickinson

Detoxification of preservatives: Tri-n-butyltin oxide as a biocide
1982 - IRG/WP 1156
T E Dudley-Brendell, D J Dickinson

Characterisation of growth and stain of different groups of sapstain fungi on lodgepole pine
1999 - IRG/WP 99-10326
Canada is the world's largest exporter of softwood lumber. These softwood shipments are susceptible to a variety of wood-inhabiting fungi that can lead to sapstain discolouration, which in turn decrease the product value. Furthermore, the presence of these microorganisms may be unacceptable to the importing countries. The objective of this work is to assess the sapstaining capability and basic nutrition of thirty-four fungi isolates representing nine species that were isolated from sawmills across western Canada. The isolates were infected onto fresh lodgepole pine billets and assessed for staining ability, longitudinal growth, host-nutrient consumption, and host viability. The results indicated that the most aggressive saptain species on fresh logs was Ceratocystis coerulescens, followed consecutively by Leptographium spp, Ophiostoma minus, O. piliferum, O. piceae, Ophiostoma spp (D and E) and Aureobasidium pullulans. Preliminary HPLC analysis of soluble sugars indicated that mannose was the free monomer carbohydrate of choice for most of the staining fungi, followed by glucose. Arabinose and galactose were not well utilised. Gas chromatography of infected wood extracts that Leptographium sp. and C. coerulescens significantly reduced the triglyceride fraction.
C Fleet, C Breuil, A Uzunovic, A Byrne

The use of chlorothalonil for protection against mold and sapstain fungi. Part 1: Laboratory evaluation
1989 - IRG/WP 3515
Laboratory screening of chlorothalonil alone and in combination with other fungicides was conducted against six mold and sapstain fungi. The most promising treatments appear to be chlorothalonil supplemented with CCA or copper-8-quinolinolate. Field tests have been implemented.
J A Micales, T L Highley, A L Richter

Laboratory evaluation of chlorothalonil formulation for stain and mold control on rubberwood and maple
1998 - IRG/WP 98-30175
We evaluated the efficacy of several chlorothalonil and carbendazim fungicides (F1 and F2), etc. in the control of mold and stain fungi on rubberwood and maple. The results showed that these formulations effectively inhibited the selected fungal species such as Aspergillus niger, Penicillium sp., Trichoderma sp. (P71H), Aureobasidium pullulans, Ceratocystis minor (C-188), Ceratocystis pilifera (RWD 9472) in laboratory tests.
Mingliang Jiang, T L Highley, L Ferge, T L Woods

Association of contents of nitrogen and sugars in rubberwood (Hevea brasiliensis) clones with susceptibility to sapstain by Botryodiplodia theobromae, Aureobasidium pullulans and Aspergillus niger
1999 - IRG/WP 99-10307
The purpose of this study was to determine if nitrogen and sugar contents in rubberwood from three selected varieties (clones) rubber trees would affect the rate of colonisation by Botryodiplodia theobromae (a tropical sapstain fungus), Aureobasidium pullulans (a temperate sapstain fungus) and Aspergillus niger (a common mould fungus). Sapstain growth was rated daily until at least 50% mycelial coverage was achieved for 40x20x5mm3 samples from three rubberwood clones (GT1, PB217 and RRIM600) inoculated with the test fungi and incubated in a humidified petri dish assembly under aseptic conditions. All samples had more than 50% coverage of mycelium after 9 days of incubation. The results indicate that the nitrogen content of clone RRIM600 (1.06lmg/g) was significantly higher when compared to that of clone GTI (0.73mg/g) and clone PB217 (0.78mg/g). After oven drying (45°C), clone PB217 contained significantly higher amounts of fructose (5.55mg/g), glucose (2.30mg/g) and total sugar (13.15mg/g), as compared to clone GTI and RRIM600. Clone GTI had the lowest fructose (0.23mg/g), glucose (0.14mg/g), sucrose (2.20mg/g) and total free sugar (2.56mg/g). RRIM600 however had the highest amount of sucrose. B. theobromae spread significantly faster on RRIM600 (4.3 days when >50% mycelial coverage is achieved) than PB217 (5.6 days) or GTI (5.8 days) which is associated with the overall higher nutrient availability in clone RRIM600. Light sanding (down to 1-2mm depth) of sapstain infected samples revealed that deep sapstain typical of B. theobromae remained in the wood, contrasting with the absence of stain by A. pullulans and A. niger (both are typical superficial stainers) in the sanded material.
A J Ashari, J W Palfreyman, A H H Wong

Black Stain of Western red-cedar by Aureobasidium pullulans and its Relationship with Tropolone Depletion
2005 - IRG/WP 05-10564
Western red-cedar is valued for its natural durability conferred by fungicidal tropolone chemicals. However, weathered surfaces of WRC products are still susceptible to ‘black stain’ caused by fungi such as Aureobasidium pullulans. The effect of weathering on the tropolone content of heartwood was characterized and correlated with the ability of this fungal species to colonize the same weathered surfaces. UV plus water spray severely reduced tropolone content but did not lead to increased fungal colonization compared to un-weathered wood. When WRC was treated with UV only, the tropolone content was less affected but the fungal colonization increased significantly. A.pullulans exhibited high tolerance to the tropolone β-thujaplicin in vitro; thus loss in tropolone content may not be required for colonization. In addition to UV resistance from melanization and ability to use lignin breakdown products as a carbon source, resistance to tropolones may confer considerable competitive advantage to A.pullulans growing on WRC exposed to weather. The application of water spray most likely washed away products of lignin photo-degradation, leaving the wood surface void of accessible carbon sources which resulted in decreased colonization.
R Chedgy, R Daniels, P I Morris, C Breuil

Evaluation of a new anti-sapstain formulation
1994 - IRG/WP 94-30035
A new anti-sapstain mixture, which consists of 2% IPBC (3-iodo-2-propynylbutyl carbamate) and 1.5% DCOI (4,5-dichloro-2-n-octylisothiazolin-3-one), was evaluated by three methods in the laboratory. A standardized test (JWPA standard 2) demonstrated that the new anti-sapstain formulation was highly effective in controlling growth of monocultures of five test fungi on wood substrate. When exposed to mixed spore suspension, the formulation performed better than TCP-based commercial product. A larger scale laboratory tests and supplemental trials at sawmills also supported a satisfactory performance of the formulation to protect freshly sawn timber from moulds and sapstain fungi.
K Tsunoda, H Kumagai, M Sakurai

Blue stain in service on wood surface coatings. Part 1: The nutritional requirements of Aureobasidium pullulans
1992 - IRG/WP 92-1556
The nutritional requirements of Aureobasidium pullulans was examined with regard to nutrient sources that are potentially available in fresh and weathered wood. The study was designed to determine how far wood cell wall components need to be broken down during weathering before they provide a useful nutrient source for Aureobasidium pullulans. Various carbon sources were tested, with eight different isolates of Aureobasidium pullulans. It was found that the organism could utilise the simple sugars well, but not the oligosaccharides. The organism was also able to utilise well several lignin precursor compounds.
P R Sharpe, D J Dickinson

Effect of thickened boron in preventing conidial germination of sapwood-inhabiting fungi
1994 - IRG/WP 94-30059
We evaluated the efficacy of thickened boron preservative "Diffusolä" in preventing conidia germination of sapwood-inhabiting fungi using plate bioassay, Southern Yellow Pine and sweetgum block tests, and green pine log sections. The test fungi were sapstain fungi Ceratocystis coerulescens, Ceratocystis minor, and Aureobasidum pullulans and mold fungi, Aspergillus niger, Penicillium spp, and Trichoderma spp. Conidial germination were inhibited in plate bioassay by Diffusol. Treatment of Southern Pine and sweetgum blocks and green pine log sections with a 10 percent boric acid equivalent of Diffusol inhibited conidial germination of sapstain and mold fungi. In the field exposure, the same Diffusol treatment of green pine log sections inhibited natural basidiospore and conidial germination of forest-inhabiting fungi, thus preventing wood discoloration and deterioration
S C Croan

Characteristics of the pigments produced by sap-staining fungi
1997 - IRG/WP 97-10223
The present study was conducted to characterize the pigments produced by sap-staining fungi, Aureobasidium pullulans (de Bary) Arnaud and Ceratocystis piceae (Münch) H. and P. Sydow. The pigments isolated from tested fungi were identified as melanins by spectroscopic methods (UV and infrared spectra). The UV and the infrared spectra of the fungal pigments were similar to those of the synthetic melanin. The spectra also indicated the similar structures of both fungal melanins. The adsorption bands at aliphatic CH in infrared spectra were stronger in fungal melanins than in synthetic melanin. From elemental analyses, H/C ratio of fungal melanins were higher than that of synthetic melanin. These results suggested that both fungal melanins had a high amount of aliphatic structures.
M Mori, M Takahashi

Gaseous treatment of timber with allyl isothiocyanate. Fungicidal and insecticidal effects
1996 - IRG/WP 96-30108
Gaseous treatment with allyl isothiocyanate (AIT) was tested for its effects on the growth of microorganisms on the wood substrate and the mortality of subterranean termites and powder-post beetles. Toxic limits of AIT were determined as concentrations in the air when an AIT-treated filter paper was placed in a sealed container with fungus-inoculated wood specimens. Those were <3.8 ppm for Aureobasidium pullulans, 7.5-15 ppm for Aspergillus niger, 30-59 ppm for Gliocladium virens, 59-118 ppm for Penicillium funiculosum and >118 ppm for Rhizopus stolonifer. LD 50 values were determined for insects. Twenty workers of Coptotermes formosanus or 10 adults of Lyctus brunneus were placed in an air-circulated glass bottle, and the mortality of the test insects was recorded after 24 hours. LD 50/24h were 10-13 ppm and approximately 80 ppm in the air for Coptotermes formosanus and Lyctus brunneus, respectively.
K Tsunoda, T Yoshimura

Colonisation of painted wood by Aureobasidium pullulans - Analysis of features and consequences for failure in service
1996 - IRG/WP 96-10144
Wooden blocks of spruce were painted with different paint formulations. Water- and solvent-borne model and commercial paints were used. The painted wooden blocks were inoculated with a spore suspension of Aureobasidium pullulans (de Bary) Berkg. and placed in an environment of high humidity for 14 months. Different colonisation and growth patterns were observed on the different paint surfaces. The susceptibility to staining and/or degradation of tested painted wood blocks are discussed. The commercial paints with and without a fungicide became heavily stained. Model paints were not as susceptible to blue stain as the commercial paints tested. Estimation of degree of staining was done with digital image analysis. Further observations were done with a stereo microscope and scanning electron microscopy.
S L Bardage

Fungal detoxification of organotin biocides
1985 - IRG/WP 1258
The ability of a range of wood decaying fungi to inactivate bis(tri-n-butyltin) oxide (TnBTO) in the extracellular growth medium, in stationary liquid culture was determined. A distinction between the ability to tolerate the fungicide and to inactivate it was made: the white-rot organism Coriolus versicolor being the most efficient inactivator. In an attempt to determine the extracellular agents responsible for any detoxification, Coriolus versicolor was shown to produce significantly greater levels of extracellular free radicals/peroxidase. Preliminary tests have shown the nature of the associated anion on the fungicide effects the susceptibility of tributyltin compounds to free radical attack in a chemical system. The ability of an free radical scavenger to reduce detoxification in such a system has also been demonstrated.
P S Belford, D J Dickinson

The development of blue stain in service in sapwood of European softwood species
1997 - IRG/WP 97-10236
The utilisation of softwood species for window joinery applications is gaining importance in countries frequently using tropical hardwood species up to now. Since wood preservation is not always part of the total concept of window frames manufactured from sapwood containing wood elements it is essential to know the impact of durability on the performance. Moisture content control is a major tool in achieving an acceptable service-life regarding wood rot. The development of blue stain can be prevented in a similar way but other parameters than natural durability are to be considered, too. This study evaluated the development of blue stain in service in sapwood of four European softwood species: scots pine, spruce, douglas fir and larch. The results were compared with data on porosity of the same samples. As a conclusion it can be stated that not only the physical protection from water penetration is important but that the wood water relationship of a softwood species plays a major role in the development of blue stain in service, as well.
J Van Acker, R Klaassen, H Militz, M Stevens

Growth inhibitory effects on blue-stain fungi of applied electricity fields
1996 - IRG/WP 96-10167
Exploratory laboratory experiments on the effects of electricity on two blue stain fungi Aureobasidium pullulans and Ceratocystis piceae on wood revealed that a potential gradient of 1 V/cm corresponding to a current of 15 mA (DC), applied without interruption during a 2 week experimental period, leads to an inhibition of the growth of these fungi. Germination is somewhat more sensitive than mycelial growth. Experiments also revealed that a potential gradient of 10-25 V/cm applied for 30 sec, 3 times every 24 h also inhibited the growth of Aureobasidium pullulans. The mechanism by which electricity exerts its growth inhibiting effect on blue stain fungi on wood is presently unclear.
J Bjurman

Utilisation of carbohydrates by stain fungi in agar culture
1998 - IRG/WP 98-10248
Stain fungi are often defined by their ability to utilise the starch and free sugars found in ray parenchyma cells, and their inability to utilise other wood constituents. However, several species of stain fungi produce bore holes in wood cell walls. This suggests that enzymatic activity capable of degrading structural polysaccharides and/or lignin is associated with the growth of the appressorium and transpressorium structures developed by these stain fungi. This pilot study examined possible base media for growth of three common blue-stain fungi isolated from hardwood sawmills in Victoria, Australia. Alternaria alternata (Fr.) Keissler, Graphium/Ophiostoma sp., and Aureobasidium pullulans (de Bary) Arnaud were grown on agar supplemented with a variety of carbon sources, and mycelial growth rates were measured to determine which carbohydrates can be used by these fungi.
J Snow, P Vinden, S M Read

Biological control of sapwood-inhabiting fungi by living bacterial cells of Streptomyces rimosus as a bioprotectant
1992 - IRG/WP 92-1564
The objective of this study is to evaluate the efficacy of antifungal activity of living bacterial cells for the protection of wood against sapwood-inhabiting fungi. The following sapwood-inhabiting fungi were selected: sapstain --Ceratocystis coerulescens, Ceratocystis minor, Ceratocystis pilifera, and Aureobasidum pullulans; mold fungi --Aspergillus niger, Penicillium spp, and Trichoderma spp. Living bacteria cells as a bioprotectant were studied in the laboratory using Southern pine and sweetgum block tests, and in field exposure trials with green pine log sections. Living bacterial cells inhibited spore germination, and therefore discoloration in laboratory wood block tests and in pine log sections exposed in field tests.
S C Croan, T L Highley

Analysis of the degradation of carbohydrates by blue-stain fungi
1990 - IRG/WP 1457
The topic of the degradative ability of blue-stain fungi literature is not clear. Many authors support the idea that those fungi don&apos;t have enzymatic capability to decay the wall of the ligneous cells. However, others have found some decay activity. In this study, we have attempted to analyze which wall cells soluble carbohydrates are metabolized by some blue-stain fungi, found in Spain. The tested fungi have been: Pullularia pullulans and Sclerophoma pityophila (wind-dispersed), and Ceratocystis huntii and Ceratocystis ips (bark beetles transmitted). This study has been realized through the determination of: the growing rate, the amount of biomass produced by these fungi and the chemical analysis of the culture media, with six different carbohydrates (glucose, mannose, rhamnose, arabinose, galactose and xylose).
M T De Troya, A M Navarrete, E Relano

Laboratory evaluation of anti-sapstain formulations
1989 - IRG/WP 3510
Four formulations were tested for their efficacy in controlling molds and sapstaining fungi on wood according to the Standard-II of Japan Wood Preserving Association (JWPA). Relative efficacy was compared on the basis of visual rating of the fungal growth on the treated and untreated wood surfaces when the wood specimens were exposed to each monoculture of 5 test fungi at 26±2°C and 70-80% for 4 weeks. The results indicated that the formulations containing IF-1000 (p-chlorophenyl-3-iodopropargyl formal)+ TBZ[2-(4-thiazolyl)benzimidazole], TCMTB [2-(thiocyanomethylthio) benzothiazole]+MBT (methylene bis thiocyanate) and TCMTB + IPBC (3-iodo-2-propynyl butyl carbamate) were superior in anti-sapstaining effectiveness to commercial trichlorophenol-base products. However, the mixture of IF-1000+Mergal HS-100 [ 2-(methoxy-carbonyl-amino)benzimidazole-4-n-dodecyl-benzene sulfonic acid] was not satisfactorily effective against Gliocladium and Rhizopus.
K Tsunoda

Influence of the nutritional elements on pigmentation and production of biomass of bluestain fungus Aureobasidium pullulans
1997 - IRG/WP 97-10198
The effect of the carbon source and the amount of the nitrogen on the melanization and the production of mycelial mass of bluestain fungus Aureobasidium pullulans was studied by using solid state cultivations. The carbon sources used varied from easily soluble sugars to structural polysaccharides existing in lignocellulosic material. The amount of melanin was evaluated by using partial purification and the conventional measurement methods. The production of melanin was clearly dependent on the amount and quality of carbon source as well as the amount of nitrogen. A. pullulans has a high tendency to produce melanin on nitrogen poor media supplemented with easily soluble sugars (eg. glucose, sucrose, mannose and xylose). The production was restricted on nitrogen rich media. The production of melanin was totally inhibited on the media containing celluloses or lignin as sole carbon sources.
A-C Ritschkoff, M Rättö, F Thomassin

Direct analysis from wood of the blue stain fungi Aureobasidium pullulans and Hormonema dematioides by denaturing gradient gel electrophoresis
2006 - IRG/WP 06-10595
Aureobasidium pullulans and Hormonema dematioides are the two organisms used in the EN 152 laboratory method for determining the effectiveness of preservatives against blue stain in service. The literature concerning the disfigurement of surface coatings and exposed timber in-service states that A. pullulans is the dominant blue stain fungus, due to its frequent isolation from stained material. Inaccuracies when differentiating morphologically between isolates of these two related species have previously been highlighted bringing the conclusion concerning the dominance of A. pullulans to question. PCR-DGGE has been used to determine the environmental profile of the two blue stain fungi; A. pullulans and H. dematioides and to establish which of these organisms is most prevalent on a range of stained timber samples taken from trials and in service. The DGGE analysis indicated that a member of the Dothioraceae family, most likely H. dematioides was more commonly present on stained timber samples under five years old. No blue stain fungi were detectable by PCR-DGGE in older samples over 25 years old. With the correct primer choice PCR-DGGE is able to differentiate between different species and the level of abundance at which those species are represented in a sample. It is clear from this study that A. pullulans is not as dominant on stained timber as would have been expected. On this basis it is suggested that H. dematioides should be the preferred species for research and development work in this field.
M J Ray, D J Dickinson

Mechanical and chemical properties of blue stained wood
2009 - IRG/WP 09-10686
Discoloration of wood is frequently caused by blue-staining fungi. In the previous researches there was general opinion that blue-stain fungi do not influence mechanical properties. On the other hand, there were some opposite results reported as well. In order to elucidate this issue, specimens made of Pinus sylvestris sapwood were exposed to two the most frequent and important blue stain fungi Aureobasidium pullulans and Sclerophoma pithyophila for various periods between two and eight weeks. Before and after exposure, FTIR, weight, colour and modulus of elasticity measurements were performed. The data obtained showed, that blue stain fungi besides considerable discoloration, does not cause any significant damage to wood. Surprisingly the non-destructive MoE analysis showed, that modulus of elasticity even slightly increase after the experimental exposure.
M Humar, V Vek, B Bučar

Degradation of Carbon-Based Preservatives by Black-stain Fungi
2010 - IRG/WP 10-30533
Some black-stain fungi are known to exhibit resistance to carbon-based preservatives. However, the cause of this resistance is unclear. The fungi may have mechanisms to tolerate these chemicals, or they may be able to degrade and detoxify them. Fungi capable of degrading carbon-based preservatives are of interest for possible bioremediation of carbon-based preservative-treated wood at the end of its service. Moreover, such fungi could potentially facilitate the colonization of carbon-based preservative treated wood by decay fungi. Two strains of Aureobasidium pullulans and two strains of Epicoccum purpurascens were evaluated for their ability to degrade three commonly used carbon-based preservatives: propiconazole, tebuconazole, and DDAC. At low and medium concentrations propiconazole and tebuconazole were found to be vulnerable to degradation by one of the Epicoccum purpurascens isolates. Where biocides are needed to control the growth of black-stain fungi on wood, a combination of biocides with different chemistries should be used to minimize the risk posed by resistant strains.
R Stirling, P I Morris

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