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Animal blood protein as a component of new, non-toxic wood preservatives fixing organic active compounds in wood
2003 - IRG/WP 03-30312
Application of natural organic compounds and simple physico-chemical treatments in wood conservation has, as its objective, not only its protection against biological agents but, equally importantly, protection of natural environment by gradual abandonment of toxic constituents still applied commonly on a wide scale. The introduction into wood of new, non-toxic impregnation agents manufactured on the basis of animal proteins aims to create an effective protection against biological factors, primarily the reduction of the degree of leaching out of the impregnation agent from wood. Experiments were carried out on the effectiveness of mixtures of carboxyl acids with animal blood protein against Coniophora puteana fungus. The object of the performed investigations was sapwood of Scots pine (Pinus sylvestris L.) impregnated with mixtures of carboxyl acids and animal blood plasma. Directly after impregnation, wood was subjected to thermal treatment with the aim to denaturate proteins. Mycological investigations were carried out using an accelerated agar-block method according to procedures described in EN 113 and EN 84 standards. The objective of the performed studies was to assess the effectiveness of blood protein as a constituent reducing the process of washing out of preservatives from wood. A low weight loss of samples saturated with the formulation containing propionic and ethyl buthyl acids and animal protein applied in the form of SOLUTEINTM preparation was demonstrated. The degree of leaching of carboxyl acids as active constituents from wood saturated with tested mixtures was estimated applying a mycological test as well as on the basis of chemical analysis of aqueous extracts from the leaching process. The performed experiments showed good protection properties of protein, which reduced effectively the extent of leaching of the active substance from wood.
B Mazela, I Polus

Reduction of preservatives leaching from wood by the application of animal proteins
2005 - IRG/WP 05-30387
The investigations consisted in the estimation of the effectiveness of protein borates and protein propionates in wood preservation against Coniophora puteana. The estimation was made on the basis of mass losses caused by the test fungus action against treated wood which was subjected to leaching with water. The investigations were performed for Scots pine wood (Pinus sylvestris L.) treated with preservatives containing solutein, albumin or casein and subjected to appropriate thermal or chemical treatment in order to protein denaturation. The objective of the investigations was to develop the method for fixing in wood boric acid or propionic acid. On the basis of the mycological tests as well as boron content in water extracts it was found that the retreatment with tannic acid was the most effective method in respect to active substance leaching. The method let to fix up to 95% of boric acid in treated wood despite the intensive leaching.
B Mazela, I Ratajczak, M Bartkowiak

La rôle de l'expert dans l'évaluation toxicologique
1990 - IRG/WP 3589
C Boudene

Potentialities of protein borates as low-toxic, long-term wood preservatives - Preliminary trials
1999 - IRG/WP 99-30212
Boron compounds are efficient wood preservatives, as well as safe for the mammals and environmentally acceptable. Their natural solubility allows them to treat almost any wood species, but is also the cause of their high depletion from treated timber in outside exposure. In order to reduce this leachability, potentialities of proteinic polymer networks retaining boron within the wood have been investigated. Several mixtures of boric acid and proteins (including ovalbumin, collagen, casein, soya flour) have been used to treat pine sapwood miniblocks. The insoluble networks were obtained by protein gelation or coagulation, induced by a physical and/or a chemical factor. These systems appeared to retard boron leaching, the decrease of the leachability rate depending on the protein and the denaturing agent involved in the network creation. The best results have generally been observed for the irreversible heat-induced protein gels. These associations are also able to conserve some boron mobility and activity. Accelerated biological tests of leached wood samples showed good durability performances against Basidiomycetes. The use of protein borates seems to be an interesting basis for low-toxic wood preservatives. Furthermore, in some cases, proteins could add their anti-nutritional factors to boron efficacy to enhance wood protection.
M-F Thévenon, A Pizzi, J P Haluk

Molecular studies on isolates of Serpula lacrymans
1989 - IRG/WP 1421
The major protein species present in detergent extracts of 14 different Serpula lacrymans isolates have been compared, by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), with a standard strain, viz. Serpula lacrymans FPRL 12C. Following silver staining of SDS gels the major protein species identified in 12 isolates were similar to those found in the standard strain. However differences were found when the final two isolates were compared with FPRL 12C, both isolates had extra molecular species not present in 12C and both were lacking some present in the standard strain. Comparison of the protein species identified in Serpula lacrymans isolates with those identified in extracts of other fungal organisms, viz. brown and white rot causing basidiomycetes and non-basidiomycetes indicated that the Serpula isolates were more similar to each other than to other organisms. Some molecular differences could be identified when individual isolates were cultured on different media, i.e. liquid culture or agar, only minor differences were seen when individual isolates were subcultured. These results indicate that whilst care must be taken to ensure as near identical conditions as possible for culture of organisms if their molecular species are to be compared by SDS-PAGE and silver staining, consistent results can be obtained using this technique. The technique may therefore offer a method of distinguishing between isolates, strains and species of wood decay basidiomycetes, and identifying new isolates.
A Vigrow, D Button, J W Palfreyman, B King, B M Hegarty

Effect of light and ventilation condition on the rate of wood decay by the brown rot basidiomycete, Tyromyces palustris
1991 - IRG/WP 1517
Effect of light and the ventilation conditions of incubation jars on the wood decay by Tyromyces palustris (Berk. et Curt.) Murr. FFPRI 0507 was investigated. Under no irradiation of light, the ventilation conditions gave extensive effect on mass loss of the test pieces when the culturing was performed with culture medium designated in Japanese Industrial Standard (JIS) A 9302 (Medium A; glucose 4.0%, malt extract 1.5%, peptone 0.3%). On the other hand, no such kind of effect for ventilation conditions was recognized under light conditions, and the wood decay by the fungus accelerated by additional light irradiation of a considerably small intensity. Next, we investigated the relationship between light and the culture medium composition during the wood decay by the fungus. It was found that almost equivalent mass loss occurred after 60-90 days of cultures when the culturings were performed under light-shield conditions with medium A, the culture medium designated in Japan Wood Preserving Association (JWPA) standard No.1 (medium B; malt extract 2.0%, peptone 1.0%), and another culture medium diluted medium B by two times (medium C). Under the irradiation of light, the mass loss on the cultures in medium B and C was markedly less than that in the same media under no irradiation conditions. These results suggested that effect of light on the wood decay by Tyromyces palustris depended on the concentration of glucose in the culture medium. Further, we also investigated the activities of several extracellular and cell wall bound enzymes in wood meal medium contained medium A. From our experimental results, the activities of cellulase (b-1,4-glucan 4-glucanohydrolase, E.C. and mannanase (b-1,4-mannan mannanohydrolase) depended on light irradiation during the wood decay and these enzyme activities may give extensive effect on the mass loss by Tyromyces palustris.
T Suzuki, M Higaki

Transformation of Ophiostoma picea and Trichoderma harzianum with green fluorescent protein (GFP)
2003 - IRG/WP 03-10477
While microbial colonization of wood is presumed to be characterized by a myriad of interactions between numerous organisms, studying these processes is often difficult owing to the opaque nature of the wood and the inability to readily distinguish among the many species colonizing the material. One method for enhancing the ability to distinguish organisms is to induce specific proteins in one or more organisms that can be detected using fluorescence or other light microscopic techniques. The insertion of genes for the production of green fluorescent proteins produced by the jellyfish, Aequora victoria, has been widely used to visualize a variety of organisms. In this report, we describe transformation of two fungi, Ophiostoma picea and Trichoderma harzianum using a green fluorescent protein (SGFP) gene under the control of the ToxA promoter of Pyrenophora tritici-repentis. The growth and wood colonization by two transformed fungi were compared to their non-transformed strains. The expression of gfp was particularly useful for studying the spatial distribution of young hyphae in wood.
Ying Xiao, L M Ciuffetti, J J Morrell

Effect of Volatiles from Trichoderma species on the regulation of protein synthesis in Serpula lacrymans isolates
2002 - IRG/WP 02-10440
The growth of wood decay fungi has previously been shown to be inhibited by volatile organic compounds (VOCs) from selected Trichoderma isolates. No mechanism of action has however been established for such inhibition. This paper reports the effects of VOCs produced by Trichoderma pseudokoningii, T. viride and T. aureoviride on the growth of four dry rot isolates and corresponding protein synthesis in two of these Serpula lacrymans strains. Volatile inhibition of growth was measured using a simple plate bioassay with S.lacrymans mycelium subsequently extracted and protein profiles analysed by SDS-PAGE. Two S.lacrymans strains isolated from building timbers were generally more sensitive to the Trichoderma VOCs compared with a strain isolated from the Himilayas or a standard lab culture strain. VOCs from T. aureoviride and T. viride affected protein synthesis in both target S. lacrymans strains, but those from T. pseudokoningii.had no effect. These results mirrored the patterns of growth inhibition in the target fungi with VOCs from T.pseudokoningii having little effect on growth of the dry rot fungi. Interestingly, when the antagonists were removed growth of the S. lacrymans isolates resumed and the original protein patterns were restored. This suggests that the VOCs act on the basic expression of proteins by the S. lacrymans rather than interfering with protein function after synthesis.
S Humphris, R Wheatley, E Buultjens, A Bruce

Biological Safety Evaluation of Animal Contact of Preservative-Treated Wood
2003 - IRG/WP 03-50196
Biological safety of preservative-treated woods that could be contacted to human and animal was evaluated for rat and rabbit exposed to CCA-, ACQ- and CCFZ-treated woods. The accumulation of preservatives ingredient in the rat’s plasma and viscera, and the transforming function of replacing preservatives were examined in this study. The result indicated that preservative-treated wood did not bring changes for the enzyme related with replacing function of preservative components in the plasma. The accumulated amount of chrome from the CCA-treated wood was detected to be very small quantity, which was less than the measurement range. However, the presence of arsenic was detected in lung, liver, and heart of rats. Furthermore, the amount of accumulated arsenic in rat's viscera was greater than that of the untreated control. Copper, a common as ingredient of all CCA-, CCFZ- and ACQ-treated woods was detected in the liver of rat exposed to the CCA-treated sample. And the copper was also detected in the lung and liver of rat exposed to the ACQ-treated samples, while it was also present in the both liver and heart of rats exposed to the CCFZ-treated sample. To evaluate dermal safety of preservative-treated wood, clinical formulations were prepared by dissolving ACQ, CCA, and CCFZ in saline solution at five times concentration of normal treatment. Dermal irritation test was repeated for nine rabbits using the prepared solution. No changes were detected for rabbits exposed to daily dermal irritation using the solution for 14 days. And cumulative dermal irritation (CDI) score using the solution for 14 days was equal to zero. This result demonstrated that the local irritation of the preservatives tested to the intact skin was negligible. Histopathological examination revealed no hemorrhage and edematous change of the subcutaneous tissue in the rabbits treated with ACQ, CCA and CCFZ.
Dong-heub Lee, Dong-won Son, Myung Jae Lee, Chang Ho Kang, Cheon Ho Kim, Eui-Bai Jeung

Effects of Prior Establishment of Trichoderma harzianum on Ophiostoma picea Growth in Freshly Sawn Douglas-fir Sapwood
2003 - IRG/WP 03-10476
Trichoderma harzianum has been shown to be an effective biocontrol agent against a number of wood inhabiting fungi under laboratory conditions, but this fungus has performed poorly in field trials. Understanding the interactions between biocontrol agents and their intended targets in wood may provide important clues for developing improved approaches to biocontrol, potentially reducing our reliance on pesticides. One particularly difficult problem with studying biocontrol in wood is the inability to accurately resolve individual organisms as they interact. The opaque nature of wood makes it difficult to observe interactions more than a few cells from the surface. Wood can be cut into thin sections, but the structural similarity of many fungal hyphae makes it difficult to determine which fungus is present. Transformation using genes for the synthesis of green fluorescent protein (GFP) has proven useful for visualizing specific microorganisms in their hosts and could be useful for biocontrol studies. In this study, interactions between gfp transformed Ophiostoma picea and non-transformed T. harzianum were studied on freshly sawn Douglas-fir sapwood blocks. Growth of O. picea was significantly retarded when applied to wood blocks where T. harzianum was well-established. Interestingly the Graphium state of O. picea was still observed on surfaces of the blocks, but the fungus was unable to penetrate deeply into the wood. The results suggest that failure of the biocontrol may not represent an inability to protect the wood beneath the surface and implies that a more detailed study of the causes of previous failures would be useful.
Ying Xiao, J J Morrell, L M Ciuffetti

Simulation test of subchronic inhalation toxicity of TBTO vapours in the air
1987 - IRG/WP 3454
The test was carried out in a wainscotted room of a normal size. Wooden panelling had been treated with one coat of basic preserving agent and, after the installation, with 2 coats of stain. Laboratory animals (rats) were kept in this room for 90 days. For the same period a control group of test animals was kept in a room of equal size where there was no panelling. During the test, concentration of tin (Sn) in the air (originating from TBTO) was measured and the weight of the animals was taken. When the test was completed, typical characteristics of animal tissue likely to be affected by TBTO, were determined. Throughout the test, the quantity of Sn in 1 m³ of air had been approx. 1 µg. No changes i.e. differences in typical parameters of both the test and control animals have been observed (histological tests have not been made yet).
V Skubic, S Kobal, J Stupar, R Ajlec, J Korošin, G R Pecenko

Extracellular carbohydrate production by isolates of Postia (=Poria) placenta
1989 - IRG/WP 1388
A monokaryotic strain of Postia (=Poria) placenta, ME20, which is unable to degrade wood, also failed to produce extracellular polysaccharide when grown in liquid culture, regardless of carbon source or concentration. Other isolates of Postia placenta, including another monokaryon and a hybrid of this monokaryon with ME20, produced large quantities of this material. The polysaccharide consisted primarily of glucose upon acid hydrolysis and resembled the glucan reported in culture filtrates of other wood decay fungi. It was produced primarily during the logarithmic phase of growth. Isolate ME20 formed high levels of laminarinase and glucan-degrading enzymes compared to the other isolates; the glucan of ME20 may be prematurely degraded. This study supports the importance of the extracellular matrix in the wood-decay process.
J A Micales, A L Richter, T L Highley

Characterization and differentiation of wood rotting fungi by protein and enzyme patterns
1999 - IRG/WP 99-20177
Standardized tests for wood preservatives are performed with defined fungal strains to ensure comparability between laboratories. However, changes of virulence and variation of results are well known events. Suitable and reliable measures to control the stability of the test organisms are necessary.Comparison of protein patterns produced by SDS-electrophoresis was already described by several authors as a possible way to identify and to characterize fungal species and strains, i.e. for Serpula, Coniophora and Poria. We compared protein patterns of several strains of Antrodia vaillantii, Poria placenta, Gloeophyllum trabeum, Lentinus lepideus, and Coniophora puteana. Isoelectric focussing and detection of esterase isoenzymes proved to be an alternative method. The investigations resulted in species-specific protein and enzyme patterns. By comparing the strains of single species it was possible to form groups with similar patterns. Possible misidentifications could be detected. The described methods will be further developed and used to follow possible changes in our test strains.
U Schoknecht

Characterization of protein patterns from decayed wood of loblolly pine (Pinus taeda L.) by proteomic analysis
2008 - IRG/WP 08-10654
The primary biotic decomposers of wood belong to the basidiomycetes. The members of this group can attack and biodegrade both wood in the forest and in service. By the time wood decay is visible, there has already been a significant loss of strength. The identification of basidiomycetes and other organisms on wood only tell us what is present, not what is actively decaying the wood. When organisms are metabolically active, such as during wood decay, they produce proteins, some of which are unique to the decay process. Detection and identification of the fungal proteins involved in wood biodegradation would be an advantage in helping to understand the complex biodegradation pathways. In this study, we concentrated on proteomics as a tool to decipher biodeterioration-linked proteins. Proteomic analysis of fresh wood (southern yellow pine), decayed wood, inoculated decayed wood, and Gloeophyllum trabeum were performed. More than 170 proteins from four treatments were visualized on Commassie-stained two-dimensional polyacrylamide gels with a high resolution and reproducibility. These protein spots were subjected to in-gel digestion with trypsin for peptide fingerprint analysis by MALDI-TOF-MS. The tryptic peptides were identified with the aid of a BLAST homology search which found 76 unique proteins from inoculated decayed wood. No proteins were detected from fresh wood. Over 110 proteins were visualized from Gloeophyllum trabeum grown in culture. Among the proteins identified were oxidative enzymes and hydrogen peroxidases. Only actin was identified from decayed wood, but inoculated decayed wood contained wood degradation proteins such as alcohol oxidase, lipoxygenase, and catalases.
Young-Min Kang, L Prewitt, S Diehl

Structural changes, basidiomycete richness, enzyme activity and proteomic profiling of decay resistant and non-resistant woods over 18 months in soil contact
2010 - IRG/WP 10-10733
Wood decay fungi damage wood by production of enzymes that attack the structural components. The objective of this study was to better understand which suite of decay genes and proteins are expressed during biodeterioration of three different wood types in forest soil over time. Variation in decay genes and proteins were determined for pine (non-resistant), cedar (naturally durable), and ACQ-treated pine (chemically resistant) in a soil decay bed. Decay was assessed by visual decay ratings, dynamic MOE, and microscopy. There were no significant difference in decay between cedar and ACQ-treated pine over the 18 month period. However, there were significant differences in decay between pine and cedar and between pine and ACQ-treated pine. The fungal mycelia penetrated the cell walls of pine and were continually observed over 18 months, but not in cedar or ACQ-treated pine. Basidiomycetes containing decay genes were detected on pine which also had a greater diversity of fungi compared to cedar and ACQ-treated pine. No basidiomycete genes were expressed and only a few basidiomycetes were identified on cedar which also showed little decay. ACQ-treated pine also showed a little decay however basidiomycetes were present and active. Proteins were first detected on pine and ACQ-treated pine at 6 months and the numbers continued to increase through 18 months, but were not detected on cedar until 14 months. There was greater number of total proteins on pine than cedar and ACQ-treated pine at each time period. From these results, the natural durability of cedar reduced the wood decay community and its activities. It appears that ACQ-treated wood did not stop the growth of the decay fungi and the production of the decay enzymes but the chemical treatment did inhibit the effectiveness of the enzymes thus decay.
Youngmin Kang, S Diehl, L Prewitt, D Nicholas

Information on a project about the conditions of admissibility of wood-protecting agents in connection with environmental protection in Poland
1974 - IRG/WP 57
Wood-protecting agents are compounds acting toxically on fungi and insects. If handled improperly or carelessly, they may exert an action harmful to health and safety of men. This action is concerning: a) workers employed at the production of wood-protecting agents; b) workers employed at the impregnation, or at the transport and handling of impregnated wood; c) inhabitants of buildings with impregnated wooden elements, or which have been treated against fungi. At the suggestion of the Scientific and Technical Committee for Wood Protection, medical institutes started investigations concerning the influence of wood-protecting agents on human health. A draft has been worked out on the terms of admissibility for the use of these agents. The toxicological specification of a wood-preserving agent has to include the results of the following tests: 1) Determination of hypertoxicity LD50 per os for rats; 2) Determination of toxicity LD50 per coeliacus for rats; 3) Determination of irritant action on the eye mucosa and the skin of the rabbit; 4) Determination of allergenic action on the skin of the guinea pig; 5) Determination of injuring action by histopathological method; 6) Determination of toxicity LD50 by inhalation after 4 hours by rats, given the content of the active substance in mg in 1 litre of air; 7) Determination of the quantity of wood-protecting agents in the air, by means of the cabin method, and their disappearance. The above mentioned determinations are to be carried out according to methods generally accepted for toxicological tests. In certain cases, they have to be adapted to the requirements of the wood-protecting agents tests. As a fundamental criterior for the evaluation of wood-protecting agents in relation to toxical noxiousness we take the toxicity per os of the whole compound, expressed in mg per kg of weight of the living experimental animal (LD50). The other determinations are of complementary character, and in the case of unfavourable results, they cause the agent to be classified at a lower class than having been classified by the fundamental criterion LD50 alone. A classification of 5 degrees has been worked out for the wood-protecting agents, based on the classification of Hodge-Sterner: Degree I: hypertoxic agent (LD50: below 50 mg/kg); Degree II: toxic agent (LD50: 51-150 mg/kg); Degree III: noxious agent (LD50: 151-500 mg/kg); Degree IV: less noxious agent (LD50: 501-5000 mg/kg); Degree V: practical not noxious agent (LD50: above 5000 mg/kg). In accordance with the accepted criteria the toxicity of some wood-protecting agents is as follows: 1) Arsenic compounds - Degree I of toxicity; 2) Sodium fluoride - Degree II of toxicity; 3) Sodium fluoride + chromium salts - Degree II of toxicity; 4) Fluoride/borates + chromium salts - Degree II of toxicity; 5) Zinc fluosilicate - Degree III of toxicity; 6) Bifluorides - Degree III of toxicity; 7) Borax, boric acid - Degree V of toxicity. In Poland the use of wood-protecting agents of the Degree I of toxicity is prohibited. It is planned to withdraw from use progressively wood-protecting agents having higher degrees of toxicity. The present draft and the suggested classification are of preliminary character. They are being submitted for further investigation and discussion to the authorities concerned with health protection and environmental protection.
J Wazny

Protein extraction from wood decay fungus Postia placenta
2014 - IRG/WP 14-10827
Wood decay fungi (often distinguished as white rot and brown rot) belonging to the basidiomycetes, are common inhabitants of forest litter, where they play an important role in carbon cycling. Brown rot fungi are perhaps the most important organism involved in the degradation of wood products, and of considerable economic importance. Brown rotted wood loses strength very early because of the rapid depolymerisation of cellulose. Identification of specific genes and enzymes involved in conversion of lignocellulose is of growing interest not only in the field of wood preservation but also to bioenergy process development. To determine which proteins are being uniquely expressed during the decay process, proteomic profiles can be used to compare fungi growing on different carbon sources, for example untreated wood and modified wood. With a better understanding of the fungal decay mechanisms, wood protection processes and product properties could be further improved and the underlying mechanisms of inhibition or delay of biological degradation in modified wood could be revealed. The aim of this paper was to develop a straight forward method for protein extraction suitable for the filamentous fungus Postia placenta - with low health hazards. We conclude that so far, the most promising method with regard to simplicity and health issues, is a sodium chloride based extraction procedure followed by a precipitation based concentration with trichloroacetic acid (TCA). Another concentration protocol based on filtration is also looking very promising and will be further investigated.
A Pilgård, P Arnold, K Richter