CA2047445A1 - Method for protection of lumber against sapstain - Google Patents
Method for protection of lumber against sapstainInfo
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- CA2047445A1 CA2047445A1 CA 2047445 CA2047445A CA2047445A1 CA 2047445 A1 CA2047445 A1 CA 2047445A1 CA 2047445 CA2047445 CA 2047445 CA 2047445 A CA2047445 A CA 2047445A CA 2047445 A1 CA2047445 A1 CA 2047445A1
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- gliocladium
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B27—WORKING OR PRESERVING WOOD OR SIMILAR MATERIAL; NAILING OR STAPLING MACHINES IN GENERAL
- B27K—PROCESSES, APPARATUS OR SELECTION OF SUBSTANCES FOR IMPREGNATING, STAINING, DYEING, BLEACHING OF WOOD OR SIMILAR MATERIALS, OR TREATING OF WOOD OR SIMILAR MATERIALS WITH PERMEANT LIQUIDS, NOT OTHERWISE PROVIDED FOR; CHEMICAL OR PHYSICAL TREATMENT OF CORK, CANE, REED, STRAW OR SIMILAR MATERIALS
- B27K3/00—Impregnating wood, e.g. impregnation pretreatment, for example puncturing; Wood impregnation aids not directly involved in the impregnation process
- B27K3/002—Impregnating wood, e.g. impregnation pretreatment, for example puncturing; Wood impregnation aids not directly involved in the impregnation process employing compositions comprising microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
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- Agricultural Chemicals And Associated Chemicals (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
ABSTRACT
The present invention consists of a method of protecting wood or wood products against unwanted sapstain by treating said wood with one or more biological control microorganisms selected from the genus Gliocladium, (fungi: Hyphomycetes), that either prevents the growth of undesirable sapstaining organisms, or prevents the formation of discolouration by these organisms.
The present invention consists of a method of protecting wood or wood products against unwanted sapstain by treating said wood with one or more biological control microorganisms selected from the genus Gliocladium, (fungi: Hyphomycetes), that either prevents the growth of undesirable sapstaining organisms, or prevents the formation of discolouration by these organisms.
Description
~047~
BACKGROUND OF THE INVENTION
Sapstain of unseasoned lumber is a cosmetic defect that is considered objectionable by many buyers. These discolorations, caused by a variety of microfungi, are a serious problem on lumber stored in lumber yards after sawing, but prior to planing and chemical treatment, and also on untreated lumber that is exported abroad. For the Canadian forest products industry, hem-fir products, spruce-pine-fir or white pine products are particularly prone to sapstain.
Several phenomena combine to create the discolorations.
Sapstaining fungi generally discolour the wood brown, grey or black. The stain is caused by the pigmented fungal hyphae that accumulate in the cells of the sapwood, particularly in the rays.
There is also evidence that dense accumulations of unpigmented hyphae in the wood tissue can cause similar discolorations. Some microfungi discolour wood by the production of coloured spores or sporulating structures. In addition, some species discolour wood red, purple, green or yellow by producing extracellular pigments that diffuse into the wood tissues.
Sapstaining fungi are primary colonizers of wood that 20 subsist mainly on soluble nutrients. Although they cause little structural damage, they are perceived as forerunners of decay fungi by many consumers, and thus the objection to sapstain dis-coloration may have a more practical basis than just aesthetics.
Many different chemicals have been used to control sapstain. In Canada, the most widely used chemical formulations incorporate the chemicals, 2( thiocyanomethyl)benzothiazole, 2 ~ ~ 7 ~ ~ ~
copper-8-quinolinolate, borax or didecyldimethyl ammonium chloride. The Canadian lumber industry has stated its intention of eLiminating the use of toxic chemicals for sapstain control.
Biological control is a relatively new concept in forest products. In biological control, a "harmless organism", in this case one that does not decay or discolour wood, is deliberately added to a product in order to prevent, retard or stop the growth of undesirable organisms. The most widely used biological control product is the bacterium Bacillus thuringiensis, better known as BT, which is used to control spruce budworm in Ontario and parts of Quebec. The bacterium produces a toxic crystal that is eaten by the budworm as it eats the leaves, eventually killing the pest.
Approximately half a dozen biological control systems have been marketed for agricultural use, for example DeVineTM, a fungal control of parasitic vines in citrus orchards. Below, some exam-ples of biological control related to wood products pathology are reviewed.
The best known example of biological control in forest products relates to the control of decay in wooden transmission poles by the injection into the wood tissue of a dart containing spores or mycelium of so-called "immunising commensals", as described by J. Ricard in Canadian patents nos. 963387 and 1106201. Ricard claims a wide range of applications for his invention, but all claims of said invention relate to the possibility of inoculating biological control agents into wood tissue, and none of the claims relate to the prevention of sap-stain.
~' 20~7~
BACKGROUND OF THE INVENTION
Sapstain of unseasoned lumber is a cosmetic defect that is considered objectionable by many buyers. These discolorations, caused by a variety of microfungi, are a serious problem on lumber stored in lumber yards after sawing, but prior to planing and chemical treatment, and also on untreated lumber that is exported abroad. For the Canadian forest products industry, hem-fir products, spruce-pine-fir or white pine products are particularly prone to sapstain.
Several phenomena combine to create the discolorations.
Sapstaining fungi generally discolour the wood brown, grey or black. The stain is caused by the pigmented fungal hyphae that accumulate in the cells of the sapwood, particularly in the rays.
There is also evidence that dense accumulations of unpigmented hyphae in the wood tissue can cause similar discolorations. Some microfungi discolour wood by the production of coloured spores or sporulating structures. In addition, some species discolour wood red, purple, green or yellow by producing extracellular pigments that diffuse into the wood tissues.
Sapstaining fungi are primary colonizers of wood that 20 subsist mainly on soluble nutrients. Although they cause little structural damage, they are perceived as forerunners of decay fungi by many consumers, and thus the objection to sapstain dis-coloration may have a more practical basis than just aesthetics.
Many different chemicals have been used to control sapstain. In Canada, the most widely used chemical formulations incorporate the chemicals, 2( thiocyanomethyl)benzothiazole, 2 ~ ~ 7 ~ ~ ~
copper-8-quinolinolate, borax or didecyldimethyl ammonium chloride. The Canadian lumber industry has stated its intention of eLiminating the use of toxic chemicals for sapstain control.
Biological control is a relatively new concept in forest products. In biological control, a "harmless organism", in this case one that does not decay or discolour wood, is deliberately added to a product in order to prevent, retard or stop the growth of undesirable organisms. The most widely used biological control product is the bacterium Bacillus thuringiensis, better known as BT, which is used to control spruce budworm in Ontario and parts of Quebec. The bacterium produces a toxic crystal that is eaten by the budworm as it eats the leaves, eventually killing the pest.
Approximately half a dozen biological control systems have been marketed for agricultural use, for example DeVineTM, a fungal control of parasitic vines in citrus orchards. Below, some exam-ples of biological control related to wood products pathology are reviewed.
The best known example of biological control in forest products relates to the control of decay in wooden transmission poles by the injection into the wood tissue of a dart containing spores or mycelium of so-called "immunising commensals", as described by J. Ricard in Canadian patents nos. 963387 and 1106201. Ricard claims a wide range of applications for his invention, but all claims of said invention relate to the possibility of inoculating biological control agents into wood tissue, and none of the claims relate to the prevention of sap-stain.
~' 20~7~
Several research teams around the world have published results of screening programs for biological control organisms for the prevention of wood decay. The concept of Ricard, using mix-tures of Trichoderma spp. and Scytalidium sp. to control decay in transmission poles, has been investigated by other research teams in the United Kingdom, the ~nited States and the Federal Republic of Germany. Antifungal metabolites of Scytalidium sp. have been isolated and chemically characterised, along with antifungal metabolites from Hyalodendron sp. and Cryptosporiopsis sp., and these metabolites have been applied to wood in an attempt to prevent decay.l Another application of biological control organisms is to inhibit decay in round wood in storage. Shields2 reported that decay by Bjerkandera adusta, Coriolus hirsutus and C. versicolor was inhibited in wood blocks precolonized with Trichoderma haræianum or an unidentified strain (now known to be Scytalidium lignicola). The strain of T. harzianum was later used in a field test on birch bolts3 where a conidial suspension was sprayed onto freshly cut ends of birch bolts. After a two week precolonization period, Blerkandera adusta was inoculated onto the bolts. After six months, very little B. adusta was re-isolated from the bolts.
Stilwell4 isolated a strain of Cryptosporiopsis sp. from yellow birch that inhibited the growth of 31 decay fungi in agar interactions. Decay of blocks by Fomes fomentarius was inhibited in precolonization experiments. In a field test, decay was reduced in peeled birch logs inoculated with a water suspension of Cryptosporiopsis sp., but no significant difference was noted in 20~74~5 unpeeled logs. Culture filtrates of Cryptosporiopsis also inhibi-ted growth of F. fomentarius. The antibiotic metabolite was puri-fied, characterized and given the name cryptosporiopsin.5 Decay of wood chips during storage was also considered as a possible target for antagonistic microorganisms. Bergman and ~ilsson6 tested several mould fungi isolated from wood chips for their ability to inhibit chip decay in laboratory experiments, and found that most decay fungi were inhibited. Gliocladium viride, a mycoparasite frequently isolated from chips, was tested on spruce chips in the field, and inhibited decay at temperatures less than 30C, but failed at higher temperatures.
Conifer chips inoculated with an antibiotic-producing ryptosporiopsis sp. and stored outdoors for 12-15 months yielded an improved quality of pulp although decay was not completely inhibited.7 The results of trials using the antibiotic as a chemical preservative, and of a proposed field test, have not been published.
Bacteria were also tested as biological control agents in chip piles. Some bacteria isolated from hardwood chips were inhibitory to selected decay fungi in agar interactions, but the antagonism was only effective on wood when the bacteria were in-oculated onto the wood several weeks before the decay fungi.8 The results of a planned field trial were not published.
The possibility of controlling sapstain by using antago-nistic organisms has also received some attention. The early work of Stilwell and his colleagues9 demonstrated the antagonism of some microorganisms towards some sapstain fungi. Stranks10 found 2~7~5 that 0.25% and 0.50% solutions of the antibiotic hyalodendrin, applied to white pine blocks by dipping, were effective at preventing sapstain by Graphium sp., while cryptosporiopsin was ineffective. Seifert et al.ll screened a variety of microfungi for their abilities to prevent sapstain precolonization experi-ments, and identified Nectria cinnabarina, Gliocladium roseum, Trichoderma spp. and Tympanis sp. as promising candidates.
Russian workersl2 have demonstrated in vitro inhibition of sapstain fungi by unidentified bacteria, but have not demons-trated efficiency on wood. Bernier and colleaguesl3 showed that an isolate of Bacillus subtilus prevented sapstain when wooden -blocks dipped into a cell suspension were placed on agar plates inoculated with sapstaining fungi, but subsequent work at Forintek Canada Corp. in Ottawa, Canada with the same culture showed that it did not inhibit sapstain when the wood was not placed on agar.
The bacterium colonized wood very poorly and this prevented effective biological control. Benkol4 has recently screened many bacteria for antagonism towards sapstain fungi in agar inter-actions, and has selected some strains of Pseudomonas for further study.
Some innovative approaches towards biocontrol of saps-tain have also been tried. Johnsonl5 studied polyoxin, an anti-biotic that inhibits the synthesis of chitin, a major component of fungal cell walls. The eight sapstain and mould species tested were sensitive to this compound but at concentrations too high to be economical on a commercial scale. Benkol6 demonstrated that crude culture extracts of some antibiotic producing mycorrhizal fungi prevented growth of several sapstaining fungi on blocks of pine.
FIELD OF THE INVENTION
The present invention consists of a method of protecting unseasoned softwood lumber against unwanted sapstain by inocula-tion of said lumber with the unique biological control microorga-nisms from the genus Gliocladium, (fungi: Hyphomycetes), that either prevents the growth of undesirable sapstaining organisms, or prevents the formation of discolouration by these organisms.
The biological control organism is a fungus that does not itself decay or discolour the wood to any objectionable extent, and comprises one or more of the following strains:
Giocladium aureum (Forintek Culture Collection, FTK 784A), Gliocladium roseum (FTK 321A, 321M), Gliocladium solani (FTK 810A), Gliocladium viride (FTK 623E), Gliocladium virens (FTK 258C, FTK 258D).
SUMMARY OF THE INVENTION
The present invention relates to a method for the protection of wood or wood products against unwanted discoloration caused by sapstain fungi. It is a feature of the present inven-tion to provide a method of controlling sapstain in wood and wood products comprising treating the wood or wood product with an inoculum comprising one or more fungi of the genus Gliocladium.
20~7~
Stilwell4 isolated a strain of Cryptosporiopsis sp. from yellow birch that inhibited the growth of 31 decay fungi in agar interactions. Decay of blocks by Fomes fomentarius was inhibited in precolonization experiments. In a field test, decay was reduced in peeled birch logs inoculated with a water suspension of Cryptosporiopsis sp., but no significant difference was noted in 20~74~5 unpeeled logs. Culture filtrates of Cryptosporiopsis also inhibi-ted growth of F. fomentarius. The antibiotic metabolite was puri-fied, characterized and given the name cryptosporiopsin.5 Decay of wood chips during storage was also considered as a possible target for antagonistic microorganisms. Bergman and ~ilsson6 tested several mould fungi isolated from wood chips for their ability to inhibit chip decay in laboratory experiments, and found that most decay fungi were inhibited. Gliocladium viride, a mycoparasite frequently isolated from chips, was tested on spruce chips in the field, and inhibited decay at temperatures less than 30C, but failed at higher temperatures.
Conifer chips inoculated with an antibiotic-producing ryptosporiopsis sp. and stored outdoors for 12-15 months yielded an improved quality of pulp although decay was not completely inhibited.7 The results of trials using the antibiotic as a chemical preservative, and of a proposed field test, have not been published.
Bacteria were also tested as biological control agents in chip piles. Some bacteria isolated from hardwood chips were inhibitory to selected decay fungi in agar interactions, but the antagonism was only effective on wood when the bacteria were in-oculated onto the wood several weeks before the decay fungi.8 The results of a planned field trial were not published.
The possibility of controlling sapstain by using antago-nistic organisms has also received some attention. The early work of Stilwell and his colleagues9 demonstrated the antagonism of some microorganisms towards some sapstain fungi. Stranks10 found 2~7~5 that 0.25% and 0.50% solutions of the antibiotic hyalodendrin, applied to white pine blocks by dipping, were effective at preventing sapstain by Graphium sp., while cryptosporiopsin was ineffective. Seifert et al.ll screened a variety of microfungi for their abilities to prevent sapstain precolonization experi-ments, and identified Nectria cinnabarina, Gliocladium roseum, Trichoderma spp. and Tympanis sp. as promising candidates.
Russian workersl2 have demonstrated in vitro inhibition of sapstain fungi by unidentified bacteria, but have not demons-trated efficiency on wood. Bernier and colleaguesl3 showed that an isolate of Bacillus subtilus prevented sapstain when wooden -blocks dipped into a cell suspension were placed on agar plates inoculated with sapstaining fungi, but subsequent work at Forintek Canada Corp. in Ottawa, Canada with the same culture showed that it did not inhibit sapstain when the wood was not placed on agar.
The bacterium colonized wood very poorly and this prevented effective biological control. Benkol4 has recently screened many bacteria for antagonism towards sapstain fungi in agar inter-actions, and has selected some strains of Pseudomonas for further study.
Some innovative approaches towards biocontrol of saps-tain have also been tried. Johnsonl5 studied polyoxin, an anti-biotic that inhibits the synthesis of chitin, a major component of fungal cell walls. The eight sapstain and mould species tested were sensitive to this compound but at concentrations too high to be economical on a commercial scale. Benkol6 demonstrated that crude culture extracts of some antibiotic producing mycorrhizal fungi prevented growth of several sapstaining fungi on blocks of pine.
FIELD OF THE INVENTION
The present invention consists of a method of protecting unseasoned softwood lumber against unwanted sapstain by inocula-tion of said lumber with the unique biological control microorga-nisms from the genus Gliocladium, (fungi: Hyphomycetes), that either prevents the growth of undesirable sapstaining organisms, or prevents the formation of discolouration by these organisms.
The biological control organism is a fungus that does not itself decay or discolour the wood to any objectionable extent, and comprises one or more of the following strains:
Giocladium aureum (Forintek Culture Collection, FTK 784A), Gliocladium roseum (FTK 321A, 321M), Gliocladium solani (FTK 810A), Gliocladium viride (FTK 623E), Gliocladium virens (FTK 258C, FTK 258D).
SUMMARY OF THE INVENTION
The present invention relates to a method for the protection of wood or wood products against unwanted discoloration caused by sapstain fungi. It is a feature of the present inven-tion to provide a method of controlling sapstain in wood and wood products comprising treating the wood or wood product with an inoculum comprising one or more fungi of the genus Gliocladium.
20~7~
The inoculum is of sufficient concentration and vigour to allow rapid colonization of the wood tissue by the inoculated biological control fungus. The actively growing and metabolizing biological control fungus does not itself damage or discolour the wood, but, likely through antibiotic facilities or mycoparasitism, protects against discoloration of the wood by undesirable organisms already present in the wood tissue, or that may be introduced to the wood tissues during handling of the lumber. Because the biological control fungus must subsist only on nonstructural wood carbohydrates, the duration of control is of necessity finite, and can be expected to become less effective as easily assimilable nutrients are depleted, perhaps up to one year after inoculation of the wood with the biological control agent. The wood or wood product is preferably unseasoned softwood lumber such as conifer wood.
The invention also relates to a wood or wood product treated with a Gliocladium sp. Accordingly, it is another feature of the present invention to provide a wood or wood product treated with a fungus of the genus Gliocladium that is essentially free of sapstain as the result of the activity of said fungus.
The invention is intended for use primarily in situa-tions where sapstain is prevalent, but for which chemical protec-tion is impractical or impossible. Suggested applications include the protection of freshly sawn timber during seasoning, prior to planing and subsequent chemical treatment, protection of export lumber where chemical treatment, or specific chemical treatments, are forbidden by the importing countries, or treatment of wood ~0~'74~
The invention also relates to a wood or wood product treated with a Gliocladium sp. Accordingly, it is another feature of the present invention to provide a wood or wood product treated with a fungus of the genus Gliocladium that is essentially free of sapstain as the result of the activity of said fungus.
The invention is intended for use primarily in situa-tions where sapstain is prevalent, but for which chemical protec-tion is impractical or impossible. Suggested applications include the protection of freshly sawn timber during seasoning, prior to planing and subsequent chemical treatment, protection of export lumber where chemical treatment, or specific chemical treatments, are forbidden by the importing countries, or treatment of wood ~0~'74~
chips during storage.
The genus Gliocladium is a biologically diverse group of moulcls (Hyphomycetes) that includes species that are parasites of other fungi (mycoparasites), parasites of slime moulds, parasites of plant roots, and species that grow in soil and on wood.
Gliocladium roseum is commonly isolated from soil in many parts of the world and is one of the most aggressive mycoparasites known.
All the tested isolates are effective biological control agents.
The isolates that we have employed are identified below. All of these were collected independently from each other as follows:
0 FTK 784A: Gliocladium aureum Rader, isolated by W.E. Rader from stored root of Daucus carota (carrot), received from H.
Bruckner, (Germany) (Centraalbureau voor schimmel-cultures 226.48, American Type Cult~re Collection 10406).
FTK 623E: Gliocladium viride Matr., isolated by M. Hawara from hardwood chip, Thurso, Quebec, May 30, 1988, identified by K.A. Seifert.
FTK 321A: Gliocladium roseum Bainier, isolated by C.K.J. Wang from soil debris, Natural Bridge, N.Y. July 11, 1980.
0 FTK 321M: Gliocladium roseum Bainier, isolated from yellow poplar stump, West Virginia, 1953/HL Barnett 914 (University of Alberta Microfungus and Herbarium 419).
FTK 810A: Gliocladium solani (Harting) Petch, isolated by T.
Benedek. identified by W. Gams, (Centraalbureau voor 2~47~
The genus Gliocladium is a biologically diverse group of moulcls (Hyphomycetes) that includes species that are parasites of other fungi (mycoparasites), parasites of slime moulds, parasites of plant roots, and species that grow in soil and on wood.
Gliocladium roseum is commonly isolated from soil in many parts of the world and is one of the most aggressive mycoparasites known.
All the tested isolates are effective biological control agents.
The isolates that we have employed are identified below. All of these were collected independently from each other as follows:
0 FTK 784A: Gliocladium aureum Rader, isolated by W.E. Rader from stored root of Daucus carota (carrot), received from H.
Bruckner, (Germany) (Centraalbureau voor schimmel-cultures 226.48, American Type Cult~re Collection 10406).
FTK 623E: Gliocladium viride Matr., isolated by M. Hawara from hardwood chip, Thurso, Quebec, May 30, 1988, identified by K.A. Seifert.
FTK 321A: Gliocladium roseum Bainier, isolated by C.K.J. Wang from soil debris, Natural Bridge, N.Y. July 11, 1980.
0 FTK 321M: Gliocladium roseum Bainier, isolated from yellow poplar stump, West Virginia, 1953/HL Barnett 914 (University of Alberta Microfungus and Herbarium 419).
FTK 810A: Gliocladium solani (Harting) Petch, isolated by T.
Benedek. identified by W. Gams, (Centraalbureau voor 2~47~
schimmel cult~res 187.29).
FTK 258C: Gliocladium virens. Miller et al., from W.H. Weston T-l (American Type Culture Collection 9645).
FTK 258D: Gliocladium virens. Miller et al. from Dr.S. Gyorgy, Budapest, Hungary, identified by J. Bisset (All-Union Collection of Non-Pathogenic Organisms, Institute of Microbiology, USSR Academy of Sciences - 1117).
Cultures of the isolates referred to above are available upon request made to Forintek Culture Collection of Forintek Canada Corp., 800 Montreal Road, Ottawa, Ontario, Canada, KlG
3Z5. Please note that any culture described in the specification that is available from the Forintek Culture Collection possesses a number that is preceded by the letters, FTK.
DETAILED DESCRIPTION OF THE INVENTION
The examples below describe the effectiveness of Glio-cladium spp. in preventing or inhibiting sapstain or sapstain fungi, and demonstrate that Gliocladium spp. does not itself damage wood.
An inoculum of the biological control agent is prepared by removing plugs of agar from stock cultures and growing them on agar in petri dishes. The growth medium employed is typically DifcoTM malt extract with 2~ agar (2% MA) for the Gliocladium spp. and for the sapstaining fungi.
Stock cultures are maintained at 4C on agar media 20~7~
FTK 258C: Gliocladium virens. Miller et al., from W.H. Weston T-l (American Type Culture Collection 9645).
FTK 258D: Gliocladium virens. Miller et al. from Dr.S. Gyorgy, Budapest, Hungary, identified by J. Bisset (All-Union Collection of Non-Pathogenic Organisms, Institute of Microbiology, USSR Academy of Sciences - 1117).
Cultures of the isolates referred to above are available upon request made to Forintek Culture Collection of Forintek Canada Corp., 800 Montreal Road, Ottawa, Ontario, Canada, KlG
3Z5. Please note that any culture described in the specification that is available from the Forintek Culture Collection possesses a number that is preceded by the letters, FTK.
DETAILED DESCRIPTION OF THE INVENTION
The examples below describe the effectiveness of Glio-cladium spp. in preventing or inhibiting sapstain or sapstain fungi, and demonstrate that Gliocladium spp. does not itself damage wood.
An inoculum of the biological control agent is prepared by removing plugs of agar from stock cultures and growing them on agar in petri dishes. The growth medium employed is typically DifcoTM malt extract with 2~ agar (2% MA) for the Gliocladium spp. and for the sapstaining fungi.
Stock cultures are maintained at 4C on agar media 20~7~
containing 2% DifcoTM malt extract as a nutrient source.
All other incubations described in this example are at 27C, 75%
relative humidity, in the dark.
The fungi used as biological control agents in this example are selected from the following strains maintained in the Forintek culture collection of wood-inhabiting fungi: Gliocladium roseum 321M, Gliocladium aureum 784A, Gliocladium solani 810A
Sapstaining fungi employed are: Ophiostoma piceae FTK 387I, O.
piliferum (Fr.) H. & P. Sydow FTK 55F, FTK 55H and Ophiostoma sp.
FTK C28.
After 1-3 weeks, a plug from the colony of the biologi-cal control agent is placed on one side of 2% MA in a 9 cm petri dish, and a plug from the colony of the sapstaining fungus is placed on the opposite side of the petri dish, such that the two fungi will grow together near the centre of the plate. Each possible combination of biological control agent and sapstaining fungus is set up. The plates are examined periodically and the interactions between the organisms are observed.
In all cases, the biological control agents inhibit the growth of the sapstaining fungus when the two colonies make contact. None of the Gliocladium isolates inhibit the sapstaining fungi before contact, suggesting that diffusible antifungal metabolites are not produced in this experimental design.
Inocula of the biological control fungi are prepared by transferring plugs of stock cultures onto DifcoTM potato dextrose agar in 6 cm petri dishes as in Example 1. Stock culture 2 OD~ l~fl ~ ~a - ll - 28113-3 maintenance and incubation conditions are as in Example 1.
The biological control agents are selected from the following strains: Gliocladium roseum 321A, 321M, Gliocladium viride 623E, Gliocladium aureum 784A, and Gliocladium solani 810A, and Gliocladium virens 258C, 258D.
The wood blocks used in this example are Jack Pine sap-wood 3 cm long and l cm x 0.5 cm in cross section. These are sterilized by gamma irradiation and placed in glass petri dishes, eight blocks per dish, upon w-shaped glass bars fashioned from 3 mm glass tubing, that rest upon 2 sheets of filter paper in which 5 mL sterile distilled water has been absorbed.
A spore suspension from 1-3 week old agar cultures is prepared. The colonies from G. roseum 321A and 321M, G aureum 784A, and G. virens 258D are transferred into a sterile WaringTM blender and homogenized for 30 seconds in 75 mL
sterile distilled water. Spore suspensions of G. viride 623E, G.
solani 810A and G. virens 258C are prepared by flooding the agar plates with 6 mL of sterile distilled water and liberating the spores with an L shaped glass rod.
The spore suspension of each Gliocladium strain is squirted onto the surface of 64 blocks, (8 per petri dish) using a sterile syringe such that the entire length of the block, though not necessarily the entire width, receives some liquid.
The blocks are then incubated for l week. Staining fungus inocula are grown in 6 cm petri dishes on appropriate agar media for 1-2 weeks. Spore suspensions are prepared in the same way as described for the biological control strains above. The 20~7~
sapstaining fungi employed are: Ophiostoma piceae FTK 387I, O. piliferum FTK 55F, FTK 55H and Ophiostoma sp. FTK C28, plus a "80Up" mixture of Cephaloascus fragrans FTK 307I, Ophiostoma piliEerum FTK 55H, Black Yeast FTK 86-010-1-1-1, Aureobasidium pullulans FTK 132Q, Leptodontidium elatius FTK 268A, Cladosporium cladosporioides FTK 273D, Ophiostoma populinum FTK 671A, Ophiostoma perfectum FTK 703A, Phialophora botulispora FTK 707A, Leptographium sp. FTK 2A2, Phoma sp. FTK 86-8-3-2-1, Alternaria alternata FTK 2G and FTK 2H.
The sapstaining fungi spore suspensions are then inocu-lated onto the surface of the wood blocks in a manner identical to that used for the biological control fungi. Each sapstaining fungi spore suspension is inoculated onto 2 groups of 8 blocks for each Gliocladium strain.
The wood blocks are incubated a further four weeks. The surface and interior of the wood blocks thus treated are free from discolorations caused by the sapstaining fungi, while control blocks inoculated at the same time with only sapstaining fungi become darkly discoloured after only 1-2 weeks. The results of this experiment are illustrated in Table 1.
In this example, the ability of the isolate Gliocladium roseum 321A to cause weight loss in Jack Pine blocks is tested.
The standard ASTM soil block test (D1413-76) is used. In this method, 200 g of a 3:1 soil-sand mixture are placed in a 500 mL
glass jar with 60 mL distilled water. A feeder strip made of red pine sapwood, 41 x 29 x 3.0 mm, is placed on the surface of the 2047~
soil. The jars are sterilized for one hour, cooled, then re-sterilized for a second hour. The lids are then replaced with sterile culture lids with a microbiological filter with a 0.2 ~ m pore size fitted over a 5 mm hole in each lid. The soil is inocu-lated with an agar plug from a growing colony of Gliocladium roseum 321A and incubated for 3 weeks. Then, two sterilized 19 mm cubes of Jack Pine sapwood of known dry weight are added to each jar. After 12 weeks incubation, the blocks are dried and re-weighed.
The weight loss caused by Gliocladium roseum 321A was 2~, and was not significantly different than the weight loss in the blank control. A typical decay fungus, Poria carbonica FTK 120AM, incubated under the same conditions, caused a weight loss of 33%.
In this example, the ability of Gliocladium roseum 321A
to cause strength loss in wood is determined. Jack Pine wood beams are incubated in a modified soil block test and the impact bending strength is measured using the Toughness Testing (ISOD) machine, as detailed below.
The ISOD impact bending machine measures the force required to break a span of wood. A weight attached to a pendulum is released using a foot pedal, which pulls a chain attached to a vertical metal bar. The bar then is pulled into the sample (=
impact), and the wood is broken. The force required to break the sample is determined by converting the value recorded by the pendulum of the machine (in degrees and minutes) to inch-pounds.
2047~
The [SOD machine was modified for the smaller wood beams by reducing the weight on the pendulum and modifying the sample holder.
The soil block test was modified from ASTM stand and D-1413 to allow for the different block sizes. Rather than using glass jars, 1 L NalgeneTM polypropylene jars are used. Each jar contains 400 g of a 3:1 soil:sand mixture and 120 mL of distilled water. Three red pine feeder strips, 4.5 x 2.5 x 0.5 cm, are placed side by side on the surface of the soil. The jars are autoclaved for one hour, cooled overnight, then autoclaved again the next day for one hour. The jars are cooled in a biological safety hood, and the lids replaced with culture lids. The culture lids are modified lids with a central hole, 5/16 of an inch in diameter, covered on the inner surface with a GelmanTM filter to allow air exchange.
The wood beams, 9.0 x 0.75 x 0.75 cm, are prepared from green Jack Pine (Pinus banksiana) sapwood. The beams are sorted into sets with more or less the same number of growth rings. Only beams with the grain more or less parallel to the long axis are selected. The beams are sterilized by gamma radiation and frozen until use.
The inoculum for Gliocladium roseum 321A is a spore suspension in water made from a 1-2 week old 2% MA culture. The spore suspension is inoculated onto the wooden beams, and the beams are preincubated for 1 week in a 1 L Nalgene polypropylene jar. At the bottom of the jar is filter paper moistened with distilled water, then alternating layers of glass rods and wood ~47~
beams. All beams used in the experiment are from a matched set.
After the preincubation period, five test beams are aseptically added to each soil jar. For controls, uninoculated test beams are aseptically added to jars. Five jars are set up for each treat-ment, for a total of 25 beams per treatment. All incubations are at 27C.
After four weeks incubation, the beams are removed from the jars and placed on screen racks. The samples are then placed at a constant temperature and humidity and allowed to equilibrate for 7 days. The force required to break each beam is then measur-ed with the ISOD toughness testing machine. The values areconverted using the equation:
Toughness (inch-pounds) = pendulum weight x (cosA2-cosAl) where Al is the initial angle, and A2 is the angle of the pendulum at failure.
The readings given by the machine when no specimen was present are converted using the same equation, and this value is subtracted from the converted test values to give a corrected value.
Wood specimens colonized with G. roseum require 12.76 +
0.32 inch-pounds to break. The control blocks, with no added fungus, require 11.93 + 0.44 inch-pounds to break while the decay control blocks required 8.87 + 0.45 inch-pounds. Statistical analysis revealed that test blocks incubated with G. roseum 321A
do not undergo significantly more strength loss than uninoculated Jack Pine test blocks under the conditions employed.
20~7~a In this example, the ability of the candidate strain Gliocladium viride 623E to prevent decay by known decay fungi Gloeophyllum trabeum FTK 47D and Merulius tremellosus FTK 52H is determined. Retained strength, a measure of a candidate's decay-prevention ability, is the ratio of impact bending strength of test beams incubated with both the candidate biocontrol fungus and the decay fungi to the impact bending strength of test beams in-cubated with the candidate biocontrol fungi alone. This figure is expressed as a percentage. The experimental set-up is the same as the set-up in Example 4 except that the blocks containing the biological control fungi were added to a NalgeneTM
jar/incubator which had been inoculated one week earlier with a decay inoculum.
The decay inoculum utilized was prepared by blending 150 mL of sterile water and growing agar cultures of two 9 cm petri plates from each of the aforementioned decay organisms. Five drops of this inoculum were placed on Jack Pine feeder strips in each ~algene jar/incubator. After an incubation period of one week, the beams colonized with the biological control fungus were added and allowed to incubate a further four weeks. The impact bending strength for each test beam was determined (as in Example 4) and the retained strengths calculated.
The test beams inoculated with Gliocladium viride 623E
had a retained strength of 94%, compared to 87% for the control test beams which contained no biological control fungi. G. viride 623E, therefore, prevented a significant amount of decay from occuring in the test blocks.
20~7~
This example examines the ability of the Gliocladium 8pp. to protect larger pieces of lumber from sapstaining organisms in a small scale trial under conditions closely related to those in field trials. The Gliocladium strain was grown on two 14 cm agar plates (2% MA see Example 1) and allowed to sporulate. A
spore suspension of each strain was prepared by flooding the sur-face of the plates with 50 mL of sterile distilled water, pooling the suspensions and diluting them to 4 liters. The final spore concentration was measured using a haemocytometer. This concentration ranged from 2 x 104 to 1 x 106 spores/mL. Freshly sawn 1" x 6" x 15" white pine lumber, 28 pieces per strain, was dipped in these spore suspensions and placed in plastic bins, 14 pieces per bin. Control bins containing lumber with no biocontrol organisms were also set up. Humidity was maintained in these bins by the addition of lL of sterile distilled water. The bins were covered with tight fitting lids equipped with ventilation holes covered with 0.22 ~L GelmanTM filters. The bins were incuba-ted in a ventilated temperature monitored shed at ambient tempera-ture. After 6 and 14 weeks, each piece of lumber was rated for the evidence of surface mold, decay and sapstain fungi. A visual rating system for the prsence of the specified fungi is used. A
piece of lumber is rated as acceptable if it contains less than 10% surface area covered with stain. The results of these trials are summarized in Table 2. A statistical analysis of this data (based on the analysis of the Chi-squared test of homogeneity of proportions) reveals that G. roseum 321A and 321M, G. solani 810A, ~74~
and G aureum 784A afford the lumber a significant protection from sapstain, mould and decay while wood treated with G. viride 623E
was not significantly different than the control.
2 0 ~ S
Table 1 Results summarizing the ability of the candidate biological control fungi to prevent sapstain on Jack Pine.
____._____________________________________________________________ Number of Biocontrol Fungus Sapstainer blocks (FTK No.) (FTK No.) stained Gliocladium roseum (321A) Ophiostoma picea (387I) 0/8 , 0/8 Ophiostoma sp. (C28) 0/8 , 0/8 Aureobasidium pullulans (132Q)0/8 , 0/8 Alternaria alternata (2G) 0/8 , 0/8 __________________________________________________________________ Gliocladium aureum (784A) soup (see Example 2) 0/8 , 0/8 Ophiostoma piliferum (55H) 0/8 , 0/8 Phialophora botulispora (707A)0/8 , 0/8 Black Yeast (86-010-1-1-1) 0/8 , 0/8 __________________________________________________________ _______ Gliocladium roseum (321M) soup (see Example 2) 0/8 , 1/8 Ophiostoma piliferum (55H) 0/8 , 0/8 Phialophora botulispora (707A)0/8 , 0/8 Black Yeast (86-010-1-1-1) 0/8 , 0/8 __________________________________________________________________ Gliocladium solani (810A) soup (see Example 2) 0/8 , 1/8 Ophiostoma piliferum (55H) 0/8 , 0/8 Alternaria alternata (2H) 0/8 , 0/8 Black Yeast (86-010-1-1-1) 0/8 , 0/8 ___________________________ ______________________________________ Gliocladium viride (623)E soup (see Example 2) O/4*
__________________________________________________________________ Gliocladium virens (258C) soup (see Example 2) 0/8 , 0/8 (258D) soup 0/8 , 0/8 __________________________________________________________________ *readings taken from wood chips 2~7~
able 2. Summary of data from small scale field trials detailing the number of acceptable pieces of lumber after 6 and 14 week incubation periods.
Treatment Acceptable Pieces (~) 6 weeks 14 weeks Control (no fungi) 71 32 Gliocladium aureum 784A 100 82 Gliocladium roseum 321A 79 71 Gliocladium roseum 321M 100 82 Gliocladium solani 810A 96 86 Gliocladium viride 623E 54 32 -.
2~7~
REFERENCES
1. Unligil, H.H. 1978. Decay resistance of wood treated with fungal antibiotics: cryptosporiopsin, hyalodendrin, and scytalidin. Wood. Sci. 11: 30-32.
2. Shields, J.K. 1966. Wood Pathology. Rept. For. Prod. Res.
Branch, Dept. For. Can., April 1964-March 1965: 48-52.
3. Shields, J.K. 1968. Role of Trichoderma viride in reducing storage decay of birch logs. Bimonthly Res. Notes Dept. For.
Can. 24: 9-10; Hulme, M.A. and J.K. Shields. 1972. Effect of primary fungal infection upon secondary colonization of birch bolts. Mat. und Org. 7: 177-188.
4. Stilwell, M.A. 1966. A growth inhibitor produced by Crypto-spor opsis sp., an imperfect fungus isolated from yellow birch, Betula alleghaniensis Britt. Can. J. Bot. 44: 249-267.
5. Strunz, G.M., A.S. Court, J. Komlossy and M.A. Stilwell.
1969. Structure of cryptosporiopsin: a new antibiotic subs-tance produced by a species of Cryptosporiopsis. Can. J.
Chem. 47: 2087-2094.
6. Bergman, O. and T. Nilsson. 1979. Outdoor chip storage--methods of reducing deterioration during OCS. pp. 245-271.
In: Chip Quality Monograph. Edited by J.V. Hatton. Pulp and Paper Tech. Ser. no. 5.
7. Stilwell, M.A. and G.M. Strunz. 1967. Antibiotic produced by a fungus. Rept. CFS For. Res. Lab., Fredericton. N.B.
8. Lapetite, D. 1970. Etude sur bois de l'action antagoniste des bactéries vis-à-vis des champignons lignivores. Mat. und Org. 5: 229-237.
9. Supra 4; Supra 7; Stilwell, M.A., R.E. Wall and G.M. Strunz.
1973. Production, isolation and antifungal activity of scytalidin, a metabolites of Scytalidium sp. Can. J. Micro-biol. 19: 597-602; O.K. Strunz, G.M., M. Kakushima and M.A.
Stilwell. 1973. Scytalidin: a new fungitoxic metabolite produced by a Scytalidium species. J. Chem. Soc. Perkin Trans. 1: 2280-2283.
10. Stranks, D. 1976. Scytalidin, hyalodendrin, cryptosporiop-sis-- Antibiotics for prevention of blue stain in white pine sapwood. Wood. Sci. 9: 110-112.
All other incubations described in this example are at 27C, 75%
relative humidity, in the dark.
The fungi used as biological control agents in this example are selected from the following strains maintained in the Forintek culture collection of wood-inhabiting fungi: Gliocladium roseum 321M, Gliocladium aureum 784A, Gliocladium solani 810A
Sapstaining fungi employed are: Ophiostoma piceae FTK 387I, O.
piliferum (Fr.) H. & P. Sydow FTK 55F, FTK 55H and Ophiostoma sp.
FTK C28.
After 1-3 weeks, a plug from the colony of the biologi-cal control agent is placed on one side of 2% MA in a 9 cm petri dish, and a plug from the colony of the sapstaining fungus is placed on the opposite side of the petri dish, such that the two fungi will grow together near the centre of the plate. Each possible combination of biological control agent and sapstaining fungus is set up. The plates are examined periodically and the interactions between the organisms are observed.
In all cases, the biological control agents inhibit the growth of the sapstaining fungus when the two colonies make contact. None of the Gliocladium isolates inhibit the sapstaining fungi before contact, suggesting that diffusible antifungal metabolites are not produced in this experimental design.
Inocula of the biological control fungi are prepared by transferring plugs of stock cultures onto DifcoTM potato dextrose agar in 6 cm petri dishes as in Example 1. Stock culture 2 OD~ l~fl ~ ~a - ll - 28113-3 maintenance and incubation conditions are as in Example 1.
The biological control agents are selected from the following strains: Gliocladium roseum 321A, 321M, Gliocladium viride 623E, Gliocladium aureum 784A, and Gliocladium solani 810A, and Gliocladium virens 258C, 258D.
The wood blocks used in this example are Jack Pine sap-wood 3 cm long and l cm x 0.5 cm in cross section. These are sterilized by gamma irradiation and placed in glass petri dishes, eight blocks per dish, upon w-shaped glass bars fashioned from 3 mm glass tubing, that rest upon 2 sheets of filter paper in which 5 mL sterile distilled water has been absorbed.
A spore suspension from 1-3 week old agar cultures is prepared. The colonies from G. roseum 321A and 321M, G aureum 784A, and G. virens 258D are transferred into a sterile WaringTM blender and homogenized for 30 seconds in 75 mL
sterile distilled water. Spore suspensions of G. viride 623E, G.
solani 810A and G. virens 258C are prepared by flooding the agar plates with 6 mL of sterile distilled water and liberating the spores with an L shaped glass rod.
The spore suspension of each Gliocladium strain is squirted onto the surface of 64 blocks, (8 per petri dish) using a sterile syringe such that the entire length of the block, though not necessarily the entire width, receives some liquid.
The blocks are then incubated for l week. Staining fungus inocula are grown in 6 cm petri dishes on appropriate agar media for 1-2 weeks. Spore suspensions are prepared in the same way as described for the biological control strains above. The 20~7~
sapstaining fungi employed are: Ophiostoma piceae FTK 387I, O. piliferum FTK 55F, FTK 55H and Ophiostoma sp. FTK C28, plus a "80Up" mixture of Cephaloascus fragrans FTK 307I, Ophiostoma piliEerum FTK 55H, Black Yeast FTK 86-010-1-1-1, Aureobasidium pullulans FTK 132Q, Leptodontidium elatius FTK 268A, Cladosporium cladosporioides FTK 273D, Ophiostoma populinum FTK 671A, Ophiostoma perfectum FTK 703A, Phialophora botulispora FTK 707A, Leptographium sp. FTK 2A2, Phoma sp. FTK 86-8-3-2-1, Alternaria alternata FTK 2G and FTK 2H.
The sapstaining fungi spore suspensions are then inocu-lated onto the surface of the wood blocks in a manner identical to that used for the biological control fungi. Each sapstaining fungi spore suspension is inoculated onto 2 groups of 8 blocks for each Gliocladium strain.
The wood blocks are incubated a further four weeks. The surface and interior of the wood blocks thus treated are free from discolorations caused by the sapstaining fungi, while control blocks inoculated at the same time with only sapstaining fungi become darkly discoloured after only 1-2 weeks. The results of this experiment are illustrated in Table 1.
In this example, the ability of the isolate Gliocladium roseum 321A to cause weight loss in Jack Pine blocks is tested.
The standard ASTM soil block test (D1413-76) is used. In this method, 200 g of a 3:1 soil-sand mixture are placed in a 500 mL
glass jar with 60 mL distilled water. A feeder strip made of red pine sapwood, 41 x 29 x 3.0 mm, is placed on the surface of the 2047~
soil. The jars are sterilized for one hour, cooled, then re-sterilized for a second hour. The lids are then replaced with sterile culture lids with a microbiological filter with a 0.2 ~ m pore size fitted over a 5 mm hole in each lid. The soil is inocu-lated with an agar plug from a growing colony of Gliocladium roseum 321A and incubated for 3 weeks. Then, two sterilized 19 mm cubes of Jack Pine sapwood of known dry weight are added to each jar. After 12 weeks incubation, the blocks are dried and re-weighed.
The weight loss caused by Gliocladium roseum 321A was 2~, and was not significantly different than the weight loss in the blank control. A typical decay fungus, Poria carbonica FTK 120AM, incubated under the same conditions, caused a weight loss of 33%.
In this example, the ability of Gliocladium roseum 321A
to cause strength loss in wood is determined. Jack Pine wood beams are incubated in a modified soil block test and the impact bending strength is measured using the Toughness Testing (ISOD) machine, as detailed below.
The ISOD impact bending machine measures the force required to break a span of wood. A weight attached to a pendulum is released using a foot pedal, which pulls a chain attached to a vertical metal bar. The bar then is pulled into the sample (=
impact), and the wood is broken. The force required to break the sample is determined by converting the value recorded by the pendulum of the machine (in degrees and minutes) to inch-pounds.
2047~
The [SOD machine was modified for the smaller wood beams by reducing the weight on the pendulum and modifying the sample holder.
The soil block test was modified from ASTM stand and D-1413 to allow for the different block sizes. Rather than using glass jars, 1 L NalgeneTM polypropylene jars are used. Each jar contains 400 g of a 3:1 soil:sand mixture and 120 mL of distilled water. Three red pine feeder strips, 4.5 x 2.5 x 0.5 cm, are placed side by side on the surface of the soil. The jars are autoclaved for one hour, cooled overnight, then autoclaved again the next day for one hour. The jars are cooled in a biological safety hood, and the lids replaced with culture lids. The culture lids are modified lids with a central hole, 5/16 of an inch in diameter, covered on the inner surface with a GelmanTM filter to allow air exchange.
The wood beams, 9.0 x 0.75 x 0.75 cm, are prepared from green Jack Pine (Pinus banksiana) sapwood. The beams are sorted into sets with more or less the same number of growth rings. Only beams with the grain more or less parallel to the long axis are selected. The beams are sterilized by gamma radiation and frozen until use.
The inoculum for Gliocladium roseum 321A is a spore suspension in water made from a 1-2 week old 2% MA culture. The spore suspension is inoculated onto the wooden beams, and the beams are preincubated for 1 week in a 1 L Nalgene polypropylene jar. At the bottom of the jar is filter paper moistened with distilled water, then alternating layers of glass rods and wood ~47~
beams. All beams used in the experiment are from a matched set.
After the preincubation period, five test beams are aseptically added to each soil jar. For controls, uninoculated test beams are aseptically added to jars. Five jars are set up for each treat-ment, for a total of 25 beams per treatment. All incubations are at 27C.
After four weeks incubation, the beams are removed from the jars and placed on screen racks. The samples are then placed at a constant temperature and humidity and allowed to equilibrate for 7 days. The force required to break each beam is then measur-ed with the ISOD toughness testing machine. The values areconverted using the equation:
Toughness (inch-pounds) = pendulum weight x (cosA2-cosAl) where Al is the initial angle, and A2 is the angle of the pendulum at failure.
The readings given by the machine when no specimen was present are converted using the same equation, and this value is subtracted from the converted test values to give a corrected value.
Wood specimens colonized with G. roseum require 12.76 +
0.32 inch-pounds to break. The control blocks, with no added fungus, require 11.93 + 0.44 inch-pounds to break while the decay control blocks required 8.87 + 0.45 inch-pounds. Statistical analysis revealed that test blocks incubated with G. roseum 321A
do not undergo significantly more strength loss than uninoculated Jack Pine test blocks under the conditions employed.
20~7~a In this example, the ability of the candidate strain Gliocladium viride 623E to prevent decay by known decay fungi Gloeophyllum trabeum FTK 47D and Merulius tremellosus FTK 52H is determined. Retained strength, a measure of a candidate's decay-prevention ability, is the ratio of impact bending strength of test beams incubated with both the candidate biocontrol fungus and the decay fungi to the impact bending strength of test beams in-cubated with the candidate biocontrol fungi alone. This figure is expressed as a percentage. The experimental set-up is the same as the set-up in Example 4 except that the blocks containing the biological control fungi were added to a NalgeneTM
jar/incubator which had been inoculated one week earlier with a decay inoculum.
The decay inoculum utilized was prepared by blending 150 mL of sterile water and growing agar cultures of two 9 cm petri plates from each of the aforementioned decay organisms. Five drops of this inoculum were placed on Jack Pine feeder strips in each ~algene jar/incubator. After an incubation period of one week, the beams colonized with the biological control fungus were added and allowed to incubate a further four weeks. The impact bending strength for each test beam was determined (as in Example 4) and the retained strengths calculated.
The test beams inoculated with Gliocladium viride 623E
had a retained strength of 94%, compared to 87% for the control test beams which contained no biological control fungi. G. viride 623E, therefore, prevented a significant amount of decay from occuring in the test blocks.
20~7~
This example examines the ability of the Gliocladium 8pp. to protect larger pieces of lumber from sapstaining organisms in a small scale trial under conditions closely related to those in field trials. The Gliocladium strain was grown on two 14 cm agar plates (2% MA see Example 1) and allowed to sporulate. A
spore suspension of each strain was prepared by flooding the sur-face of the plates with 50 mL of sterile distilled water, pooling the suspensions and diluting them to 4 liters. The final spore concentration was measured using a haemocytometer. This concentration ranged from 2 x 104 to 1 x 106 spores/mL. Freshly sawn 1" x 6" x 15" white pine lumber, 28 pieces per strain, was dipped in these spore suspensions and placed in plastic bins, 14 pieces per bin. Control bins containing lumber with no biocontrol organisms were also set up. Humidity was maintained in these bins by the addition of lL of sterile distilled water. The bins were covered with tight fitting lids equipped with ventilation holes covered with 0.22 ~L GelmanTM filters. The bins were incuba-ted in a ventilated temperature monitored shed at ambient tempera-ture. After 6 and 14 weeks, each piece of lumber was rated for the evidence of surface mold, decay and sapstain fungi. A visual rating system for the prsence of the specified fungi is used. A
piece of lumber is rated as acceptable if it contains less than 10% surface area covered with stain. The results of these trials are summarized in Table 2. A statistical analysis of this data (based on the analysis of the Chi-squared test of homogeneity of proportions) reveals that G. roseum 321A and 321M, G. solani 810A, ~74~
and G aureum 784A afford the lumber a significant protection from sapstain, mould and decay while wood treated with G. viride 623E
was not significantly different than the control.
2 0 ~ S
Table 1 Results summarizing the ability of the candidate biological control fungi to prevent sapstain on Jack Pine.
____._____________________________________________________________ Number of Biocontrol Fungus Sapstainer blocks (FTK No.) (FTK No.) stained Gliocladium roseum (321A) Ophiostoma picea (387I) 0/8 , 0/8 Ophiostoma sp. (C28) 0/8 , 0/8 Aureobasidium pullulans (132Q)0/8 , 0/8 Alternaria alternata (2G) 0/8 , 0/8 __________________________________________________________________ Gliocladium aureum (784A) soup (see Example 2) 0/8 , 0/8 Ophiostoma piliferum (55H) 0/8 , 0/8 Phialophora botulispora (707A)0/8 , 0/8 Black Yeast (86-010-1-1-1) 0/8 , 0/8 __________________________________________________________ _______ Gliocladium roseum (321M) soup (see Example 2) 0/8 , 1/8 Ophiostoma piliferum (55H) 0/8 , 0/8 Phialophora botulispora (707A)0/8 , 0/8 Black Yeast (86-010-1-1-1) 0/8 , 0/8 __________________________________________________________________ Gliocladium solani (810A) soup (see Example 2) 0/8 , 1/8 Ophiostoma piliferum (55H) 0/8 , 0/8 Alternaria alternata (2H) 0/8 , 0/8 Black Yeast (86-010-1-1-1) 0/8 , 0/8 ___________________________ ______________________________________ Gliocladium viride (623)E soup (see Example 2) O/4*
__________________________________________________________________ Gliocladium virens (258C) soup (see Example 2) 0/8 , 0/8 (258D) soup 0/8 , 0/8 __________________________________________________________________ *readings taken from wood chips 2~7~
able 2. Summary of data from small scale field trials detailing the number of acceptable pieces of lumber after 6 and 14 week incubation periods.
Treatment Acceptable Pieces (~) 6 weeks 14 weeks Control (no fungi) 71 32 Gliocladium aureum 784A 100 82 Gliocladium roseum 321A 79 71 Gliocladium roseum 321M 100 82 Gliocladium solani 810A 96 86 Gliocladium viride 623E 54 32 -.
2~7~
REFERENCES
1. Unligil, H.H. 1978. Decay resistance of wood treated with fungal antibiotics: cryptosporiopsin, hyalodendrin, and scytalidin. Wood. Sci. 11: 30-32.
2. Shields, J.K. 1966. Wood Pathology. Rept. For. Prod. Res.
Branch, Dept. For. Can., April 1964-March 1965: 48-52.
3. Shields, J.K. 1968. Role of Trichoderma viride in reducing storage decay of birch logs. Bimonthly Res. Notes Dept. For.
Can. 24: 9-10; Hulme, M.A. and J.K. Shields. 1972. Effect of primary fungal infection upon secondary colonization of birch bolts. Mat. und Org. 7: 177-188.
4. Stilwell, M.A. 1966. A growth inhibitor produced by Crypto-spor opsis sp., an imperfect fungus isolated from yellow birch, Betula alleghaniensis Britt. Can. J. Bot. 44: 249-267.
5. Strunz, G.M., A.S. Court, J. Komlossy and M.A. Stilwell.
1969. Structure of cryptosporiopsin: a new antibiotic subs-tance produced by a species of Cryptosporiopsis. Can. J.
Chem. 47: 2087-2094.
6. Bergman, O. and T. Nilsson. 1979. Outdoor chip storage--methods of reducing deterioration during OCS. pp. 245-271.
In: Chip Quality Monograph. Edited by J.V. Hatton. Pulp and Paper Tech. Ser. no. 5.
7. Stilwell, M.A. and G.M. Strunz. 1967. Antibiotic produced by a fungus. Rept. CFS For. Res. Lab., Fredericton. N.B.
8. Lapetite, D. 1970. Etude sur bois de l'action antagoniste des bactéries vis-à-vis des champignons lignivores. Mat. und Org. 5: 229-237.
9. Supra 4; Supra 7; Stilwell, M.A., R.E. Wall and G.M. Strunz.
1973. Production, isolation and antifungal activity of scytalidin, a metabolites of Scytalidium sp. Can. J. Micro-biol. 19: 597-602; O.K. Strunz, G.M., M. Kakushima and M.A.
Stilwell. 1973. Scytalidin: a new fungitoxic metabolite produced by a Scytalidium species. J. Chem. Soc. Perkin Trans. 1: 2280-2283.
10. Stranks, D. 1976. Scytalidin, hyalodendrin, cryptosporiop-sis-- Antibiotics for prevention of blue stain in white pine sapwood. Wood. Sci. 9: 110-112.
11. Seifert, K.A., C. Breuil, M. Best, L. Rossignol and J.N.
Saddler. Screening of microorganisms with the potential for biological control of sapstain on unseasoned lumber. Mat.
und Org.: 23 BD Heft 2.
2~7~
REFERENCES (cont'd) 12. Vasiliev, O.A. 1968. (On the question of using the antago-nism of fungi and bacteria for the protection of wood).
Mach. Trudy Leningr. Lesotekh Akad. 110: 28-33 (in Russian).
Saddler. Screening of microorganisms with the potential for biological control of sapstain on unseasoned lumber. Mat.
und Org.: 23 BD Heft 2.
2~7~
REFERENCES (cont'd) 12. Vasiliev, O.A. 1968. (On the question of using the antago-nism of fungi and bacteria for the protection of wood).
Mach. Trudy Leningr. Lesotekh Akad. 110: 28-33 (in Russian).
13. Bernier, R. Jr., M. Desrocher and L. Jurasek. 1986. Antago-nistic effect between Bacillus subtilus and wood staining fungi. J. Inst. Wood. Sci. 10: 214-2I6.
14. Benko, R. 1988. Bacteria as possible organisms for biologi-cal control of blue stain. Int. Res. Group on Wood Preserv., Document No. IRG/WP/1339.
15. Johnson, B.R. 1986. Sensitivity of some wood stain and mold fungi to an inhibitor of chitin synthesis. For. Prod. J.
36(3): 54-56.
36(3): 54-56.
16. Benko, R. 1987. Antagonistic effect of some mycorrhizal fungi as biological control of sapstain. Int. Res. Group on Wood Preserv., Document No. IRG/WP/1314.
Claims (26)
1. A method of controlling sapstain in wood and wood products comprising treating the wood or wood product with an inoculum comprising one or more fungi of the genus Gliocladium.
2. The method according to claim 1 wherein said fungi is selected from one of the following species of the genus Glio-cladium: Gliocladium aureum, Gliocladium viride, Gliocladium roseum, Gliocladium solani, and Gliocladium virens.
3. The method according to claim 2 wherein the Gliocladium aureum is FTK 784A.
4. The method according to claim 2 wherein the Gliocladium viride is FTK 623E.
5. The method according to claim 2 wherein the Gliocladium roseum is FTK 321M.
6. The method according to claim 2 wherein the Gliocladium solani is FTK 810A.
7. The method according to claim 2 wherein the Gliocladium virens is selected from FTK 258C or FTK 258D.
8. The method according to claims 1, 2, 3, 4, 5, 6 or 7 wherein said wood or wood product is softwood or conifer lumber.
9. The method according to claims 1, 2, 3, 4, 5, 6 or 7 wherein said wood or wood product is wood chips.
10. The method according to claims 1, 2, 3, 4, 5, 6 or 7 wherein said wood or wood product is pine wood.
11. A wood or wood product treated with at least one fungus of the genus Gliocladium said product being essentially free of sapstain as the result of said treatment of said fungus.
12. A wood or wood product according to claim 11 wherein the fungus is selected from one of the following species of the genus Gliocladium: Gliocladium aureum, Gliocladium viride, Gliocladium roseum, Gliocladium solani, and Gliocladium virens.
13. A wood or wood product according to claim 12 wherein the Gliocladium aureum is FTK 784A.
14. A wood or wood product according to claim 12 wherein the Gliocladium viride is FTK 623E.
15. A wood or wood product according to claim 12 wherein the Gliocladium roseum is FTK 321M.
16. A wood or wood product according to claim 12 wherein the Gliocladium solani is FTK 810A.
17. A wood or wood product according to claim 12 wherein the Gliocladium virens is FTK 258C and 258D.
18. A wood or wood product according to claim 12, 13, 14, 15, 16, or 17 wherein said wood or wood product is softwood or conifer lumber.
19. A wood or wood product according to claim 12, 13, 14, 15, 16, or 17 wherein said wood or wood product is wood chips.
20. A wood or wood product according to claim 12, 13, 14, 15, 16, or 17 wherein said wood or wood product is pine wood.
21. A method of preventing weight loss in wood and wood products comprising treating the wood or wood product with an inoculum comprising one or more fungi of the genus Gliocladium.
22. A method according to claim 21 wherein said fungi is selected from one of the following species of the genus Glio-cladium: Gliocladium aureum, Gliocladium viride, Gliocladium roseum, Gliocladium solani, and Gliocladium virens.
23. A method of preventing strength loss in wood and wood products comprising treating the wood or wood product with an inoculum comprising one or more fungi of the genus Gliocladium.
24. The method according to claim 23 wherein wherein said fungi is selected from one of the following species of the genus Gliocladium: Gliocladium aureum, Gliocladium viride, Gliocladium roseum, Gliocladium solani, and Gliocladium virens.
25. A method of preventing decay in wood and wood products comprising treating the wood or wood product with an inoculum comprising one or more fungi of the genus Gliocladium.
26. A method of claim 25 wherein said fungi is selected from one of the following species of the genus Gliocladium: Glio-cladium aureum, Gliocladium viride, Gliocladium roseum, Glio-cladium solani, and Gliocladium virens.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2047445 CA2047445A1 (en) | 1991-07-19 | 1991-07-19 | Method for protection of lumber against sapstain |
| AU23010/92A AU2301092A (en) | 1991-07-19 | 1992-07-16 | Method for protection of lumber against sapstain |
| PCT/CA1992/000299 WO1993001923A1 (en) | 1991-07-19 | 1992-07-16 | Method for protection of lumber against sapstain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2047445 CA2047445A1 (en) | 1991-07-19 | 1991-07-19 | Method for protection of lumber against sapstain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2047445A1 true CA2047445A1 (en) | 1993-01-20 |
Family
ID=4148036
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2047445 Abandoned CA2047445A1 (en) | 1991-07-19 | 1991-07-19 | Method for protection of lumber against sapstain |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU2301092A (en) |
| CA (1) | CA2047445A1 (en) |
| WO (1) | WO1993001923A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4142286C2 (en) * | 1991-12-20 | 1995-01-05 | Siempelkamp Gmbh & Co | Three-layer gypsum-based building material board and manufacturing method |
| CA2101485C (en) * | 1993-07-28 | 1999-10-12 | Brenda J. Mcafee | Two stage method for the protection of lumber against sapstain |
| ES2088200T3 (en) * | 1993-08-09 | 1996-08-01 | Trierische Kork Ind Fr Weber & | PROCEDURE FOR THE TREATMENT OF CORK BARK OBTAINED FROM THE CORK FOR THE MANUFACTURE OF BOTTLE PLUGS. |
| FI101631B1 (en) * | 1994-09-30 | 1998-07-31 | Kemira Agro Oy | Cliocladium catenulatum fungal strains for biological control of plant diseases |
| US6083537A (en) * | 1998-05-26 | 2000-07-04 | Forintek Canada Corp. | Integrated method for protecting logs and green lumber from sapstain and mold |
| WO2001093665A1 (en) * | 2000-06-07 | 2001-12-13 | University Of Waikato | Fungi for improvements of wood and pulp appearance and qualities |
| EP1537967A1 (en) | 2003-12-01 | 2005-06-08 | Nederlandse Organisatie voor toegepast-natuurwetenschappelijk Onderzoek TNO | Ecologically protected material |
| US8716001B2 (en) | 2009-02-06 | 2014-05-06 | Cornell University | Trichoderma strains that induce resistance to plant diseases and/or increase plant growth |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1274292A (en) * | 1969-04-29 | 1972-05-17 | Jacques Jean Louis Ricard | Device for use in counteracting harmful micro-organisms in a substrate with microbial commensals and other agents |
| SE373527B (en) * | 1971-05-21 | 1975-02-10 | Binab Bio Innovation Ab | KIT FOR TREATMENT OF TREYTOR WITH SUBSTANCES AT THE SAME TIME AS A PRIMER MECHANICAL PROCESSING OF THE WOOD MATERIAL AND DEVICE FOR PERFORMING THE PROCEDURE |
| US4996157A (en) * | 1988-11-14 | 1991-02-26 | Cornell Research Foundation, Inc. | Biological control of phytophthora by trichoderma |
-
1991
- 1991-07-19 CA CA 2047445 patent/CA2047445A1/en not_active Abandoned
-
1992
- 1992-07-16 AU AU23010/92A patent/AU2301092A/en not_active Abandoned
- 1992-07-16 WO PCT/CA1992/000299 patent/WO1993001923A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| AU2301092A (en) | 1993-02-23 |
| WO1993001923A1 (en) | 1993-02-04 |
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