Patent Application: US-8479998-A

Abstract:
the present invention relates to a method for controlling sapstain and mold in wood using a biocontrol agent having a high ph tolerance and an alkaline solution . biocontrol agents that do not discolor wood may be introduced onto wood or wood products to prevent sapstain and mold . the biocontrol agent grows on the wood surface and protects the wood against attack by stain or decay fungi . the application of an alkaline solution containing a biocontrol agent having a high ph tolerance to the wood product is very effective in controlling stain or decay fungi . the alkaline solution raises the ph of the wood to a level which impedes growth of other wood - inhabiting fungi but does not inhibit colonisation of the wood by the biocontrol agent .

Description:
hydrogen ion concentration ( ph ) of the substrate is one of the main factors that affect fungal growth . the growth of fungi is favored at a ph range of about ph 3 to 6 . some wood decay basidiomycetes do not grow at a ph of above 6 . 5 . the alkaline solution of the invention has a ph value in the range of about ph 10 . 5 - 11 . 5 and , in any event , is sufficiently alkaline to raise the ph of the wood surface to within the range of ph 7 - 10 . 5 , preferably within the range of ph 8 . 5 to 10 . to obtain the appropriate ph level , an alkaline solution formed by the addition of sodium carbonate and sodium bicarbonate to an aqueous solution was used . other suitable alkalis are sodium borate , sodium phosphate , sodium sulphate and the like . the concentration of the alkaline solution applied to the wood allows for rapid colonisation of the biocontrol agent on the wood tissue . rapid colonisation of the wood tissue is achieved by raising the surface ph of the wood to a level sufficient to favor growth of the biocontrol agent over growth of competing wood inhabiting fungi . the invention combines the application to wood of both an alkaline solution and a biocontrol agent . the biological agent must be able to tolerate the surface ph value of the wood following application of the alkaline solution . a preferred biological agent is gliocladium roseum or glyocladium aureum but other biocontrol agents having a high ph tolerance could also be used . gliocladium roseum bainier , also known as gliocladium aureum ( forentek culture collection fcc 321u ) isolated from root of carrot has been deposited with the centraalbureau voor schimmelcultures , cbs 226 . 48 and the american type culture collection , atcc 10406 . this fungus has demonstrated antagonistic activity to many other fungi . because it is a colorless fungus and does not cause significant weight and strength losses of colonized wood , this microorganism has high potential to control sapstain in wood as well as tree diseases . as previously indicated , gliocladium roseum has been identified as a biological anti - sapstain agent . however , the performance of this microorganism has been reported to be poor on green lumber in field testing . the following examples describe in detail how the performance of this biocontrol agent is improved by the simultaneous application of a selected alkaline solution . wood wafers in size of 4 cm × 2 cm × 0 . 5 cm were cut from defect - free green sapwood of white pine and jack pine purchased from local sawmills . g . roseum was subcultured in petri plates containing 2 % malt extract agar ( 20 g malt extract and 20 g difco agar in 1 l distilled water ). the plates were incubated at 26 ° c . with continuous light for 14 days . conidia formed were collected by adding 2 ml of sterile distilled water to each plate and gently agitating it with a glass rod . the final spore suspension was adjusted into 1 × 10 6 spores per ml of water . a mixed spore suspension ( 1 × 10 6 spores / ml ) was also prepared in the same way as with g . roseum using equal amounts of spores from the following ten sapstaining fungi : ophiostoma piceae 387e , aureobasidium pullulans 132s , ceratocystis adiposa 251e , alternaria alternata 2g , cladosporium sphaerospermum 806b , hormonema dematioides 742d , rhinocladiella atrovirens 135e , ophiostoma minus 864a , phialophora botulispora 707c and ophiostoma piliferum 55b . wood wafers were dipped for 30 seconds in the following solutions : 1 ) g . roseum spore suspension at 1 × 10 6 spores per ml of water ; 2 ) an aqueous solution containing 4 % sodium carbonate and 1 % sodium bicarbonate ; 3 ) an aqueous solution containing 4 % sodium carbonate , 1 % sodium bicarbonate and g . roseum spores at a concentration of 1 × 10 6 spores / ml ; and 4 ) a reference anti - sapstain chemical containing didecyl dimethyl ammonium chloride ( 64 %) and 3 - iodo - 2 - propynyl butyl carbamate ( 7 . 6 %) in a 1 : 80 dilution of the product in water . untreated wafers served as control . after the treatment , each of the treated wafers , ten pieces per treatment , was immediately inoculated with 0 . 1 ml of the mixed sapstaining spore suspension and was placed , two pieces per plate , on a w - shaped glass supporter sitting on two layers of wet filter paper in a petri plate . these plates were incubated in a growth chamber set at 26 ° c . and 75 % rh . wood wafers were inspected for mold and sapstain growth after 8 and 16 weeks incubation based on a visual rating scale of 0 to 5 where : 0 = no growth , 1 = trace of growth , 2 = little growth , 3 = moderate growth , 4 = heavy growth , and 5 = very heavy growth . a wood wafer of lumber was rated as acceptable if it had a score of 2 or less . results showed that wood wafers treated with the solution containing 4 % sodium carbonate , 1 % sodium bicarbonate and spores of g . roseum gave a satisfactory level of protection with 100 % acceptable pieces up to sixteen weeks , which was as good as for wood wafers treated with the reference chemical ( table 1 ). the treatment of both white pine and jack pine wood wafers with this mixture doubled the number of acceptable pieces as compared to the treatment with spores of g . roseum alone . this example examined the efficacy of the different treatments on the protection of wood under a favorable environmental condition but without artificial inoculation of mold and sapstaining fungi . white pine and jack pine wood wafers and inoculum of g . roseum were prepared as in example 1 . wood wafers were treated in the same way as with the samples in example 1 but without the inoculation of sapstaining fungi after the treatment . samples in this test , 30 wood wafers per treatment , were randomly placed in three replicate covered plastic bins , three layers of paper towels were placed in the bottom of the bin and were wetted with 2 l of water . plastic grids which supported the samples were put on the top of the paper , and the relative humidity inside the container was measured as 100 %. these bins were placed in a growth chamber set at 26 ° c . and 75 % rh , and wood wafers inside were inspected after 8 and 16 weeks . mold and sapstain developed on each wafer was visually rated in the same way as that described in example 1 . data were subjected to analysis of variance ( anova ) to compare the level of mold and sapstain amongst treatments . following the anova , the individual means were compared and ranked using scheffe &# 39 ; s test for multiple comparison . in this test , wood wafers of white pine and jack pine treated with spores of g . roseum alone only provided partial protection from fungal sapstain during a sixteen - week testing period . however , adding 4 % sodium carbonate and 1 % sodium bicarbonate into the g . roseum spore suspension significantly increased the number of acceptable pieces of treated wood wafers ( table 2 ). field trials were performed on white pine and jack pine in forintek eastern laboratory at sainte - foy , quebec . g . roseum was subcultured on 2 % malt extract agar at 26 ° c . for 7 days . mycelium discs , 5 mm in diameter , were cut from the periphery of the colonies and transferred to 1 - l flasks containing 500 ml of 1 . 5 % malt extract broth ( 15 g malt extract broth in 1 l water ). the culture was incubated for 7 days at 26 ° c . with agitation on a rotary shaker ( 100 rpm ). thereafter , spores and mycelia produced in the flasks were ground by a homogenizer at a low speed . the suspension was then centrifuged at 5 , 000 rpm for 15 minutes . the supernatant was poured off and the cell pellet was washed with sterile distilled water and collected by centrifugation . four aqueous solutions were prepared : a ) g . roseum cell suspension adjusted to 1 × 10 6 propagules per ml of water , b ) an aqueous solution containing 4 % sodium carbonate and 1 % sodium bicarbonate , c ) g . roseum suspension ( 1 × 10 6 propagules / ml ) in 4 % sodium carbonate and 1 % sodium bicarbonate solution , and d ) a reference anti - sapstain chemical in a 1 : 80 dilution of the product in water . freshly sawn , ungraded and clean lumber of white pine and jack pine was obtained from local sawmills and was cut into the size of 1 &# 34 ;× 4 &# 34 ;× 16 &# 34 ;. the boards were dipped in the different solutions separately one piece at a time . each treatment contained 120 boards in three different packs ( 40 boards per pack ), and untreated boards served as controls . the sets of boards treated with different solutions were piled side by side in the yard of forintek . the pile was covered with a plastic sheet to prevent wood dry up and to create a favorable condition for the fungal growth . after four months storage , each board was rated for the development of mold , sapstain and decay individually based on the visual rating system describe in example 1 . the board was considered as acceptable if the sum of scores of the three defects in this board was 2 or less . the results of this test are shown in fig1 . on white pine wood , g . roseum alone or alkaline solution alone did not provide protection from sapstain while the combination of both agents significantly reduced the wood infection and increased the number of acceptable pieces . on jack pine wood , application of g . roseum alone only provided partial protection from fungal attack . however , adding 4 % sodium carbonate and 1 % sodium bicarbonate into the spore suspension significantly increased acceptable pieces of treated lumber . on both wood species , g . roseum in 4 % sodium carbonate and 1 % sodium bicarbonate solution gave better protection than the reference chemical did . the evaluation of the combination of alkalis and the biocontrol agent against sapstain on logs was also conducted in field conditions . black spruce logs were obtained from a local sawmill in the summer of 1997 and were cut into 80 cm in length . these logs were dipped in either a g . roseum cell suspension , or a suspension containing both g . roseum inoculum and the alkalis described in example 3 . logs treated with the reference chemical in a 1 : 40 dilution of the product in water as well as untreated logs served as controls . each treatment contained 7 replicate logs , and all logs were closely piled in the forintek backyard and covered with a plastic sheet to prevent dry up . extent of sapstain in logs was assessed after 4 months storage . logs were cut into four 3 cm - thick discs from each end . surface cover and maximal penetration of sapstain on the cross section was measured for each disc . a mean value was calculated from a total of 56 discs sawed from 7 replicate logs in each treatment . a statistical analysis was employed in the same way as in example 2 , and the result is summarized in table 3 . logs treated with the suspension containing both g . roseum and alkalis resulted in 2 . 2 times and 4 . 1 times less stained wood than that treated with g . roseum alone and untreated logs respectively . no significant difference existed between the treatments with the mixed solution and the reference chemical . table 1______________________________________growth of mold and sapstain on wood wafers of white pineand jack pine treated with different agents in a laboratorytest under conditions of inoculation with sapstaining fungi acceptable pieces (%) white pine jack pinetreatment 8 weeks 16 weeks 8 weeks 16 weeks______________________________________na . sub . 2 co . sub . 3 + nahco . sub . 3 + 100 100 100 100g . roseumg . roseum 80 50 90 80na . sub . 2 co . sub . 3 + nahco . sub . 3 50 20 40 30reference chemical ( 1 : 80 ) 100 100 100 90untreated 10 0 50 10______________________________________ table 2______________________________________growth of mold and sapstain on wood wafers of white pineand jack pine treated with different agents in a laboratorytest under conditions of natural infection acceptable pieces (%) white pine jack pinetreatment 8 weeks 16 weeks 8 weeks 16 weeks______________________________________na . sub . 2 co . sub . 3 + nahco . sub . 3 + 100 100 100 100g . roseumg . roseum 70 40 97 87na . sub . 2 co . sub . 3 + nahco . sub . 3 17 10 80 67reference chemical ( 1 : 80 ) 67 47 97 90untreated 7 3 63 47______________________________________ table 3______________________________________development of mold and sapstain in logs of black spruce treatedwith different agents in a field test % stain maximal penetrationtreatment coverage ( mm ) ______________________________________na . sub . 2 co . sub . 3 + nahco . sub . 3 + g . roseum 6 . 1 7 . 2g . roseum 13 . 2 13 . 6reference chemical ( 1 : 40 ) 5 . 7 7 . 5untreated 25 . 0 13 . 9______________________________________ alexander , m . 1977 . introduction to soil microbiology . 2nd ed ., john wiley and sons , new york . bergman , o , t . nilsson and p . jerkeman . 1970 . reduction of microbial deterioration in outside chip storage by alkali treatment . svensk papperstidning 73 : 653 - 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