Efficient use of sapstain control products requires information on the effectiveness of the actives and formulations against different types of fungal challenges. The present work examines sapstain control product efficacy against spores, approaching mycelia, and established mycelia in laboratory experiments, and against preinfection in field experiments. Laboratory experiments found that 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT)/2-methyl-4-isothiazolin-3-one (MIT), 3-iodo-2-propynylbutylcarbamate (IPBC), and propiconazole applied prior to exposure to mold fungi were most effective against spores, less so against approaching mycelium, and least effective against live mycelia already present in wood prior to treatment. Field tests confirmed that sapstain was more difficult to control on wood preinfected with sapstain and mold. Although short periods of exposure prior to treatment may not affect efficacy, any delays between manufacture and treatment increase the risk of preinfection and should be avoided.ABSTRACT
Fungal infection of wood can occur at many points along the wood-processing value chain, including in the living tree, in logs at various stages after harvest, during postharvest storage and processing into lumber or other wood products, and during subsequent storage and transport. This infection normally appears as a visible discoloration of wood, termed sapstain. This occurs predominantly on the sapwood and is caused by fungi from various groups, including blue stain fungi, black yeasts, colored molds, and/or decay fungi (Uzunovic et al. 1999). The predominance of one type of the fungal group over others may vary substantially depending on location, substrate, and time of the year and is often linked with the type of wood used, storage conditions, moisture content, and tolerance to specific sapstain control formulations (Tsunoda and Nishimoto 1985, Williams et al. 1998, Strong et al. 2005). The presence of these fungal infections reduces the value of the wood. Because terminology is sometimes confusing and used inconsistently, the terminology we use is described below. We do not synonymize sapstain with blue stain; sapstain is considered discoloration of sapwood caused by the fungal groups mentioned above, whereas blue stain is discoloration caused by a specific group of fungi called blue stain fungi. The most common blue stain fungi affecting commercially important species in temperate zones belong to the Ceratocystis, Ophiostoma, Grosmannia, Leptographium, and Sphaeropsis genera (Uzunovic et al. 1999). These fungi colonize and cause penetrating blue stain in standing trees and stored logs (also called deep penetrating blue stain) and lumber (called surface blue stain; Uzunovic et al. 1999). The surface blue stain that occurs on clean lumber can be planed off, similar to discoloration caused by molds, while deep blue stain that develops in logs cannot. The black yeasts include species such as Aureobasidium pullulans and Hormonema dematioides (Uzunovic et al. 1999). The dark molds may include species from Alternaria, Cladosporium, and Aspergillus. Other mold genera such as Trichoderma and Penicillium can also discolor the surface of wood, but in general do not penetrate deep into the sapwood. Some studies have shown that a majority of mold species preferentially attack dead wood while live wood (e.g., freshly cut, green lumber with living host cells) is primarily colonized by blue stain species and very few molds, including most commonly Trichoderma (Williams et al. 1998, Strong et al. 2005). All of these fungi have the potential to exist as preinfections, and because they also have different biology, their identification plays an important role in developing control strategies. Certain formulations may be more effective against one fungal group but ideally should have a sufficient spectrum of activity to control all of the fungal groups that can cause sapstain.
Sapstain control products (antisapstain treatments) may be applied to logs or green lumber to prevent the formation of stain during processing and delivery to market. Sapstain control products may also be used to protect kiln-dried lumber from sapstain fungi when there is a risk of rewetting (Melencion and Morrell 2007). Treatments are typically applied either by dipping or spraying.
Older, broad-spectrum antisapstains, including organo-mercurials and chlorophenates, were capable of preventing already existing and established infection by blue stain, mold, and decay fungi (preinfection) from manifesting itself at the surface of the lumber. This was most likely because of their toxicity, and possibly better penetration below the surface, since they were typically applied by soaking rough cut wood in dip tanks (Roff et al. 1980, Byrne 1997). Sapstain control products used in recent years are generally prophylactic—that is, they are not intended to arrest or kill existing fungal infections, but rather stop new ones. Such antisapstain formulations have been shown to be less effective against mycelia than against spores (Xiao and Kreber 1999). The efficacy of these treatments therefore supposes that the wood being treated is substantially free of preinfection. This is not always the case, and the degree to which infection prior to treatment affects treatment efficacy is poorly understood.
Minimizing the time between felling or manufacture and treatment will limit the risk of preinfection (Butcher 1980). In field studies, logs could be stored for much longer in winter than in summer without preinfection limiting the efficacy of sapstain control products (Eden et al. 1997, Kreber et al. 2001). Stringent log inventory management and minimizing the time between felling and lumber production have been found to be critical in minimizing discolorations on southern hardwoods (Irby 2008).
Mobile sapstain control products that can diffuse into the wood may enhance activity against preinfections (Singh 2008). However, this also depletes actives from the surface of the wood, which could increase vulnerability to new infections (Williams et al. 1985). The vapor action and diffusibility of methylene-bis-thiocyanate (MBT) have been implicated in its efficacy (Singh et al. 2002). Eden et al. (1999) reported that a formulation of MBT and 2-n-octyl-4-isothiazolin-3-one killed two sapstain fungi up to 2.5 mm deep when applied up to 10 days after cutting. Borates are well known for their ability to diffuse into wood (Schoeman et al. 1998), which would likely be valuable in combating preinfection; however, at the concentrations used they typically did not provide sufficient activity alone to control mold fungi (Byrne 1990). Antisapstain formulations with borates added have been shown to be effective in preventing brown rot decay fungal colonization that was not controlled by carbendazim and prochloraz (Adams et al. 1997). Relatively little is known about the mobility of other actives and their effect on preinfection.
More information is needed on performance against preinfection to ensure the efficient use of sapstain control products. The present work examines the efficacy of a combination of two isothiazolones (5-chloro-2-methyl-4-isothiazolin-3-one [CMIT] and 2-methyl-4-isothiazolin-3-one [MIT]), 3-iodo-2-propynylbutylcarbamate (IPBC), and propiconazole against spores, established mycelia from an outside source, and mycelia from wood colonized prior to treatment (preinfection). We hypothesized that some fungal isolates/species may be more tolerant to chemicals used, and certain types of fungal challenges would be more difficult to control. The test fungi used in the laboratory test are those repeatedly found on treated wood in the field at the time of the experiments. It happened that they included several mold fungi and not blue stain or decay fungi. The present work also examines sapstain control product efficacy in a field test on fresh and preinfected material where both blue stain and mold fungi were present but blue stain fungi happened to be predominant. We hypothesized that longer delays in product application would allow more fungi to grow and become established, making them more difficult to control.
Materials and Methods
Laboratory test
Kiln-dried Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) sapwood was cut into 1-mm-thick radial strips, autoclaved, and conditioned to equilibrium moisture content (approximately 8%). Veneer strips were then laminated on one side with a polyethylene plastic through the application of heat to reduce the risk of chemicals from the wood diffusing into the growth medium (Woo et al. 2010). Test wood disks (6-mm diameter) were then aseptically punched from the laminated veneer strips. Actives concentrates, including propiconazole (20%), IPBC (20%), and a mixture of isothiazolones (10.5% CMIT and 3.5% MIT), were diluted to 10, 30, and 100 ppm (actives basis) with distilled ethanol (100%). Ten microliters of each of the treating solutions and of an ethanol control were aseptically dispensed onto the surface of each wood disk. Four replicates were set up for each isolate with each inoculation method. In addition, four wood disks were left uninoculated to serve as sterile controls. Wood disks were exposed to filtered air in a biosafety cabinet for 30 minutes after treatment to allow for ethanol evaporation.
Test fungi were molds that had been isolated from treated wood, tentatively identified based on the internal transcribed spacer region of extracted DNA and partial beta tubulin gene sequence, and deposited in FPInnovations culture collection (Table 1). An additional isolate was tested as a reference—a Penicillium species isolated from moldy bread. After initial results were obtained, two additional concentration levels (300 and 600 ppm) were added to the study using five of the initial seven test isolates. Two Penicillium test species were dropped from the test: isolate AU 312-28 because it was thought to be the same as AU 312-29, and isolate AU 312-27 because it was thought to be more sensitive to the actives based on the initial results.
Eggins and Pugh cellulose medium (Eggins and Pugh 1962) was prepared and 2 mL was dispensed into each well (16-mm diameter) of a 24-well sterile polystyrene multiwell plate. Treated wood disks were placed in each well, laminated side down, prior to inoculation with fungi (except for precolonized wood disks). Three sources of inoculum were used for testing: fungal spore suspensions (spore inoculum) applied to test wood disks, mycelia established on agar near test wood disks (approaching mycelia inoculum), and test wood disks precolonized with mycelia prior to chemical application (precolonized wood substrate).
Spores were harvested from test isolates that had grown on 1 percent malt extract agar at 21°C for 7 or 8 days. Spores were scraped off the agar with a sterile scalpel, and the spore solution was then filtered through sterile glass wool to remove mycelial fragments. The solution was thoroughly vortexed and diluted to 5,000 spores per mL. Twenty microliters of the solution (approximately 100 spores) was then pipetted onto each treated wood disk in the center of the multiwell plates. For approaching mycelial inoculation, a plug (6 mm in diameter) of fungal tissue and agar was sampled from the margin of actively growing mycelia and was dipped and washed in a 0.5 percent agar solution and dabbed on a sterile Kim-Wipe to remove loose spores, thus preventing cross-contamination between wells. The plug was then cut in half with a sterile scalpel, a 10-mm stainless steel pin was inserted into each half, and each half plug was fixed mycelia side down on one side of a well. The wood disk was placed on the opposite side of the well, approximately 4 mm apart from the inoculum.
For precolonized disk inoculation, wood disks were placed with the wood side in contact with mycelia on the growing margin of two species, the Penicillium isolate AU 312-22 and the Fusarium isolate. Cultures were incubated for a further 8 days prior to chemical application to allow colonization of the wood. Wood disks were then removed from cultures, and excess mycelium was scraped off with a sterile scalpel prior to placing disks onto the agar surface in the multiwell plates with the mycelia side facing up. Ten microliters of treatment solution at a given concentration was then administered onto the surface of the disk. All multiwell plates were incubated at 25°C.
Assessments for growth were made on days 2, 5, 8, and 12 for the first set of concentrations (10, 30, and 100 ppm) and on days 5 and 12 for the second set of concentrations (300 and 600 ppm). Growth was rated on a 0 to 5 ordinal scale (Table 2). Differences between fungal species were assessed by averaging the growth rating for day 12 for each set of replicates. Differences in rating distributions between inoculum types were assessed using the Kruskal-Wallis test (α = 0.05). Minimum inhibitory concentrations (MICs) were defined as the concentrations at which growth of all replicates of an isolate were inhibited on the wood disk.
Field test
The antisapstain field test followed the AWPA E29‐13 method (American Wood Protection Association 2013) with the following modifications: nontest boards were used as outer pieces on both sides, an acrylic plastic frame was used to support a nontreated plywood roof over the package, storage was above soil rather than on an asphalt surface, and a soaker hose was used to keep the ground moist underneath the pallets that supported test boards. Coastal Douglas-fir logs were felled and milled within 4 to 6 weeks. Nominal 1 by 4-inch (19 by 89-mm) boards were cut to include as much sapwood as possible and stored in a freezer within 24 to 72 hours of milling until used. Boards were cut into 0.9-m lengths and thawed under cover in the shade for 4 to 16 hours prior to treatment. Samples with pure heartwood, extensive wane, and with sapstain already present were excluded.
Artificial preinfection inoculum was prepared from a selection of sapstain fungi that were previously isolated from Douglas-fir. The fungi included Ophiostoma setosum (AU160‐16, AU312‐1), Ophiostoma piceae (AU160‐10, AU160‐5, AU160‐2, and AU160‐1), Ophiostoma floccosum (AU312), Ophiostoma quercus (AU312‐2), and Penicillium spp. (AU311, AU312‐21, and AU312‐26). Each isolate was first grown for 10 days on 1 percent malt extract agar, and profuse sporulation was confirmed by observing the cultures under microscope. Ten milliliters of sterile water was poured into each plate, and the surface mycelium was scraped to form a slurry that was decanted into a larger collection flask containing sterile water. Each plate after scraping was flushed one or two more times with additional sterile water into a collection jar. The collection jar was then shaken vigorously and poured through two layers of sterile gauze to remove any large mycelial networks. The average spore count was 2.05 × 106 per mL of spray liquid. The mix contained numerous spores of the above-mentioned Ophiostoma species in addition to distinct, round, and greenish spores of Penicillium spp.
For artificially preinfected piles, each lumber piece was first sprayed on all four sides with the spore mix. Approximately 3,000 mL of the spore mix solution was used to spray 180 boards. Inoculated boards were left closely packed (no stickers) and were loosely wrapped on the top and sides (not bottom) with a layer of lumber wrap (white side facing out). The piles were stored in a shaded area to incubate for 3 or 7 days.
Test packs were treated without artificial preinfection (fresh), or after 3 or 7 days of incubation following artificial preinfection. A commercial sapstain control product was evaluated at a low and high concentration that contained a 4:3 ratio of propiconazole to IPBC. Boards were individually treated by dipping for 1 minute followed by dripping and stacking into test piles. Treating solutions were analyzed for propiconazole before and after treatment, and five 625-mm2 surface samples were taken from each treatment group for propiconazole analysis by high-performance liquid chromatography using methods similar to AWPA A28‐08 (AWPA 2008). Test packs were constructed with close-packed and stickered sections as described in AWPA E29‐13 (AWPA 2013). Because the performance of close-packed and stickered sections was similar (Stirling and Uzunovic 2012), only data from close-packed sections are reported here. There were 25 test boards for each variable; however, replication ranged from 8 to 19 because many boards were excluded for having sapwood content less than 25 percent of the face. Samples were exposed for 4 months above soil and shaded by small trees and underbrush in Vancouver, British Columbia. The maximum, minimum, and mean temperatures during the exposure period were 27°C, −5°C, and 11°C, respectively. The total precipitation was approximately 375 mm.
Each board face and edge with substantial sections of sapwood was visually rated on the 0 to 5 scale specified in AWPA E29‐13 for blue stain and mold, where 0 represents no growth and 5 represents very heavy fungal growth. Only data from the wide faces are presented in this article (edge data showed similar trends). Faces with less than approximately 25 percent sapwood were not considered. Means and medians were calculated for each treatment group. The Mann-Whitney U and associated asymptotic P values were calculated to determine whether there were statistically significant differences between median ratings from selected groups.
Results and Discussion
Laboratory test
All fungal isolates were able to grow on the untreated wood disks. Test groups were compared on the basis of their 12-day ratings, since high growth ratings (2 or greater) were found on all control samples at this time (Tables 3 and 4). Controls inoculated with spores and mycelia were not fully covered because the ratings were made before they were able to fully colonize the test wood disks. Although mycelial inoculation was associated with the lowest median growth ratings, possibly due to the need to grow 4 mm over the agar before contacting the disk, most isolates were able to establish some growth on the disk. After the application of actives, growth was least affected on disks that were precolonized prior to treatment. Inoculum source had a significant effect on ratings distribution (χ2 = 108.5, P < 0.001). This indicates that the treatments were better able to control spore germination than prevent approaching mycelial colonization from media or mycelia present in precolonized wood.
MICs were calculated for each isolate and each chemical treatment (Table 5). None of the evaluated concentrations of isothiazolones were able to control all isolates as approaching mycelia or when present in precolonized disks. However, the isothiazolones completely inhibited spore germination for all replicates of all isolates at 100 ppm. Only 4 of 56 test samples inoculated as spores (1 Fusarium and 3 Penicillium replicates) were able to germinate at the lower concentrations (10 and 30 ppm). It is unlikely that this difference was due to species resistance, because growth was not confined to a single species. The individuals that germinated achieved moderate growth, suggesting that the active may interfere with the germination process rather than slowing the growth of mycelia once the spore germinated. This is supported by the data showing that growth from the mycelia inoculum was not slowed substantially for most isolates against these actives.
IPBC prevented growth from spores and mycelium for all tested isolates except Fusarium. MICs were not reached for wood precolonized by either Fusarium or Penicillium AU 312‐22 (Table 5). MICs were 100 ppm for all spore inocula and between 30 and 300 ppm for approaching mycelial inocula. Although generally not as effective against spores as the isothiazolones, IPBC was more effective against mycelia.
Propiconazole had limited efficacy against the test fungi at the concentrations tested. MICs were only found for two strains with spore (AU312‐26, AU312‐27) and mycelial (AU312‐26, AU312‐29) inocula. As with the other chemical treatments, the MIC was not reached for wood precolonized by either Fusarium or Penicillium AU 312‐22.
The growth of Fusarium on precolonized disks was generally very high (Table 6). The highest concentration of isothiazolones showed some potential inhibition, with an average growth rating of 2.5. All other actives had average growth ratings of 3.5 or greater, suggesting limited activity. Higher concentrations, or different actives, would be needed to control Fusarium on precolonized wood in this test.
The growth of Penicillium on precolonized disks was high on the untreated controls, and none of the chemical treatments fully prevented the growth of this fungus. However, there was a clear dose response for each active(s), with IPBC and the isothiazolones showing the greatest potential to control this fungus. None of the differences observed between the Penicillium and Phoma isolates were substantial, while Fusarium had higher overall tolerance to the actives and could possibly be used as a test species to determine effective concentrations against molds. Performance against sapstain fungi may be different. The use of dry wood could have affected the growth of fungi on the wood and uptake and distribution of antisapstain chemicals compared with green lumber (Terziev 1997). There was therefore a need to evaluate the effect of preinfection in a field test using green lumber.
Field test
Treating solutions exhibited low levels of propiconazole stripping following dip treatment (Table 7). Propiconazole analysis of selected wood samples after dipping showed similar results for fresh and preinfected samples (Table 7). Although samples treated with the high concentration had, on average, more propiconazole than those treated with the low concentration, there were no statistically significant (P < 0.05) differences between these groups. This was likely due to natural variability in the wood and suggests that more rigorous sampling may be needed for effective quality control.
Blue stain fungi had colonized the sapwood of most untreated boards and reached ratings of 5 after 1 month of storage (Fig. 1). Moderate levels of sapstain were observed on the samples freshly treated with the low concentration of the sapstain control product, while very low levels of sapstain were observed on the samples freshly treated with the high concentration. At the high concentration, median ratings from 3-day preinfected material were not significantly different from those of freshly treated samples; however, median ratings from the 7-day preinfected samples were significantly greater (P < 0.05). This suggests that under the conditions of this test, the high concentration of the antisapstain formulation was less effective against blue stain after inoculation and storage for between 3 and 7 days.
Citation: Forest Products Journal 63, 1-2; 10.13073/FPJ-D-13-00010
Only small amounts of mold were detected on untreated controls (Fig. 1). Mold ratings on samples treated fresh, or after 3 days of storage after preinfection, were generally similar or slightly higher. Blue stain suppression by the antisapstain treatment may have contributed to increased mold ratings in some samples. Mold ratings on 7-day preinfected samples were 0 or very low, suggesting that the experimental conditions favored the growth of blue stain fungi.
The preinfection in this experiment was intended to represent a challenge for a sapstain control product that would mimic a worst-case scenario where sapstain fungi are already present on the lumber surface for either 3 or 7 days before the product was applied. Under our field experiment conditions, 3 days of storage after inoculation was generally not associated with increased discoloration after the sapstain treatment, indicating that the fungi did not establish and grow well enough to challenge the efficacy of the control product. However, 7 days of storage after inoculation was associated with the increased presence of blue stain. This demonstrated that preinfection can greatly reduce sapstain control product efficacy. While antisapstain treatment will be most effective when applied immediately after sawing, storage for short periods of time before treatment may not result in poorer performance under some circumstances.
In regular operations, there is often some delay in converting freshly felled logs into lumber, or delay after conversion to lumber before an antisapstain treatment is applied. In most cases the delay is difficult to avoid, especially the one between felling and lumber production. In the present study we used material from logs that were 4 to 6 weeks old. This was the freshest material available. The positive control (where sapstain control products were applied on fresh lumber) did not have noticeable stain compared with untreated controls. It appears that longer delays for lumber production in some cases may be tolerated; however, a safe treatment delay period for freshly cut lumber is still unclear. From the present study it appears that a 3- to 7-day delay is not recommended, while delay of 1 to 3 days may be tolerated, although some boards in the 3-day preinfection group had noticeable sapstain. Safe delay periods would vary for different wood species, products, and exposures (season, geographic location, and microclimate). Where the weather and humidity is conducive to sapstain at almost any time of the year, the delay for antisapstain treatment must remain minimal and is most likely closer to a maximum of 1 day.
Conclusions
The two experiments (the laboratory and the field experiment) were not linked, but both addressed preinfection. The fungi used in the laboratory test section represented a range of common mold fungi that were encountered on wood treated with antisapstain products at the time of the experiments. We are not suggesting them as standard test isolates. It is likely that other fungi encountered under similar conditions and substrates and used as test isolates would react similarly. The laboratory test provided a method to examine the effect of specific fungi on chemical treatments under highly controlled conditions. In contrast, the field test replicated conditions that would be experienced in industry, something a laboratory test can never achieve. Because the aims of the tests were different, it was not necessary to use the same test fungi. Indeed, often the fungi that grow on wood in field tests are not the ones that were inoculated.
Laboratory tests using mold fungi demonstrated that the isothiazolones, IPBC, and propiconazole were less effective against mycelial growth than against spores and ineffective against growth from heavily precolonized disks at the concentrations tested. Field tests showed that established blue stain fungi were more difficult to control than new infections from spores.
Isothiazolones were best able to prevent spore germination in laboratory tests; however, IPBC was most effective against mycelia. Propiconazole was not inhibitory at the concentrations investigated in this test. The laboratory study demonstrated that there were different tolerance levels against actives among isolates used. Higher concentrations or different actives need to be tested to find inhibitory levels against more aggressive species, such as Fusarium.
The propiconazole/IPBC sapstain control product at the high concentration was largely able to control sapstain in freshly cut material. Incubation of preinfected lumber for 3 days prior to antisapstain treatment was not associated with increased fungal growth in most samples; however, incubation of preinfected lumber for 7 days prior to antisapstain treatment was associated with increased colonization by blue stain fungi.
Blue stain and mold ratings of board faces at low and high concentrations of sapstain control product over a period of 4 months.
Contributor Notes
The authors are, respectively, Principal Scientist, Scientist, and Senior Scientist, FPInnovations, Vancouver, British Columbia, Canada (adnan.uzunovic@fpinnovations.ca [corresponding author], angela.dale@fpinnovations.ca, rod.stirling@fpinnovations.ca); and R&D and Technical Manager, Diacon Technologies Ltd., Richmond, British Columbia, Canada (asidhu@diacon.com). This paper was received for publication in January 2013. Article no. 13‐00010.