CANADIAN BLUESTAIN FUNGI: VARIATION IN TOLERANCE TO SAPSTAIN CONTROL BIOCIDES.
Devaluation of Canadian wood products by sapstain accounts for large and unpredictable losses each year. A large-scale survey of bluestain fungi in Canada found that the majority of aesthetic damage to softwood lumber was caused by three sapstain fungi: Ophiostoma piceae, O. pi1iferum, and O. floccosum. This paper reviews an evaluation of the tolerance of 30 of the isolated fungal strains (10 strains of each species) to a range of concentrations of active ingredients in different sapstain control products. The tolerance was correlated to the colony diameter after 12 days of fungal growth at 18[degrees]C on "pine-twig" agar amended with the chemicals at different concentrations. Little or no variation in tolerance within the 10 strains of each species tested was observed.
As part of a collaborative project with the University of British Columbia and Universite Laval, a large number of staining fungi have recently been isolated from Canadian softwood logs and lumber. The project aimed to determine which fungi cause the greatest stain problems in logs and lumber across Canada, and further to determine the genetic diversity within the main species [7,14]. Data on growth rates and other properties of these fungi have been gathered. While most of the fungi have been identified to their genus and species, the molecular biology work to determine their genetic diversity is still ongoing. This project has resulted in the first large-scale, detailed survey of such fungi across Canada. This paper reviews an evaluation of the variability in the resistance of the strains to sapstain control chemicals. 
There have been fluctuations in the effectiveness of some of the chemicals used to control sapstain over the years when applied on lumber at their recommended levels (Forintek, unpublished data). This provokes the question of whether fungi have developed resistance to such fungicides. The same question has also been raised in New Zealand [8,15], which led to researchers examining the growth of fungi on treated crosssections of branch wood . Forintek has also previously tested fungal tolerance to chemicals on solid wood . The main difficulty with these tests is that the chemicals are usually applied by dipping the wood into a liquid solution. This results in an uneven chemical distribution and in uncertainty about the amount of chemical present on the wood surface giving an inherent variability in results that cannot be controlled. While there is recognition that testing wood-inhabiting fungi on wood is the most desirable and life-like, the current authors felt that a simple assay on a suitable agar sub strate could be used to determine variability within and between isolates of key fungal species in their resistance to sapstain control chemicals. Measuring the fungal colony diameter on agar after a set incubation period could provide a method for the rapid screening of substances that affect fungal growth.
Preliminary testing involved developing a suitable nutrient-substrate, and selecting test chemicals at suitable concentrations. In nature, sapstain fungi utilize the nutrients obtained from the sapwood and bark of trees. For this reason, we examined the following growth substrates for suitability: media prepared from defoliated twigs with the growth substrate should mimic that found in nature and require the fungi to produce the enzymes and metabolic systems that they would use in infection of logs or lumber. Inclusion of ground pine twigs into agar media has recently been used by Uzunovic and Webber  to study growth rates and patterns of growth of various bluestain species. A similar substrate has been used over an extended period by Brasier  who used elm-twig agar for his work with the Dutch elm disease fungus, Ophiostoma ulmi.
MATERIALS AND METHODS
PRELIMINARY TEST TO DETERMINE SUITABLE MEDIA
A preliminary test was done with three isolates (each representing a different fungal species) grown on several water-based media (Table 1).
Sawdust was prepared from either fresh or frozen lodgepole pine (Pinus contorta Dougl.) sapwood lumber, or lodgepole pine twigs (1 to 2 cm diameter) that were first defoliated, scrubbed clean of lichens, and dried overnight (80[degrees]C) before grinding in a Thomas mill and passing through a 2-mm screen. The sawdust was added to agar and distilled water. The various media were then autoclaved and cooled prior to pouring into petri plates.
The fungi used in this preliminary test were: 1) Ophiostoma piceae (Munch) H. & P. Syd. (AU 114-2-1) isolated from white spruce lumber (Picea glauca (Moench) Voss), Big River, SK; 2) Ophiostoma floccosum (Mathiesen) (AU 55-6), isolated from lodgepole pine lumber, Okanagan Falls, BC; and 3) Ophiostoma piliferum (Fr.) H. & P. Syd (AU 121-3), isolated from white spruce lumber, Big River, SK. These were selected as being representative of the three species in terms of morphological appearance when grown on malt extract agar.
The fungal growth rates were determined by measuring the colony diameter after 3 and 6 days of incubation at 20[degrees]C. During measurements, observations of the colony morphology and growth of reproductive structures were also made. Based on the results, a medium consisting of pine twig sawdust (6%), and agar (3%) was selected for the next phases of the test.
PRELIMINARY TEST TO DETERMINE RANGE OF CHEMICAL CONCENTRATIONS
Selection of sapstain control chemicals was based on the products used by the Canadian forest products industry in their treatment of green export lumber. There are about 10 chemical formulations registered for sapstain control use in Canada. The formulations most commonly used by the BC industry are based on the quaternary ammonium compound didecyldimethylammonium chloride (DDAC). This chemical is used with the co-biocides disodium octaborate tetrahydrate (DOT) in the formulation F2 (Walker Brothers Ltd.), or with 3-iodo-2-propynyl butyl carbamate (IPBC) in the formulation NP-1 (Kop-Coat Inc.), or without a co-biocide but mixed with a latex (a formulation called QC-3). The active ingredients DDAC and DOT, and the formulations NP-1 and F2, were included in the scope of the experiments reported here. The source of DDAC was "Maquat 4480-E," an 80 percent concentrate produced by Mason Chemical company. The DOT was from stock supplies of Tim-Bor (98% a.i. powder) from U.S. Borax Inc. In addition, Mycostat-P, a n ew formulation containing 4.5 percent propiconazole (PCZ), was obtained from Diacon Technologies Ltd. and included in the testing. The PCZ active ingredient was not tested by itself due to its insolubility in the medium used.
The same three fungal isolates were used in an experiment to determine the range of chemical concentrations to be used in the main test. In addition to control plates with no chemical added, the pine-twig medium was amended with DDAC, DOT, or Mycostat-P in the following amounts: 1) DDAC at 1, 10, 100, and 1,000 ppm; 2) DOT at 10, 100, 1,000, and 10,000 ppm; c) Mycostat-P at 1, 10, 100, and 1,000 ppm.
The appropriate amount of fungicide to achieve the desired concentrations was added to the medium after it had been autoclaved and allowed to partially cool. After further cooling, the agar was poured into petri plates with care taken to distribute the pine-twig sawdust as homogeneously as possible between the plates. Inoculation occurred the following day with a single 5-mm mycelial plug of one of the three test fungi placed centrally on each plate. The plates were incubated at 20[degreeS]C, and colony diameters were measured after 6 and 13 days.
Based on results from the first preliminary test, a medium containing 6 percent pine-twig sawdust and 3 percent agar was selected and prepared as described previously. Fungal tolerance to two active ingredients (DDAC and DOT) and three sapstain-control products (F2, Mycostat-P, and NP-1) was examined. The active ingredient concentrations were selected for the various chemicals based on the preliminary tests (Table 2).
NP-1 consists largely of DDAC, and the concentration of total actives (DDAC + IPBC) tested was the same as for DDAC alone. F2 consists largely of DOT, and the concentration of total actives (DOT + DDAC) was the same as for DOT alone. The tenfold increase in concentration of DDAC and NP-i, the twofold increase in concentration of DOT and F2, and the fivefold increase in the concentration of Mycostat-P, reflect differences in the tolerance of fungi to these chemicals seen in the preliminary test. The fungicide-amended plates were made as previously described. A total of 10 different isolates of each of the 3 fungal species were used in the main test. Apart from three isolates of 0. piliferum, the isolates were obtained in the 19971998 Canadian survey from different wood hosts (e.g., lodgepole pine, jack pine (Pinus banksiana Lamb.), white and black spruce (Picea mariana (Mill.) as well as from different geographic regions in Canada.
Each fungal strain was inoculated onto three plates of each concentration of fungicide. The inoculum source was an agar plug (5 mm) from the perimeter of an actively growing fungal colony. The plates were incubated at 18[degrees]C for 12 days, after which the colony diameters were measured along two intersecting lines.
Based on growth rate data from the main test, a small experiment was set up to determine the role that the sawdust might be playing in decreasing the bioavailibility of the chemicals (in particular DDAC). For both DOT and DDAG, three flasks were prepared containing 3.0 g of lodgepole pine-twig sawdust in 50 mL of distilled water. To these flasks 400, 800, and 1,200 ppm of the respective chemical was added. In addition, one control flask was prepared for each chemical at a concentration of 1,200 ppm in which no sawdust was added.
The flasks were mixed well and allowed to stand for 2.5 hours at room temperature. Following this period, the contents were filtered through a Buchner funnel under vacuum and the filtrate was analyzed for DOT or DDAC, respectively. Determination of DOT concentration was done by titration, where the borate was complexed with mannitol and titrated to neutral pH with potassium hydroxide. For the DDAC, 1.0 mL of the filtrate was placed in a vial and the water was evaporated off before redissolution in acetonitrile. The HPLG method developed by Daniels (5) was used to determine the DDAC concentration.
RESULTS AND DISCUSSION
The colony diameters on the different media used in the preliminary test are shown in Table 3. O. piceae, and O. pilferum generally grew at similar rates on a given medium while O. floccosum grew somewhat slower. There were only slight differences in growth rates between the different media but the appearance of the colonies was variable. On water agar, the fungal growth was sparse and colony diameters could not be measured.
PRELIMINARY TEST TO DETERMINE SUITABLE MEDIA
Sporulation and density of the colony was most pronounced on the malt extract agar and 10 percent twig sawdust medium. However, at 10 percent sawdust, the medium was extremely viscous and difficult to handle. Therefore, a medium consisting of 6 percent pine-twig sawdust and 3 percent agar was chosen for further testing. Because only slight differences were observed between the growth of the three replicates ([sim]1 mm in diameter after 10 days) of each fungus on each medium, three replicate plates per variable were continued for the rest of the work.
PRELIMINARY TEST TO DETERMINE RANGE OF CHEMICAL CONCENTRATIONS
Figures 1 through 3 show the average growth of the three test species on the selected media when amended with DDAC, DOT, or Mycostat-P. The fungi show decreased growth in response to increased concentration of these fungicides. It can be seen that the fungal growth response to DDAC occurred over a larger concentration range than the response to DOT or Mycostat-P. Based on these results, the final test concentrations for the chemicals with DDAC alone were selected to cover a broader range than those with DOT. The presence of DOT in the F2 formulation influenced the final concentrations chosen for testing.
Figures 4 through 8 show the average growth after 12 days of the 10 strains of each species tested in the main test. Error bars representing [+ or -] one standard deviation indicate that the variation in the growth rates between the different isolates of each fungal species is low for all chemicals at the concentrations tested.
In the control plates (no chemical added) after 12 days, the average colony diameters of the strains of O. piceae and O. piliferum were almost the same, 56 mm [+ or -] 1.7 mm and 56 mm [+ or -] 1.9 mm, respectively. O. floccosum grew at a slower rate with a diameter of 43 mm [+ or -] 1.2 mm. Care was taken in preparing the pine-twig agar to make the plates as uniform as possible; however, due to the coarse nature of the medium, it was difficult to distribute an even amount of sawdust into the plates. The small amount of variation within each species grown on pine-twig agar suggests that the nutrients from the sawdust were extracted into the agar during autoclaving, and that the amount of sawdust present in each agar plate may. not be as important as first thought.
With fungicide-amended media there was a consistent drop in the growth rate of all the fungi with increasing chemical concentration. The variation in growth rates within each species remained low and similar to the control plates. At 1,200 ppm DDAC, all three species still showed growth, while at the same concentration of NP-1, fungal growth was completely halted. As NP-1 contains DDAC and IPBC in the proportions 9:1, it can be assumed that the increased effectiveness is a result of the co-biocide IPBC and/or the inert ingredients in the formulation. However, there are considerations in the way active ingredients behave in this test method that limit its usefulness in comparing the efficacy of certain active ingredients such as DDAC or formulations thereof.
As DOT is generally less toxic to stain and/or mould fungi than DDAC (3), it is interesting to note that while DDAC did not stop fungal growth at 1,200 ppm, DOT at a concentration of 800 ppm did. However, it was hypothesized that this probably does not represent the true toxic thresholds for these fungicides. DDAC is a surfactant with a strong affinity to bind to solid surfaces [9,10], including wood  through an ion-exchange re action with the acidic groups [11,12]. Thus, it is probable that a large portion of the DDAC was being bound to the sawdust and was not bioavailable to the fungus at the concentration calculated. The small sawdust-adsorption experiment described previously was set up to test this hypothesis.
From the results of this small test (Table 4) it can be clearly seen that there is a negligible binding of the water-soluble DOT to the sawdust. Recoveries ranging from 92 to 95 percent DOT were measured in the filtrate of the sawdust-containing flasks, while the control flask with no sawdust showed a recovery of 94 percent DOT. However, a very different result was seen with DDAC. DDAC recoveries from solution in flasks containing sawdust ranged from 0 to 6 percent, while the control flask showed a recovery of 93 percent DDAC. This confirms that the DDAC was strongly adsorbed by the sawdust.
Mycostat-P was capable of preventing growth of all of the fungi screened at only 80 ppm of PCZ. This concentration is much lower than that of the other chemicals screened. However, the manufacturer recommends application levels approximately 10 times lower than the other formulations. While Mycostat-P lists PCZ as the active ingredient (4.5% of the concentrate), other compounds present in the formulation, including surfactants, may well play a role in affecting fungal growth.
The growth rates within each species of bluestain fungi were very consistent both on fungicide-amended and non-amended agar. This confirms that growth rates can be a valuable diagnostic tool for determining species of certain Ophiostomatoid fungi. The test method proved to be suitable for examining the variation in tolerance between fungal isolates to a particular chemical. However, due to the interaction of some chemicals with the sawdust in the medium, the test was not suitable for comparing the tolerance of an individual fungal isolate to different chemicals. The study found no evidence of variation in tolerance to the fungicides screened. Such variation might be anticipated if resistance in strains of these species is occurring. However, it should also be noted that only 30 fungal isolates out of 2,000 in the Canadian survey were chosen for this experiment, and increased tolerance to some antisapstain products may still exist.
As it appeared that the fungi were growing on the soluble nutrients from the sawdust, and that the sawdust itself could interfere with the bioavailability of some chemicals, the authors recommend using an agar medium containing the extracted nutrients without the sawdust to conduct these types of chemical screening tests.
The authors are, respectively, Environmental Scientist, Wood Protection Scientist, and Mycological Technologist, Forintek Canada Corp., Western Lab., 2665 East Mall, Vancouver, BC, Canada V6T IW5. The authors would like to recognize the advice and input of Adnan Uzunovic and the technical help of Kenneth Binnie. In addition, they thank Bob Daniels and Steven Erickson for conducting the DDAC analysis. This paper was received for publication in April 1999. Reprint No. 8967.
(1.) The mention of any commercial product used in testing by Forintek Canada Corp. does not constitute endorsement by Forintek Canada Corp.
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Media screened for suitability in preliminary test. Code Nutrient source Agar A 5% pine twig 3% agar (Difco) B 10% pine twig 3% agar (Difco) C 5% pine sapwood 3% agar (Difco) D 10% pine sapwood 3% agar (Difco) E Nil 3% agar (Difco) F 3% malt extract (Oxoid) 1.5% agar (Oxoid) Concentrations of active ingredients in the fungicide-amended media. NP-1 F2 Mycostat-P DDAC (DDAC + IPBC) DOT (DOT + DDAC) (PCZ) (ppm) 1,200 1,200 800 800 80 120 120 400 400 16 12 12 200 200 3 Colony diameter of fungi on various media after 6 days of incubation at 20 [degrees]C (5-mm agar plug inoculum substracted). Concentration Medium O. piceae O. floccosum O. piliferum (%) (ppm) 10 LP sapwood sawdust 30 25 32 5 LP sapwood sawdust 30 23 31 10 LP twig sawdust 28 23 29 5 LP twig sawdust 28 23 28 -- Malt extract agar 32 26 32 The loss of DOT and DDAC from solution by interaction with sawdust. DOT DDAC Sawdust Initial Final Recovery Initial Final Recovery (%) (ppm) (%) (ppm) (%) 6 380 360 95 390 0 0 6 750 690 92 780 40 5 6 1,130 1,040 92 1,170 75 6 None 1,160 1,090 94 1,170 1,090 93