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Inhibition of basidiospore germination by western redcedar heartwood extractives
International Biodeterioration & Biodegradation 114 (2016) 145e149
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Short communication
Inhibition of basidiospore germination by western redcedar heartwood extractives Rod Stirling*, Stacey Kus, Adnan Uzunovic FPInnovations, 2665 East Mall, Vancouver, BC, V6T 1Z4, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2016 Received in revised form 25 May 2016 Accepted 15 June 2016 Available online 27 June 2016
Understanding the relationship between extractives and decay resistance in western red cedar (WRC) is essential for breeding durable planting stock. To date, most such work has focused on resistance to mycelial attack. However, this is potentially misleading since WRC is largely used in above-ground applications where basidiospore germination is the primary way in which the wood is initially colonized. Efficacy against basidiospores is likely a critical factor affecting wood’s decay resistance in above-ground applications. In this study the effect of selected extractives on basidiospore germination was evaluated. Partially-extracted WRC veneer samples, and spruce samples treated with beta-thujaplicin, thujic acid, or plicatic acid were used. Beta-thujaplicin and thujic acid were associated with significantly reduced rates of Gloeophyllum sepiarium, Fomitopsis palustris, and Dichomitus squalens basidiospore germination. Plicatic acid was not associated with any effect on basidiospore germination. Planting stock that generates heartwood with high concentrations of thujaplicins and thujic acid should be selected to yield wood that will be resistant to basidiospore germination. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Basidiospores Extractives Germination Thuja plicata Western redcedar
1. Introduction Untreated western redcedar (Thuja plicata Donn) is used largely in above-ground, exterior applications, such as decking, fencing, and siding. These applications rely on the natural decay resistance of western redcedar (WRC) to meet service life expectations (Cartwright, 1941; Scheffer, 1957; Freitag and Morrell, 2001; Morris et al., 2011; Morris and Ingram, 2013). To date, most research investigating the relationship between extractives and decay resistance has focused on resistance to mycelial attack (Nault, 1988; DeBell et al., 1997; Taylor et al., 2006; Morris and Stirling, 2012; Kirker et al., 2013). This is potentially misleading since basidiospore germination is the primary way in which wood is initially attacked above ground (Savory and Carey, 1976; Schmidt and French, 1979; Bjurman, 1984; Fougerousse, 1984; Hegarty and Buchwald, 1988; Croan, 1994, 1995). Efficacy against basidiospores is likely a critical factor affecting the species’ decay resistance. WRC heartwood has been shown to inhibit germination and kill Gloeophyllum trabeum (Pers.) Murrill (reported as Lenzites trabea (Pers.) Fr.) basidiospores (Morton and French, 1966). It was also reported to
* Corresponding author. E-mail addresses: [email protected] (R. Stirling), stacey.kus@ fpinnovations.ca (S. Kus), [email protected] (A. Uzunovic). http://dx.doi.org/10.1016/j.ibiod.2016.06.008 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
be more resistant to colonization by basidiospores of Phellinus weirii (Murrill) Gilb. than other species (Nelson, 1976). However, the extractives responsible for this inhibitory and sporicidal activity had not been investigated. Twenty-one heartwood extractive compounds have been isolated from WRC heartwood and identified. The most abundant extractives are the thujaplicins, plicatic acid, and thujic acid (Daniels and Russell, 2007). The thujaplicins are well-known fungicides with demonstrated activity against mycelial attack (Rennerfelt, 1948; Rudman, 1962, 1963; Morita et al., 2004; Yen et al., 2008; Li et al., 2012). Plicatic acid is reported to have fungistatic activity (Roff and Atkinson, 1954), but has not shown fungicidal efficacy against mycelia at concentrations that would be present in wood (Lim et al., 2007; Stirling et al., 2007; Stirling and Morris, 2016). Thujic acid is generally believed to have low activity against decay fungi (Erdtman and Gripenberg, 1949), but has been reported to inhibit the growth of Rhinocladiella atrovirens Nannf. and Phellinus ferreus (Pers.) Bourdot & Galzin mycelia growing on artificial media (Lim et al., 2007). Established mycelia are generally more tolerant to chemicals than basidiospores (Morton and French, 1966; Choi et al., 2002; Woo and Morris, 2010), though exceptions have been noted (Savory and Carey, 1976). Chromated copper arsenate and sodium pentachlorophenate are reported to be effective against
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basidiospores at 10 to 100X lower concentrations than they are against established mycelia of Coriolopsis gallica (Fr.) Ryvarden (reported as Trametes hispida Bagl.), Rhodonia placenta (Fr.) €, K.H. Larss. & Schigel (reported as Poria placenta (Fr.) Cke., Niemela and Gloeophyllum trabeum (Pers.) Murrill (Schmidt and French, 1979). Assessment of the efficacy of copper against basidiospores showed that fungi whose mycelia were copper-tolerant produced basidiospores that were susceptible to copper (Choi et al., 2002; Woo and Morris, 2010). Aliphatic organic acids have also been shown to prevent germination and kill basidiospores (Schmidt, 1985). It was hypothesized that one or more WRC heartwood extractive compounds inhibit germination of basidiospores from wood decay fungi. Only beta-thujaplicin, plicatic acid, and thujic acid were available in sufficient quantities for testing at this stage. Understanding which extractives contribute to decay resistance is necessary for selecting planting stock that will yield heartwood that is durable when used in above-ground applications. 2. Materials and methods 2.1. Inoculum preparation Cultures of Gloeophyllum sepiarium (Wulfen) P. Karst., Fomitopsis palustris (Berk. & M.A. Curtis) Gilb. & Ryvarden, and Dichomitus squalens (P. Karst.) D.A. Reid were obtained from FPInnovations’ culture collection. Sporulating cultures were grown using the methods similar to those described by Choi et al. (2001). Some modifications to these methods have been made to encourage sporulation of G. sepiarium and F. palustris cultures. Two different zones on the growing surface of the plates were created to perturb the growth of fungi across the plate surface. Plates contained an area of malt agar surrounded by water agar, a piece of filter paper placed to one side, and a pine wood plug placed in the water agar, creating nutrient rich and deficient areas. A photograph of the setup for spore production is shown in Fig. 1. Agar plugs were used for inoculation as this allowed sporulation to occur more quickly than with wood inoculum. In addition, for G. sepiarium, pine sapwood sawdust, fertilizer (NPK 20/20/20), and oat bran were added to the nutrient media. Plates were incubated for 2 weeks at 24 C to allow mycelia to grow. The plates were then inverted and placed in a 15 C and 90% relative humidity incubator for six weeks with cycles of 12 h darkness and 12 h artificial light (fluorescent and
UV). To collect spores for inoculations, sporulating cultures were removed from the growth chamber, a fresh sterile lid was put in place, then the plate was sealed with Parafilm® to prevent contamination and placed back in the growth chamber for two days to allow collection of fresh spores. Sterilized distilled water was used to wash the spores collected from the lid into an inoculum suspension. A small portion of solution was retained to confirm the presence of spores under a compound microscope at high magnification. The number of spores present was counted with a haemocytometer and the spore solution was diluted with sterile distilled water to a 250,000 spores mL1 suspension. A 30 mL spore suspension used for individual inoculation contained approximately 8000 spores. Spore suspension viability was confirmed by growth on malt agar plates. 2.2. Test sample preparation White spruce (Picea glauca (Moench) Voss) heartwood was selected as a substrate to evaluate the impact of selected compounds on basidiospore germination because it is has few extractives, no natural durability, and very low concentrations of nutrients to support fungal growth. White spruce and WRC heartwood were cut into 10 mm 10 mm 1 mm samples. White spruce samples were conditioned at 40 C overnight, then diptreated in groups of 25 for 5 min at 20 C with the solutions specified in Table 1. Concentration targets were based on amounts typically found in WRC heartwood (Daniels and Russell, 2007). The target for copper loading was based on concentrations previously reported to control basidiospore germination (Choi et al., 2004; Stirling et al., 2015). The beta-thujaplicin and thujic acid samples were dipped twice to ensure the target uptakes were met. Ethanol was used to prepare beta-thujaplicin and thujic acid treatment solutions. An additional set of samples treated only with ethanol was included as a control in the spore germination tests. Samples were gently agitated with a vortex mixer during dipping to ensure all sample faces were exposed to the treating solution. Western redcedar heartwood samples were Soxhlet-extracted for 6 h with selected solvents as specified in Table 2. To estimate extractives content for the WRC samples, 20 random samples for each treatment were cut up, ethanol-extracted, and analyzed by HPLC (Daniels and Russell, 2007). Total thujaplicins were calculated as the sum of alpha-, beta-, and gamma-thujaplicin. All samples were conditioned at 40 C for 24 h, numbered, and randomly sorted into inoculation groups for analysis. Twelve samples from each treatment group were selected for each basidiospore type. A total of 120 samples were evaluated against basidiospores from each test fungus. The samples were double bagged in sealed polyethylene bags and sterilized by 3.4 Mrad of electron beam irradiation. 2.3. Inoculation and inspection
Fig. 1. Photograph of set up for basidiospore production. Each plate contains an area of malt agar surrounded by water agar, a piece of filter paper placed to one side, and a pine wood plug placed in the water agar.
Twenty-four multiwell plates were prepared under sterile conditions with 2 mL of water agar and a polypropylene mesh separator in each well. One sample was aseptically placed in each well and inoculated with 30 mL of basidiospore suspension. Samples were inoculated in situ to ensure placement of spore solution on the wood surface. This was repeated for each basidiospore culture. After inoculation the samples were moved to an incubator at 24 C for two weeks. Each sample was visually inspected for basidiospore germination after 14 days using a dissecting microscope with 10 to 20 magnification. After 21 days, samples without mycelial growth were removed from the well plates and individually inspected under higher magnifications (40 magnification) to ensure
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Table 1 Extractives applied to white spruce veneer samples. Extractive
Solution concentration (%)
Average solution uptake (mg g1) 105
Average actives uptake (mg g1) 103
None (control) Ethanol (solvent control) Copper (II) sulfate (reference as Cu metal) Plicatic acid Beta-thujaplicin Thujic acid
N/A N/A 0.27 2.86 0.61 2.19
N/A N/A 5.3 (0.3) 4.2 (0.3) 4.1 (0.3) 3.7 (0.4)
N/A N/A 1.4 (0.1) 11.9 (0.8) 2.5 (0.2) 8.1 (0.8)
Standard deviations appear in parentheses (n ¼ 5).
Table 2 Extractives content of western redcedar veneer samples. Extraction solvent
Extractives (mg g1) Plicatic acid
Total thujaplicins
Thujic acid
None (control) Hexanes Hexanes, ethanol Hexanes, Ethanol, water
5100 4514 3389 606
1426 1316 37 24
3515 3333 261 128
negative growth. The presence of any observable hyphae was reported as a positive result. Typically a well-developed mycelium was present in samples positive for germination. 2.4. Statistical analysis Statistical differences between the germination rates observed for each treatment and associated untreated controls were assessed by calculating Z scores for two population proportions and comparing them to critical values (a ¼ 0.05). 3. Results F. palustris, G. sepiarium, and D. squalens basidiospore germination was observed on all untreated spruce (control) samples after two weeks of incubation (Fig. 2). As a result, in this study, any treatment with a germination rate of less than 67% was significantly different from the untreated spruce controls (p < 0.05). 30e50% germination was observed on the copper-treated reference samples
100 90
(Fig. 2), a higher percentage than observed in similar previous tests, but still significantly different from the controls (Choi et al., 2004; Stirling et al., 2015). This was an aggressive test since the large numbers of spores and ideal germination conditions would seldom be encountered in the field. The absence of any growth on the reference untreated WRC samples highlights how effective WRC was in preventing germination for all three fungal species tested. All of the spruce samples treated with plicatic acid had germinating spores (Fig. 2). Thujic acid inhibited spore germination in more than half of the G. sepiarium and D. squalens samples, but only inhibited just over one third of the F. palustris samples. Beta thujaplicin inhibited almost all germination with less than 10% observed for G. sepiarium and F. palustris and none observed for D. squalens, demonstrating this to be a key extractive in basidiospore germination inhibition. Unextracted and hexane-extracted WRC completely inhibited G. sepiarium while F. palustris and D. squalens had less than 10% germination. The absence of a hexanes-extraction effect can be explained by the fact that the extraction removed less than 10% of the extractives (Fig. 3). With successive extractions more WRC extractives were removed and the ability to suppress spore germination deteriorated. G. sepiarium had the biggest increase in germination rate as almost all WRC hexanes/ethanol extracted samples and all of the WRC hexanes/ethanol/water extracted samples germinated. Analysis of these samples indicated that most of the thujaplicins and thujic acid had been removed by extraction. This further highlighted the relationship between these two extractives and basidiospore inhibition. WRC extracted samples inoculated with F. palustris and D. squalens spores exhibited low rates for germination, significantly different from untreated spruce controls. Only very low quantities of measured extractives were present after the full series of extractions. It may be that the remaining extractives are effective even at low concentrations, or that other
GerminaƟon (%)
80 70 60
100
50 40 30
80 G. sepiarium
10
Spruce
Hexanes/Ethanol/Water Extracted
Hexanes/Ethanol Extracted
Hexanes Extracted
Unextracted
Copper Treated
Beta thujaplicin Treated
Thujic acid Treated
PlicaƟc acid Treated
Ethanol Treated
Untreated
0
F. palustris D. squalens
WRC
ExtracƟves Remaining (%)
20
60 Hexanes Hexanes/Ethanol Hexanes/Ethanol/Water
40
20
0 PlicaƟc acid
Fig. 2. Percent basidiospore germination on spruce veneer samples untreated and treated with WRC extractives and WRC veneer samples untreated and extracted with hexanes, ethanol, and water.
Total thujaplicins
Thujic acid
Fig. 3. Percent WRC extractives remaining in WRC veneer samples extracted by hexanes, ethanol, and water compared to initial WRC extractives present.
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compounds resistant to extraction contribute to basidiospore germination resistance. This is difficult to assess as it is not practical to remove all extractives from the wood. Extraction of WRC sequentially with hexanes and methanol has been shown to reduce resistance to mycelial attack by the brown rot fungus, Postia placenta (Taylor et al., 2006). However, these samples retained some residual durability and were not decayed to the same extent as the non-durable controls. 4. Discussion Placing wood veneer samples on mesh atop water agar in multiwell plates created a reproducible environment conducive to basidiospore germination. Earlier basidiospore germination assessment methods used individual tubes to house each sample during incubation (Choi et al., 2001). The use of multiwell plates greatly improved the ease of handling and inspection, facilitating experiments where it is practical to assess larger numbers of samples. Concerns about greater risk of contamination proved unfounded since no contamination was observed. The methods described could be used to evaluate extractives from other naturally durable species as well as other biocides. Thujaplicins are known to deplete rapidly from WRC in service (Johnson and Cserjesi, 1980). Weathered WRC may therefore be much more susceptible to basidiospore germination. If basidiospores are able to germinate and grow on the weathered wood surface, the decay resistance of weathered wood above ground may therefore depend more on resistance to mycelial attack. However, the continued presence of high concentrations of beta-thujaplicin in surface checks could explain the slow initiation of decay in WRC decks (Morris et al., 2011) in the same way copper loadings on check surfaces prevent decay of shell-treated wood (Choi et al., 2002; Woo and Morris, 2010). It also suggests that treatments for the weathered wood surface which inhibit basidiospore germination could potentially help enhance overall decay resistance. The ability of beta-thujaplicin to inhibit basidiospore germination confirms its overall importance in protecting WRC. Planting stock that generates heartwood with high concentrations of thujaplicins and thujic acid should be selected to yield wood that will be resistant to basidiospore germination. The mechanism through which these compounds inhibit basidiospore germination are not known. Further work is needed to identify these mechanisms. 5. Conclusions Beta-thujaplicin, and to a lesser extent thujic acid, were associated with resistance to germination of G. sepiarium, F. palustris, and D. squalens basidiospores. Plicatic acid had no inhibitory effects on basidiospore germination. Acknowledgements This project was financially supported by Natural Resources Canada (Canadian Forest Service) and the Province of British Columbia. References Bjurman, J., 1984. Conditions for Basidiospore Production in the Brown Rot Fungus Gloeophyllum sepiarium in Axenic Culture. Int Res Group Wood Preservation, Stockholm, Sweden, p. 11. Document No. IRG/WP/1232. Cartwright, K.S.G., 1941. The variability in resistance to decay of the heartwood of home-grown western red cedar (Thuja plicata D. Don) and its relation to position in the log. Forestry 15 (1), 65e75. Choi, S.M., Ruddick, J.N.R., Morris, P.I., 2001. The Effect of Storage and Subculturing on in vitro Fruit Body Formation and Spore Production in Gloeophyllum
sepiarium and Oligoporus placentus. Int Res Group Wood Preservation, Stockholm, Sweden, p. 11. Document No. IRG/WP/01e20232. Choi, S.M., Ruddick, J.N.R., Morris, P.I., 2002. The Copper Tolerance of Mycelium vs Spores for Two Brown Rot Fungi. Int. Res. Group Wood Preservation, Stockholm, Sweden, p. 4. Document No. IRG/WP/02e10422. Choi, S., Ruddick, J.N., Morris, P., 2004. Chemical redistribution in CCA-treated decking. For. Prod. J. 54 (3), 33. Croan, S., 1994. Basidiosporogenesis by White-rot Basidiomycetes in vitro. Int Res Group Wood Preservation, Stockholm, Sweden, p. 14. Document No. IRG/WP/ 94e10081. Croan, S., 1995. Basidiosporogenesis by Brown-rot Basidiomycetes in vitro. Int Res Group Wood Preservation, Stockholm, Sweden, p. 9. Document No. IRG/WP/ 95e10126. Daniels, C.R., Russell, J.H., 2007. Analysis of western redcedar (Thuja plicata Donn) heartwood components by HPLC as a possible screening tool for trees with enhanced natural durability. J. Chromatogr. Sci. 45 (5), 281e285. DeBell, J.D., Morrell, J.J., Gartner, B.L., 1997. Tropolone content of increment cores as an indicator of decay resistance in western redcedar. Wood Fiber Sci. 29 (4), 364e369. Erdtman, H., Gripenberg, J., 1949. Structure of thujic acid (dehydroperillic acid). Nature 164 (4164), 316, 316. Fougerousse, M.E., 1984. Some considerations of fungal testing of treated wood with particular interest to basidiomycetes fungi. In: Proceedings American Wood Preservation Association, vol. 80, pp. 59e66. Freitag, C.M., Morrell, J.J., 2001. Durability of a changing western redcedar resource. Wood Fiber Sci. 33 (1), 69e75. Hegarty, B., Buchwald, G., 1988. The Influence of Timber Species and Preservative Treatment on Spore Germination of Some Wood-destroying Basidiomycetes. Int Res Group Wood Preservation, Stockholm, Sweden, p. 13. Document No. IRG/ WP/2300. Johnson, E.L., Cserjesi, A.J., 1980. Weathering effect on thujaplicin concentration in western redcedar shakes. For. Prod. J. 30 (6), 52e53. Kirker, G.T., Blodgett, A.B., Arango, R.A., Lebow, P.K., Clausen, C.A., 2013. The role of extractives in naturally durable wood species. Int. Biodeterior. Biodegrad. 82, 53e58. Li, S., Freitag, C.M., Morrell, J.J., Okabe, T., 2012. Antifungal effects of hinokitiol and its sodium salt for wood protection. BioResources 7 (4), 5312e5318. Lim, Y.W., Chedgy, R.J., Amirthalingham, S., Breuil, C., 2007. Screening fungi tolerant to western red cedar (Thuja plicata Donn) extractives. Part 2. Development of a feeder strip assay. Holzforschung 61, 195e200. Morita, Y., Matsumura, E., Okabe, T., Fukui, T., Shibata, M., Sugiura, M., Ohe, T., Tsujibo, H., Ishida, N., Inamori, Y., 2004. Biological activity of alpha-thujaplicin, the isomer of hinokitiol. Biol. Pharm. Bull. 27 (6), 899e902. Morris, P.I., Ingram, J.K., 2013. Twenty-year performance of decking with two levels of preservative penetration. For. Prod. J. 62 (7e8), 566e570. Morris, P.I., Ingram, J., Larkin, G., Laks, P., 2011. Field tests of naturally durable species. For. Prod. J. 61 (5), 344e351. Morris, P.I., Stirling, R., 2012. Western red cedar extractives associated with durability in ground contact. Wood Sci. Technol. 46 (5), 991e1002. Morton, H.L., French, D.W., 1966. Factors affecting germination of spores of woodrotting fungi on wood. For. Prod. J. 16 (3), 25e30. Nault, J., 1988. Radial distribution of thujaplicins in old growth and second growth western red cedar (Thuja plicata Donn). Wood Sci. Technol. 22 (1), 73e80. Nelson, E.E., 1976. Colonization of wood disks initiated by basidiospores of Phellinus weirii (laminated root rot). For. Sci. 22 (4), 407e411. Rennerfelt, E., 1948. Thujaplicin, a fungicidal substance in the heartwood of Thuja plicata. Physiol. Plant 1, 245e254. Roff, J.W., Atkinson, J.M., 1954. Toxicity tests of a water-soluble phenolic fraction (thujaplicin-free) of western red cedar. Can. J. Bot. 32, 308e309. Rudman, P., 1962. The causes of natural durability in timber. IX. The antifungal activity of heartwood Extractives in a wood substrate, extractives in a wood substrate. Holzforschung 16 (3), 74e77. Rudman, P., 1963. The causes of natural durability in timber. XI. Some tests on the fungi toxicity of wood extractives and related compounds. Holzforschung 17 (2), 54e57. Savory, J.G., Carey, J.K., 1976. Laboratory assessment of the toxic efficacy of joinery preservative treatments. In: Proceedings of the British Wood Preserving Association, pp. 3e24. Scheffer, T.C., 1957. Decay resistance of western redcedar. J. For. 55 (6), 434e442. Schmidt, E.L., 1985. Aliphatic acids as spore germination inhibitors of wood-decay fungi in vitro. Can. J. Bot. 63 (2), 337e339. Schmidt, E.L., French, D.W., 1979. CCA and sodium pentachlorophenate inhibition of basidiospore germination of decay fungi: contact agar method. For. Prod. J. 29 (5), 53e54. Stirling, R., Clark, J.E., Daniels, C.R., Morris, P.I., 2007. Methods for Determining the Role of Extractives in the Natural Durability of Western Red Cedar Heartwood. Int Res Group Wood Protection, Stockholm, Sweden, p. 12. Document No. IRG/ WP/07e20356. Stirling, R., Morris, P.I., 2016. Potential contributions of lignans to decay resistance in western red cedar. Wood Sci. Technol. 50 (2), 399e412. Stirling, R., Ruddick, J.N., Xue, W., Morris, P.I., Kennepohl, P., 2015. Characterization of copper in leachates from ACQ- and MCQ-treated wood and its effect on basidiospore germination. Wood Fiber Sci. 47 (3), 1e8. Taylor, A.M., Gartner, B.L., Morrell, J.J., Tsunoda, K., 2006. Effects of heartwood
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Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Short communication
Inhibition of basidiospore germination by western redcedar heartwood extractives Rod Stirling*, Stacey Kus, Adnan Uzunovic FPInnovations, 2665 East Mall, Vancouver, BC, V6T 1Z4, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2016 Received in revised form 25 May 2016 Accepted 15 June 2016 Available online 27 June 2016
Understanding the relationship between extractives and decay resistance in western red cedar (WRC) is essential for breeding durable planting stock. To date, most such work has focused on resistance to mycelial attack. However, this is potentially misleading since WRC is largely used in above-ground applications where basidiospore germination is the primary way in which the wood is initially colonized. Efficacy against basidiospores is likely a critical factor affecting wood’s decay resistance in above-ground applications. In this study the effect of selected extractives on basidiospore germination was evaluated. Partially-extracted WRC veneer samples, and spruce samples treated with beta-thujaplicin, thujic acid, or plicatic acid were used. Beta-thujaplicin and thujic acid were associated with significantly reduced rates of Gloeophyllum sepiarium, Fomitopsis palustris, and Dichomitus squalens basidiospore germination. Plicatic acid was not associated with any effect on basidiospore germination. Planting stock that generates heartwood with high concentrations of thujaplicins and thujic acid should be selected to yield wood that will be resistant to basidiospore germination. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Basidiospores Extractives Germination Thuja plicata Western redcedar
1. Introduction Untreated western redcedar (Thuja plicata Donn) is used largely in above-ground, exterior applications, such as decking, fencing, and siding. These applications rely on the natural decay resistance of western redcedar (WRC) to meet service life expectations (Cartwright, 1941; Scheffer, 1957; Freitag and Morrell, 2001; Morris et al., 2011; Morris and Ingram, 2013). To date, most research investigating the relationship between extractives and decay resistance has focused on resistance to mycelial attack (Nault, 1988; DeBell et al., 1997; Taylor et al., 2006; Morris and Stirling, 2012; Kirker et al., 2013). This is potentially misleading since basidiospore germination is the primary way in which wood is initially attacked above ground (Savory and Carey, 1976; Schmidt and French, 1979; Bjurman, 1984; Fougerousse, 1984; Hegarty and Buchwald, 1988; Croan, 1994, 1995). Efficacy against basidiospores is likely a critical factor affecting the species’ decay resistance. WRC heartwood has been shown to inhibit germination and kill Gloeophyllum trabeum (Pers.) Murrill (reported as Lenzites trabea (Pers.) Fr.) basidiospores (Morton and French, 1966). It was also reported to
* Corresponding author. E-mail addresses: [email protected] (R. Stirling), stacey.kus@ fpinnovations.ca (S. Kus), [email protected] (A. Uzunovic). http://dx.doi.org/10.1016/j.ibiod.2016.06.008 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
be more resistant to colonization by basidiospores of Phellinus weirii (Murrill) Gilb. than other species (Nelson, 1976). However, the extractives responsible for this inhibitory and sporicidal activity had not been investigated. Twenty-one heartwood extractive compounds have been isolated from WRC heartwood and identified. The most abundant extractives are the thujaplicins, plicatic acid, and thujic acid (Daniels and Russell, 2007). The thujaplicins are well-known fungicides with demonstrated activity against mycelial attack (Rennerfelt, 1948; Rudman, 1962, 1963; Morita et al., 2004; Yen et al., 2008; Li et al., 2012). Plicatic acid is reported to have fungistatic activity (Roff and Atkinson, 1954), but has not shown fungicidal efficacy against mycelia at concentrations that would be present in wood (Lim et al., 2007; Stirling et al., 2007; Stirling and Morris, 2016). Thujic acid is generally believed to have low activity against decay fungi (Erdtman and Gripenberg, 1949), but has been reported to inhibit the growth of Rhinocladiella atrovirens Nannf. and Phellinus ferreus (Pers.) Bourdot & Galzin mycelia growing on artificial media (Lim et al., 2007). Established mycelia are generally more tolerant to chemicals than basidiospores (Morton and French, 1966; Choi et al., 2002; Woo and Morris, 2010), though exceptions have been noted (Savory and Carey, 1976). Chromated copper arsenate and sodium pentachlorophenate are reported to be effective against
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basidiospores at 10 to 100X lower concentrations than they are against established mycelia of Coriolopsis gallica (Fr.) Ryvarden (reported as Trametes hispida Bagl.), Rhodonia placenta (Fr.) €, K.H. Larss. & Schigel (reported as Poria placenta (Fr.) Cke., Niemela and Gloeophyllum trabeum (Pers.) Murrill (Schmidt and French, 1979). Assessment of the efficacy of copper against basidiospores showed that fungi whose mycelia were copper-tolerant produced basidiospores that were susceptible to copper (Choi et al., 2002; Woo and Morris, 2010). Aliphatic organic acids have also been shown to prevent germination and kill basidiospores (Schmidt, 1985). It was hypothesized that one or more WRC heartwood extractive compounds inhibit germination of basidiospores from wood decay fungi. Only beta-thujaplicin, plicatic acid, and thujic acid were available in sufficient quantities for testing at this stage. Understanding which extractives contribute to decay resistance is necessary for selecting planting stock that will yield heartwood that is durable when used in above-ground applications. 2. Materials and methods 2.1. Inoculum preparation Cultures of Gloeophyllum sepiarium (Wulfen) P. Karst., Fomitopsis palustris (Berk. & M.A. Curtis) Gilb. & Ryvarden, and Dichomitus squalens (P. Karst.) D.A. Reid were obtained from FPInnovations’ culture collection. Sporulating cultures were grown using the methods similar to those described by Choi et al. (2001). Some modifications to these methods have been made to encourage sporulation of G. sepiarium and F. palustris cultures. Two different zones on the growing surface of the plates were created to perturb the growth of fungi across the plate surface. Plates contained an area of malt agar surrounded by water agar, a piece of filter paper placed to one side, and a pine wood plug placed in the water agar, creating nutrient rich and deficient areas. A photograph of the setup for spore production is shown in Fig. 1. Agar plugs were used for inoculation as this allowed sporulation to occur more quickly than with wood inoculum. In addition, for G. sepiarium, pine sapwood sawdust, fertilizer (NPK 20/20/20), and oat bran were added to the nutrient media. Plates were incubated for 2 weeks at 24 C to allow mycelia to grow. The plates were then inverted and placed in a 15 C and 90% relative humidity incubator for six weeks with cycles of 12 h darkness and 12 h artificial light (fluorescent and
UV). To collect spores for inoculations, sporulating cultures were removed from the growth chamber, a fresh sterile lid was put in place, then the plate was sealed with Parafilm® to prevent contamination and placed back in the growth chamber for two days to allow collection of fresh spores. Sterilized distilled water was used to wash the spores collected from the lid into an inoculum suspension. A small portion of solution was retained to confirm the presence of spores under a compound microscope at high magnification. The number of spores present was counted with a haemocytometer and the spore solution was diluted with sterile distilled water to a 250,000 spores mL1 suspension. A 30 mL spore suspension used for individual inoculation contained approximately 8000 spores. Spore suspension viability was confirmed by growth on malt agar plates. 2.2. Test sample preparation White spruce (Picea glauca (Moench) Voss) heartwood was selected as a substrate to evaluate the impact of selected compounds on basidiospore germination because it is has few extractives, no natural durability, and very low concentrations of nutrients to support fungal growth. White spruce and WRC heartwood were cut into 10 mm 10 mm 1 mm samples. White spruce samples were conditioned at 40 C overnight, then diptreated in groups of 25 for 5 min at 20 C with the solutions specified in Table 1. Concentration targets were based on amounts typically found in WRC heartwood (Daniels and Russell, 2007). The target for copper loading was based on concentrations previously reported to control basidiospore germination (Choi et al., 2004; Stirling et al., 2015). The beta-thujaplicin and thujic acid samples were dipped twice to ensure the target uptakes were met. Ethanol was used to prepare beta-thujaplicin and thujic acid treatment solutions. An additional set of samples treated only with ethanol was included as a control in the spore germination tests. Samples were gently agitated with a vortex mixer during dipping to ensure all sample faces were exposed to the treating solution. Western redcedar heartwood samples were Soxhlet-extracted for 6 h with selected solvents as specified in Table 2. To estimate extractives content for the WRC samples, 20 random samples for each treatment were cut up, ethanol-extracted, and analyzed by HPLC (Daniels and Russell, 2007). Total thujaplicins were calculated as the sum of alpha-, beta-, and gamma-thujaplicin. All samples were conditioned at 40 C for 24 h, numbered, and randomly sorted into inoculation groups for analysis. Twelve samples from each treatment group were selected for each basidiospore type. A total of 120 samples were evaluated against basidiospores from each test fungus. The samples were double bagged in sealed polyethylene bags and sterilized by 3.4 Mrad of electron beam irradiation. 2.3. Inoculation and inspection
Fig. 1. Photograph of set up for basidiospore production. Each plate contains an area of malt agar surrounded by water agar, a piece of filter paper placed to one side, and a pine wood plug placed in the water agar.
Twenty-four multiwell plates were prepared under sterile conditions with 2 mL of water agar and a polypropylene mesh separator in each well. One sample was aseptically placed in each well and inoculated with 30 mL of basidiospore suspension. Samples were inoculated in situ to ensure placement of spore solution on the wood surface. This was repeated for each basidiospore culture. After inoculation the samples were moved to an incubator at 24 C for two weeks. Each sample was visually inspected for basidiospore germination after 14 days using a dissecting microscope with 10 to 20 magnification. After 21 days, samples without mycelial growth were removed from the well plates and individually inspected under higher magnifications (40 magnification) to ensure
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Table 1 Extractives applied to white spruce veneer samples. Extractive
Solution concentration (%)
Average solution uptake (mg g1) 105
Average actives uptake (mg g1) 103
None (control) Ethanol (solvent control) Copper (II) sulfate (reference as Cu metal) Plicatic acid Beta-thujaplicin Thujic acid
N/A N/A 0.27 2.86 0.61 2.19
N/A N/A 5.3 (0.3) 4.2 (0.3) 4.1 (0.3) 3.7 (0.4)
N/A N/A 1.4 (0.1) 11.9 (0.8) 2.5 (0.2) 8.1 (0.8)
Standard deviations appear in parentheses (n ¼ 5).
Table 2 Extractives content of western redcedar veneer samples. Extraction solvent
Extractives (mg g1) Plicatic acid
Total thujaplicins
Thujic acid
None (control) Hexanes Hexanes, ethanol Hexanes, Ethanol, water
5100 4514 3389 606
1426 1316 37 24
3515 3333 261 128
negative growth. The presence of any observable hyphae was reported as a positive result. Typically a well-developed mycelium was present in samples positive for germination. 2.4. Statistical analysis Statistical differences between the germination rates observed for each treatment and associated untreated controls were assessed by calculating Z scores for two population proportions and comparing them to critical values (a ¼ 0.05). 3. Results F. palustris, G. sepiarium, and D. squalens basidiospore germination was observed on all untreated spruce (control) samples after two weeks of incubation (Fig. 2). As a result, in this study, any treatment with a germination rate of less than 67% was significantly different from the untreated spruce controls (p < 0.05). 30e50% germination was observed on the copper-treated reference samples
100 90
(Fig. 2), a higher percentage than observed in similar previous tests, but still significantly different from the controls (Choi et al., 2004; Stirling et al., 2015). This was an aggressive test since the large numbers of spores and ideal germination conditions would seldom be encountered in the field. The absence of any growth on the reference untreated WRC samples highlights how effective WRC was in preventing germination for all three fungal species tested. All of the spruce samples treated with plicatic acid had germinating spores (Fig. 2). Thujic acid inhibited spore germination in more than half of the G. sepiarium and D. squalens samples, but only inhibited just over one third of the F. palustris samples. Beta thujaplicin inhibited almost all germination with less than 10% observed for G. sepiarium and F. palustris and none observed for D. squalens, demonstrating this to be a key extractive in basidiospore germination inhibition. Unextracted and hexane-extracted WRC completely inhibited G. sepiarium while F. palustris and D. squalens had less than 10% germination. The absence of a hexanes-extraction effect can be explained by the fact that the extraction removed less than 10% of the extractives (Fig. 3). With successive extractions more WRC extractives were removed and the ability to suppress spore germination deteriorated. G. sepiarium had the biggest increase in germination rate as almost all WRC hexanes/ethanol extracted samples and all of the WRC hexanes/ethanol/water extracted samples germinated. Analysis of these samples indicated that most of the thujaplicins and thujic acid had been removed by extraction. This further highlighted the relationship between these two extractives and basidiospore inhibition. WRC extracted samples inoculated with F. palustris and D. squalens spores exhibited low rates for germination, significantly different from untreated spruce controls. Only very low quantities of measured extractives were present after the full series of extractions. It may be that the remaining extractives are effective even at low concentrations, or that other
GerminaƟon (%)
80 70 60
100
50 40 30
80 G. sepiarium
10
Spruce
Hexanes/Ethanol/Water Extracted
Hexanes/Ethanol Extracted
Hexanes Extracted
Unextracted
Copper Treated
Beta thujaplicin Treated
Thujic acid Treated
PlicaƟc acid Treated
Ethanol Treated
Untreated
0
F. palustris D. squalens
WRC
ExtracƟves Remaining (%)
20
60 Hexanes Hexanes/Ethanol Hexanes/Ethanol/Water
40
20
0 PlicaƟc acid
Fig. 2. Percent basidiospore germination on spruce veneer samples untreated and treated with WRC extractives and WRC veneer samples untreated and extracted with hexanes, ethanol, and water.
Total thujaplicins
Thujic acid
Fig. 3. Percent WRC extractives remaining in WRC veneer samples extracted by hexanes, ethanol, and water compared to initial WRC extractives present.
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compounds resistant to extraction contribute to basidiospore germination resistance. This is difficult to assess as it is not practical to remove all extractives from the wood. Extraction of WRC sequentially with hexanes and methanol has been shown to reduce resistance to mycelial attack by the brown rot fungus, Postia placenta (Taylor et al., 2006). However, these samples retained some residual durability and were not decayed to the same extent as the non-durable controls. 4. Discussion Placing wood veneer samples on mesh atop water agar in multiwell plates created a reproducible environment conducive to basidiospore germination. Earlier basidiospore germination assessment methods used individual tubes to house each sample during incubation (Choi et al., 2001). The use of multiwell plates greatly improved the ease of handling and inspection, facilitating experiments where it is practical to assess larger numbers of samples. Concerns about greater risk of contamination proved unfounded since no contamination was observed. The methods described could be used to evaluate extractives from other naturally durable species as well as other biocides. Thujaplicins are known to deplete rapidly from WRC in service (Johnson and Cserjesi, 1980). Weathered WRC may therefore be much more susceptible to basidiospore germination. If basidiospores are able to germinate and grow on the weathered wood surface, the decay resistance of weathered wood above ground may therefore depend more on resistance to mycelial attack. However, the continued presence of high concentrations of beta-thujaplicin in surface checks could explain the slow initiation of decay in WRC decks (Morris et al., 2011) in the same way copper loadings on check surfaces prevent decay of shell-treated wood (Choi et al., 2002; Woo and Morris, 2010). It also suggests that treatments for the weathered wood surface which inhibit basidiospore germination could potentially help enhance overall decay resistance. The ability of beta-thujaplicin to inhibit basidiospore germination confirms its overall importance in protecting WRC. Planting stock that generates heartwood with high concentrations of thujaplicins and thujic acid should be selected to yield wood that will be resistant to basidiospore germination. The mechanism through which these compounds inhibit basidiospore germination are not known. Further work is needed to identify these mechanisms. 5. Conclusions Beta-thujaplicin, and to a lesser extent thujic acid, were associated with resistance to germination of G. sepiarium, F. palustris, and D. squalens basidiospores. Plicatic acid had no inhibitory effects on basidiospore germination. Acknowledgements This project was financially supported by Natural Resources Canada (Canadian Forest Service) and the Province of British Columbia. References Bjurman, J., 1984. Conditions for Basidiospore Production in the Brown Rot Fungus Gloeophyllum sepiarium in Axenic Culture. Int Res Group Wood Preservation, Stockholm, Sweden, p. 11. Document No. IRG/WP/1232. Cartwright, K.S.G., 1941. The variability in resistance to decay of the heartwood of home-grown western red cedar (Thuja plicata D. Don) and its relation to position in the log. Forestry 15 (1), 65e75. Choi, S.M., Ruddick, J.N.R., Morris, P.I., 2001. The Effect of Storage and Subculturing on in vitro Fruit Body Formation and Spore Production in Gloeophyllum
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