Lignin - ACS Publications - American Chemical Society

Chapter 8

Lignin Influence on Angiosperm Sapwood Susceptibility to White-Rot Fungal Colonization: A Hypothesis Tor P. Schultz and Darrel D. Nicholas

Downloaded by CORNELL UNIV on June 9, 2012 | http://pubs.acs.org Publication Date: November 30, 1999 | doi: 10.1021/bk-2000-0742.ch008

Forest and Wildlife Research Center and Forest Products Laboratory, Box 9820, Mississippi State University, Mississippi State, MS 39762-9820

It has long been recognized that angiosperm sapwood in nature is usually degraded by white-rot fungi. This susceptibility to white-rot fungi is generally believed to be due to the structure and concentration of angiosperm lignin. However, an explicit explanation as to why lignin structure and/or concentration makes a particular wood vulnerable to white-rot fungal colonization and subsequent degradation has apparently never been given. We propose that phenolic groups in wood, such as those present in the lignin or heartwood extractives, can act as free radical scavengers (antioxidants) to inhibit the various white-rot free radical degradative mechanisms. Consequently, the presence of a relatively high phenolic "density", such as that in gymnosperm sapwood or angiosperm heartwood, may retard white-rot colonization. Conversely, white-rot fungi may rapidly colonize wood with a relatively low free phenolic content, such as angiosperm sapwood. Once a wood is colonized, subsequent antagonistic factors known to exist among fungi may give the advantage to the principal fungus present rather than fungi with fast decay rates (based on rates measured using monocultures in the laboratory). The complex structure of angiosperm wood where different cell types have different amounts and types of lignin - and consequently different phenolic "densities" - influences the susceptibility of angiosperm wood to initial white-rot colonization and, perhaps, also the subsequent decay rate. In addition to the free phenolic "density" and amount and bioactivity of any extractives present other factors undoubtedly also affect the decay resistance of a particular wood.

© 2000 American Chemical Society In Lignin: Historical, Biological, and Materials Perspectives; Glasser, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

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Introduction In this paper we propose that sapwood susceptibility to white-rot fungal colonization and subsequent degradation is related to phenolic "density" [with the decay resistance of heartwood related to both this factor and the bioactivity of the extractives]. Consequently, wood with a low phenolic "density", such as angiosperm sapwood, is more vulnerable to white-rot colonization and subsequent degradation than wood which has a high phenolic "density" such as gymnosperm sapwood or angiosperm heartwood. This hypothesis is developed in part from our earlier work on the natural durability of angiosperm heartwood where we suggested that angiosperm heartwood extractives play a dual role in natural durability: extractives have some limited fungicidal activity and are also excellent free radical scavengers and thus inhibit the various fungal free radical degradation pathways (i). This article is a discussion of our tentative hypothesis and consequently does not have experimental results followed by discussion and conclusions. Instead, an effort is made to review prior work in this area and discuss how some of these results support our hypothesis. Discussion Large volumes of wood/lumber are treated with various biocides [wood preservatives] to form a product resistant to fungal and insect degradation. The major wood preservatives used today in the US are C C A [chromated copper arsenate], pentachlorophenol and creosote. Recent environmental concerns have resulted in these biocides being scrutinized by various governmental agencies, and use of pentachlorophenol and/or C C A is restricted or banned in many countries. Consequently, many organizations are trying to develop alternative, environmentally-benign wood preservatives. The heartwood of some trees has considerable resistance against fungi. As part of our effort to develop environmentally-benign wood preservatives, we have studied the role which extractives, particularly stilbenols, play in the natural durability of angiosperm heartwood ( i , 2). Particularly intriguing to us was recent work (3) which reported that white-rot fungi are believed to initially disrupt xylem cell walls using free radicals to increase the pore size (4) so that the relatively large extracellular fungal enzymes can then penetrate the cell wall. Backa'setal. report, combined with the common knowledge that extractives are excellent antioxidants and dispersed throughout the cell wall, suggested that extractives may prevent the cell wall from being perturbed or "opened up" by free radicals during the initial attack by white-rot fungi. The above reasoning and related experimental work (5) led us to propose (i) that extractives protect angiosperm heartwood against whiterot fungal degradation by: A) extractives having some fungal activity; and B)

In Lignin: Historical, Biological, and Materials Perspectives; Glasser, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

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angiosperm phenolic extractives being effective antioxidants and protecting wood from fungal free radical oxidative-type degradations. It is well known that angiosperm sapwood in nature is highly susceptible to whiterot fungal degradation. While the exact reason for this vulnerability is not known, research has indicated that the different types of lignin in angiosperm and gymnosperm sapwoods, and possibly also the amount of lignin, are significant factors (6, 7). However, the exact reason why lignin type influences the susceptibility of a particular wood to white-rot colonization and subsequent degradation has not been proposed. In reviewing these reports we wondered if the presence of phenolic groups, which should be antioxidants, may influence the susceptibility of a particular sapwood to white-rot degradation. This susceptibility would be related to our earlier hypothesis (i) that the natural durability of angiosperm heartwood may be influenced by fungicidal extractives which are also antioxidants. Since almost all free phenolic groups present in normal sapwood are due to the lignin component, we decided to see if data in prior studies indicate that a low phenolic "density" in sapwood is related to the ease of degradation by whiterot fungi. Highley (7) found that the rate of degradation of sweetgum (Liquidambar styraciflua L.) sapwood by a white-rot fungus [Coriolus versicolor (L.) Quel.] was significantly faster than for southern pine (Pinus spp.). The lignin in normal gymnosperm xylem is composed almost entirely of guaiacyl (G) units while temperate angiosperm lignin is normally composed of both guaiacyl and syringyl (S) units. This difference influences the lignin structure, including the free phenolic content. Lignin in normal gymnosperm xylem is reported to have 15-30 phenolic groups per 100 lignin units as compared to 10-15 phenolic groups in angiosperm lignin (8). In addition, angiosperm xylem typically has a slightly lower lignin content than gymnosperms. These two factors, a lower lignin content and lower phenolic content per unit of lignin present, suggest that phenolic "density" may be lower for angiosperm than gymnosperm sapwood. A rough estimate of the phenolic density can be calculated for sweetgum and southern yellow pine sapwood given a lignin content of 20.1 and 28.0%, respectively (7), and averaging the free phenolic content from the above to give an estimated 13 and 23% free phenolic groups per 100 lignin units. Based on this, pine will have about 35 mMoles and sweetgum about 13 mMoles of phenolic groups per 100 grams of sapwood. The significantly lower level - less than half — of phenolic groups in sweetgum as compared to pine sapwood supports our tentative hypothesis that the free phenolic "density" may be correlated to resistance to white-rot fungal degradation. While angiosperm sapwood has a low phenolic "density", the heartwood of many angiosperm species has extractives which are mostly phenolic in nature. These extractives would be expected to increase the free phenolic content of the wood and


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impart enhanced resistance to white-rot fungi. For example, the highly-durable heartwood of osage orange [Madura pomifera (Raf.) Schneid.] (2) was found to have 9.9 wt.% extractives and all the extractives identified had three or more phenolic groups. From the compounds identified and the total amount present we can estimate that osage orange heartwood has about 145 mMoles of phenolic groups per 100 grams of wood due only to the extractives, or approximately an order of magnitude greater than that due to the lignin. Thus, the amount of extractives present in a particular wood has been suggested to be a good indicator of durability (2). A high level of compounds which have some fungicidal activity in addition to being antioxidants will further protect heartwood against decay fungi. Our hypothesis is dependent on the proposition that the free phenolic groups of lignin can act as free radical scavengers or antioxidants. Phenolic plant extractives are well known to be excellent free radical scavengers (9). The stilbenol resveratrol which is found in red wine and many woods is one classic example; the extensive studies on utilizing quercetin obtained from Douglas-fir bark [Pseudotsuga menziesii (Mirb.) Franco] as a commercial antioxidant is another. While the antioxidant properties of lignins have been studied less, several references have reported that lignins are antioxidants (10,11,12). When we use the term "density", we do not suggest that dense wood is more durable. Rather, we imply that the number of phenolic groups per unit mass of cell wall substance (or sample mass, since all wood cell wall substance is reported to have the same specific gravity) is related to a wood's resistance to white-rot colonization. It has been suggested that decay susceptibility is inversely related to specific gravity (13). However, the differences reported were relatively minor (decay ranged from about 16 to 19.5 wt. %) and may be related to the amount of summerwood, as the authors suggested. [An external reviewer pointed out that the researchers plotted decay susceptibility (% oven dry weight loss) versus specific gravity. He noted that the negative slope may be due to the fact that high density wood has a higher initial weight. Plotting the mass loss per unit volume versus the specific gravity gave a plot with a slight positive slope which suggested to the reviewer that the higher density samples did not have greater durability.] Furthermore, decay rates, not susceptibility, were measured. Many researchers have used decay rates as an indication of decay resistance. We are unsure, however, if small differences in decay rates indicates a difference in susceptibility to fungal attack, especially considering the inherent error in laboratory decay tests. If density is related to white-rot decay resistance, then the sapwood of a very dense angiosperm such as osage orange should be more resistant than aspen sapwood (specific gravity of 0.76 and 0.35, respectively). This is not the case - normal sapwood of all angiosperms has very little decay resistance.


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In further support of our hypothesis, Srebotnik et al. (14) found that a white-rot fungus degraded a synthetic lignin much faster when the phenolic groups were methylated as compared to the same lignin which had not been methylated. In addition, the decay rates of three angiosperm sapwoods (7) by C. versicolor (sweetgum > white oak [Quercus alba L.] > ceibo [Erythrina crista-galli L.]) appear to be related to their approximate free phenolic "density" based on the lignin content and the atypical lignin structure of ceibo wood [mainly G] as compared to the other two angiosperm woods. We are still unsure, however, of the relationship between decay rate versus susceptibility to colonization for untreated sapwood samples. Colonization is, of course, necessary before degradation can occur. Thus, it could be possible that white-rot fungi can colonize angiosperm sapwood faster than brown-rot fungi. Once colonization has occurred, antagonism (15,16) between white- and brown-rot fungi may give the upper hand to the fungus which colonized the wood first rather than the fungus with the fastest decay rate (where decay rate is the mass loss per unit time for a fungal culture on a particular wood sample under laboratory conditions with no other fungus present). Examining only the macro phenolic content of a particular wood is misleading, of course. The complex structure of angiosperms as compared to gymnosperms has also been suggested to influence decay susceptibility to white-rot fungi (6, 17). Obst et al. (6), in an excellent study, found that the decay susceptibility of different cell types in the sapwood of seven angiosperms generally followed the pattern of fibers > vessels > ray parenchyma. As discussed by Obst et al., angiosperm fiber cell walls are believed to be composed of mainly S lignin, vessel cell walls of G lignin, and ray cells of S/G lignin. The lignin concentration in birch (Betula spp.) for the secondary cell wall of these three cell types has been reported to be 19,27 and 27% lignin for fibers, vessels, and ray cells, respectively (8). It would be expected that the phenolic content of lignin would be in the order of G > G/S > S. Thus, the finding of Obst et al. that fibers were much more susceptible to white-rot fungi than vessels would agree with our hypothesis, since vessels should have a higher phenolic content than fibers based on a higher lignin concentration (27 vs. 19%) and the lignin type (G vs. S), respectively. However, the high decay resistance of ray parenchyma cells, which have a high lignin content (27%) but with the lignin probably made up of G/S units, is surprising since we would predict that ray parenchyma would have a decay resistance between that of fibers and vessels. One possibility the authors suggested is that some extractives may have been present in these sapwood samples. Extractives would influence the results since the food reserves held in the sapwood parenchyma cells can often be quickly converted - within hours for some angiosperms - into antimicrobial metabolites (phytoalexins) such as phenolic extractives once a tree is cut (18, 20). The


210 parenchyma cells will remain viable for an extended period until the log is dried, steamed or fumigated. The heterogenous distribution of the lignin units in the cell wall (21) may also affect colonization by white- and brown-rot fungi. Specifically, white-rot fungi gradually erode cell walls from the lumen inwards [the initial attack occurs at the S layer] while the entire cell wall is uniformly degraded by brown-rot fungi. It has been reported (21) that the S and S cell layers in angiosperms contain a high proportion of uncondensed S-type lignin units [i.e. high levels of ether linkages and consequently few phenolic groups] as compared to the lignin in compound middle lamella which contains larger amounts of condensed G and p-coumaryl alcohol elements. Thus, the layers of the cell wall which is first degraded by white-rot fungi may be the easiest layers for white-rot fungi to degrade, and consequently these fungi may have a relatively easy start colonizing a cell. Conversely, brownrot fungi degrade the entire cell wall and thus experience both condensed and uncondensed lignin. This heterogenous lignin distribution may result in white-rot fungi being able to colonize angiosperm wood faster than brown-rot fungi. 3


3

2

We have concentrated on white-rot fungi, but brown-rot fungi are also reported to initially degrade wood by generating free radicals (79, 22). This might suggest that angiosperm sapwood in nature should be attacked and degraded by both brown- and white-rot fungi. We believe that the susceptibility of angiosperm sapwood in nature to white-rot fungi is simply due to white-rot fungi colonizing some or all of angiosperm sapwood faster than brown-rot fungi. Once a wood is colonized, subsequent antagonistic factors may give the advantage to the fungus present at the high level rather than fungi with fast decay rates (based on decay rates measured using monocultures in the laboratory). We believe this rapid colonization may be due to the low phenolic "density" of angiosperm sapwood. Conversely, white-rot fungi are initially inhibited when colonizing wood with a relatively high phenolic "density". For example, Highley (7) reported that the white-rot fungus C. versicolor degraded the sapwood of two typical angiosperms (sweetgum and white oak) much faster than three gymnosperms (southern pine, white spruce [Picea glauca (Moench.) Voss.], and western hemlock (Tsuga heterophylla (Raf.) Sarg.]. In addition, we and others (T. Amburgey, personal communication) have observed that angiosperm sapwood is degraded by white-rot fungi in an above-ground test much faster than similar gymnosperm samples are degraded by brown-rot fungi. Furthermore, when we added the antioxidant B H T to aspen (Populus spp.) sapwood, at a level which would approximately double the phenolic "density", followed by inoculation with the white-rot fungus Trametes (formerly Coreolus ) versicolor (L.) Quel, in the soil-block test, we observed some protective effect but only during the early stages of incubation (Table 1). This relatively short protected period against white-rot fungi, however, may result in a "window of opportunity" for brown-rot fungi. (We measured strength rather than weight loss


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in this test since strength loss is a more accurate indication of incipient decay). Alternatively, it may be possible that the type and mode of action of free radicals generated by brown- and white-rot fungi during the initial colonization stage are different and, thus, that the mechanisms(s) by which wood is initially perturbed differ. Another possibility might be that syringyl lignin units are simply more susceptible to free radical oxidation/degradation than guaiacyl units (25). Consequently, free radical attack would mainly affect angiosperm wood and it's syringyl-guaiacyl lignin rather than normal gymnosperm wood which has guaiacyl lignin. Finally, it is well known that white-rot fungi degrade wood by radical mechanisms. Thus, the degradation rate of white-rot fungi may be more affected by a high phenolic "density" than that of brown-rot fungi. Interestingly, phenolic syringyl units would be expected to be a better antioxidant than a free phenolic quaiacyl. (The second 0-methoxyl would confer beneficial steric and electronic properties). Indeed, this is supported by data from a recent study (72). However, the higher phenolic quantity in gymnosperm is likely more important than the faster antioxidant rate but lower phenolic level in angiospemis (quantity vs. quality). Table 1. Average percent compression strength loss for aspen sapwood samples either untreated or treated with 5% BHT, then inoculated with F. versicolor and tested in the soil-block test for 2-, 3-, and 4-weeks incubation. The results are the average of five replicates. Avg. % Strength Loss Incubation Time, Weeks Treatment Control 5% B H T

2

3

4

70.0 34.2

87.3 56.7

89.1 78.6

We need to emphasize that even if our tentative hypothesis is correct, other factors probably also influence angiosperm sapwood susceptibility to white-rot colonization. For example, it is well known that white-rot fungi prefer wood with a higher moisture content than brown-rot fungi. The higher hydrophobicity of gymnosperm extractives, such as the terpenoid-based extractives found in pine sapwood and heartwood, would make gymnosperm wood relatively water repelling. Other decay susceptibility factors could be differences in the structure, amount and accessability of the hemicelluloses, the fungicidal activity, microdistribution and hydrophobicity of any extractives present, antagonism between different fungi, and other factors - some as yet unknown. Synergism or antagonism between two or


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more factors is also possible. Whatever the cause(s), after studying natural durability for some time we believe that multiple factors affect natural durability for both gymnosperms and angiosperms and that most wood extractives have very poor fungicidal activity when compared to commercial biocides. It is also interesting that our radical hypothesis [pun intended] suggests that wood components protect wood by a general, nonspecific mechanism. It is likely that if an extractive were to inhibit a specific part of a critical fungal metabolic pathway, and thus was highly fungicidal, a decay fungus would rapidly (in an evolutionary time frame) mutate into a strain unaffected by the extractive. A nonspecific defense mechanism such as our antioxidant hypothesis, by contrast, may be difficult for fungi to overcome by mutation. Extractives or other antioxidants may protect the wood cell wall from being disrupted or perturbed byfreeradicals during an initial attack by white-rot fungi (5). If the wood is disrupted in some manner such as by autohydrolysis, a previous attack by white-rot fungi, a limited lignin degradation by some chemical means (7) or photodegradation as discussed above, then we would predict that adding nonbiocidal antioxidants to an already-disrupted xylem would have no or minimal influence on later white-rot susceptibility. Thus, we are unable to test our hypothesis by methylating the lignin phenolic groups of a wood block, since methylation would require a pretreatment to make the cell walls permeable. Furthermore, we are unsure of the relationship between colonization, decay susceptibility, and decay rate for untreated sapwood samples. Finally it should be noted that resistance to white-rot fungi does not imply that a wood will also be resistant to brown- or soft-rot fungi. As mentioned in the beginning, our hypothesis is only tentative. We have presented these thoughts so that these ideas may lead to a better understanding of wood decay and natural durability. Conclusion Based on prior articles of angiosperm sapwood susceptibility to white-rot fungal degradation we suggest that: The resistance of sapwood to white-rot fungal colonization and subsequent degradation is related to a wood's phenolic "density", with the phenolic groups acting as antioxidants. In normal sapwood essentially all phenolic groups are in the lignin and thus phenolic "density" is related to lignin concentration and structure. This antioxidant function prevents the fungal-generated free radicals from initially perturbing the wood structure and, consequently, thwarts the relatively large


213 extracellular fungal enzymes from penetrating into and degrading the wood substance. Once the cell wall has been disrupted by some means, either biological, physical or chemical, this antioxidative protective mechanism may no longer help prevent white-rot colonization. Acknowledgments


Funding was provided by a USDA-competitive grant and the state of Mississippi. Discussions with Dr. K. Kirk, USDA-Forest Products Lab and Dr. T. Nilsson, SLU, Sweden, were much appreciated. Approved for publication as Journal Article No. FP102 of the Forest and Wildlife Research Center, Mississippi State University. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Schultz, T.P.; Nicholas, D.D.; Minn, J.; McMurtrey, K.D.; Fisher, T.H. IRG Paper WP 98-30172, 1998, and references therein. Schultz, T.P.; Harms, W.B.; Fisher, T.H.; McMurtrey, K.D.; Minn, J.; Nicholas, D.D. Holzforschung 1995, 49, 29, and references therein. Backa, S.; Gierer, J.; Reitberger, T; Nilsson, T. Holzforschung 1993, 47, 181. Flournoy, D.S.; Paul, J.A.; Kirk, T.K.; Highley, T.L. Holzforschung 1993, 47, 297. Schultz, T.P.; Nicholas, D.D. U.S. Patent 5,730,907. 1998. Obst, J.; Highley, T.L.; Miller, R.B. Biodeterioration; Research 4; Plenum Press: New York, N Y ; 1994, Vol. 4 pp. 357-374. Highley, T.L. Can. J. For. Res. 1982, 12, 435, and references therein. Sjostrom, E. Wood Chemistry: Fundamentals and Applications, 2 Ed., Academic Press: New York, N Y . 1993, pp. 83. Larson, R. A. Phytochem. 1988, 27, 969, and references therein. Han, D.N.S. In Wood and Cellulosic Chemistry; Han, D.N.S. and Shiraishi, N . , eds; Marcel Dekker, New York; 1991, chp. 11. Schmidt, J.A.; Rye, C.S.; Gurnagul, N. PolymerDegrad. Stability 1995, 49, 291. Barclay, L.R.C.; Xi, F.; Norris, J.Q.; In J. Wood Chem. Tech.; 1997, 17, 73. Schmidtling, R.C.; Amburgey, T.L. Holzforschung 1982, 36, 159. Srebotnik, E.; Jensen, K.A., Jr.; Hammel, K.E. Proc. Natl. Acad. Sci. 1994, 91, 12794. Holmer, L.; Stenlid, J. FEMS Microbiol. Ecology, 1993, 12, 169. Owens, E . M . ; Reddy, C.A.; Grethlein, H.E. FEMS Microbiol. Ecology, 1994, 14, 19. Faix, Ο.; Mozuch, M.D.; Kirk, T.K. Holzforschung 1985, 39, 203. Hillis, W.E. In Heartwood and Tree Exudates, Springer-Verlag; New York, N Y 1987. Hirano, T.; Tanaka, H.; Enoki, Α.; Holzforschung, 1997, 51, 389. Chen, Y-r; Schmidt, E.L.; Olsen, K.K. Wood Fiber Sci., 1998, 30, 18, and references therein. Lewis, N.G.; Davin, L.B. In: Lignin and Lignan Biosynthesis; Lewis, N.G.; Sarkanen, S., Eds; ACS Symp. Series: Washington, D C , 1998, Vol. 697, pp. 344, also see pp. 188. Backa, S.; Gierer, J.; Reitberger, T.; Nilsson, T. Holzforschung 1992, 46, 61. Ander, D. Holzforschung 1996, 50, 413. nd


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