Effect of sapstaining fungi on structural wood integrity of radiata pine (Pinus radiata D. Don)
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Selected sapstaining fungi belonging to the Ascomycetes and Fungi Imperfecti isolated in New Zealand were evaluated for potential degradation of unseasoned radiata pine sapwood using four independent methods: i) toughness and weight loss measurements, ii) chemical analysis of wood composition, iii) enzyme assays and iv) light and scanning electron microscopy. Several isolates of albino-strains, potential candidates for use as biocontrol agents against sapstain on radiata pine, were included in the experiments. Sets of side-matched specimens were inoculated with isolates of Ophiostoma floccosum, O. pluriannulatum, O. ips, O. piceae, Leptographium procerum and Sphaeropsis sapinea and incubated for 8 or 16 weeks, along with non-inoculated control samples. Three decay fungi, Gloeophyllum trabeum, Phlebiopsis gigantea and Schizophyllum commune, were included in the tests to serve as references for weight and toughness loss. After incubation, the samples were equilibrated to 14% moisture content before recording toughness and weight losses. Weight loss was determined by comparing the dry weights of the inoculated samples to the untreated controls rather than expressing weight loss as a percentage of the original weight of a sample. Neither toughness nor dry weight were significantly (p<0.05) different between specimens inoculated with sapstaining fungi and their controls, except for an isolate of O. ips in one out of three experiments. This isolate of O. ips caused 18% toughness loss without causing weight loss. In one out of four experiments, there was a slight, but statistically significant (p < 0.05) overall weight loss of samples inoculated with different sapstaining isolates which was attributed to the degradation of non-lignified parenchyma cells and extractives. In comparison, the brown-rot fungus G. trabeum caused up to 61% toughness loss after 4 months’ incubation, accompanied by a weight loss of 8%, and the white-rot fungus S. commune produced a toughness loss of 32% without any significant weight loss which indicates that toughness loss is a more sensitive indicator of decay than weight loss. None of the sapstaining fungi tested caused significant reductions of lignin or structural carbohydrates (arabinose, galactose, glucose, xylose and mannose) in radiata pine sapwood after 16 weeks’ incubation, but all fungal strains, with the exception of O. pluriannulatum, reduced the amount of extractives significantly. Of the isolates tested, O. floccosum degraded extractives most effectively (53.6% reduction). The fact that there were no major differences with regard to the composition of the structural cell wall components of sapstained wood and controls complements the results obtained in the weight loss experiments. Significant amylose and extractives consumption reflect the observation of sapstaining fungi growing extensively in the parenchyma cells and resin canals of radiata pine. Sapstaining fungi secrete hydrolytic enzymes into the growth substrate and subsequently absorb the products of hydrolysis in the fungal mycelium. Enzyme assays demonstrated that all sapstaining isolates tested secreted low amounts of xylanase (up to 1.64 μmoles/min/ml) and pectinase (up to 0.11 μmoles/min/ml) into the growth medium, but extracellular cellulose was not detected. Under the conditions tested, mannanase was secreted only by O. piceae (0.29 μmole/min/ml). The results suggest that although galactoglucomannans are the major hemicelluloses in softwoods, the arabino-4-methylglucuronoxylans are preferably used by sapstaining as well as decay fungi when growing in softwoods. This may be attributed to the accessibility of the xylans in the S₃-layer of the wood cell wall. Amylase activity of the fungal species tested was more significant than xylanase, mannanase and pectinase activities which confirms that sapstaining fungi preferably metabolize easily accessible, non-structural wood components like starch. A possible function of the cell-wall degrading enzymes may be the facilitation of the colonization of wood cells, specifically, pectinase may assist the fungal penetration of pit membranes. It is likely that the extracellular sheath which has been observed around hyphae of S. sapinea contains these enzymes and represents an important source of support and hyphal contact with the host cell wall. In addition, pectinase and xylanase could be involved in the pathogenesis of certain sapstaining fungi, as has been demonstrated for O. ulmi and O. novoulmi. The production of specific antibodies against xylanase and pectinase and the subsequent immunohistochemical localization in the host might resolve this question. Hyphae of sapstaining fungi pass from one wood cell to another by growing through the pit membranes. No differences were observed with regard to the spatial distribution of the sapstaining fungi. Direct cell wall penetration was not observed. The non-lignified parenchyma cell walls of the wood samples infected with sapstaining fungi appeared to be degraded. The ray tracheids in radiata pine were less colonized than the parenchyma cells. Although low amounts of hemicellulolytic enzymes and pectinase were detected, the effect of sapstaining fungi on wood quality of radiata pine has to be considered cosmetic and non-degradative which confirms the evaluation of sapstain according to the New Zealand Timber Grading Rules (NZS 3631, 1988). In general, sapstaining fungi do not affect the structural wood integrity of radiata pine. If severe sapstain occurs which obscures the grain, there is a possibility that decay fungi may also be present, and since decay fungi do affect the strength of timber, it seems justified not to allow for the use of severely sapstained timber for structural purposes, consistent with NZS 3631 (1988). The use of selected albino-strains of the naturally occurring sapstain population in New Zealand as biocontrol agents on radiata pine logs and timber can be recommended since these fungi were also not found to decrease toughness nor to cause weight loss in radiata pine.
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