Canada is the largest exporter of softwood lumber in the world. Sapstain caused by fungi is a
costly problem for the Canadian lumber industry. To maintain and increase future sales and
exports of lumber requires not only effective, but also environmentally acceptable anti-sapstain
methods. A thorough understanding of the physiological and biochemical features of
sapstaining fungi should facilitate the development of new methods of protection. This work
was carried out to determine the available lipid nutrients in wood and to understand the
biodegradation mechanisms of lipids by sapstaining Ophiostoma species.
Lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) constitutes the greatest volume
of wood harvested in British Columbia. As with all wood species, the freshly sawn sapwood is
susceptible to sapstain. The total extractives content in the sapwood at the breast height of a
35-year-old lodgepole pine tree was 1.9-2.2% of oven dry weight, which included 1.1-1.3%
triglycerides (TG), 0.02-0.03% free fatty acids (FA), 0.3-0.4% free resin acids (RA), and 0.15-
0.21% steryl esters and waxes (SEAV). A considerably higher content of extractives was
present in heartwood, i.e., 9.7-12.4%, including 0.6-0.7% TGs, 0.8-1.1% free FAs, 4.0-5.6%
free RAs, and 0.25-0.33% SE/Ws. The wood TGs were composed mainly of oleic (18:1),
linoleic (18:2), linolenic (18:3) and palmitic (16:0) acids. The dominant RA was palustric acid,
followed by abietic, neoabietic, dehydroabietic, isopimaric, pimaric, and sandaracopimaric
acids.
The ability of three sapstaining fungal species, Ophiostoma piceae 387N, O. ainoae 701 A, and
O. piliferum 55H to degrade and utilize the major lipids in the sapwood of lodgepole pine and
trembling aspen (Populus tremuloides Michx.) was investigated. The fungal growth rate in
wood was monitored by quantifying ergosterol extracted from colonized wood. After two weeks
colonization, the TGs in wood were degraded by 50% to 80%, which resulted in an
accumulation of free FAs in the wood.
Lipases (glycerol ester hydrolases, EC 3.1.1.3) are the enzymes responsible for hydrolyzing
TGs into glycerol and FAs which are assimilable by fungal cells. Extracellular lipase activity of
O. piceae 387N was detected both in colonized wood and in liquid culture. The effect of various
factors (carbon sources, nitrogen sources, and medium pH) on the growth and lipase activity of
O. piceae 387N were examined in liquid culture. The extracellular lipase secretion was
enhanced in the presence of triglycerides. The composition of a medium was optimized for a
high extracellular lipase production, which contained 2% olive oil as a carbon source and 0.5%
ammonium sulfate and 3% peptone as nitrogen sources with an initial medium pH of 5.0.
A major extracellular lipase was purified from the liquid culture filtrates of O. piceae 387N by
hydrophobic interaction chromatography and anion exchange chromatography. This lipase was
characterized as a monomer with a molecular weight of 35 kDa, and was glycosylated,
containing 10.1% carbohydrates. It was resolved as a single band on SDS-PAGE (sodium
dodecyl sulfate- polyacrylamide gel electrophoresis) gels, whereas 3 bands at pi's 4.3, 4.1 and
3.8 were observed on LEF (isoelectric focusing) gels. Lipolytic stain demonstrated that the three
bands on IEF gels were lipolytically active. The 3 isoforms were found to have a same N -
terminal sequence as D¹-V²-S³-V⁴-T⁵-T⁶-T⁷-D⁸-I⁹-D¹⁰-A¹¹-L¹²-A¹³-F¹⁴-F¹⁵-T¹⁶-Q¹⁷-W¹⁸-A¹⁹-G²⁰.
The purified O. piceae 387N lipase was stable at pH's 4 to 8 and at temperatures below 40°C.
The pH and temperature optima for activity were approximately pH 5.2 and 30°C, respectively.
Enzyme activity was not influenced by N-ethylmaleimide, P-mercaptoethanol, and
dithiothreitol, was slightly enhanced by Ca²⁺ and Mn²⁺, and was severely inhibited by Hg and
Fe³⁺, diethyl pyrocarbonate, diethyl /?-nitrophenyl phosphate, butyric acid, caproic acid, and
SDS. The lipase showed high specificity toward substrates with intermediate and long chain FA
residues, and belonged to a group of 1(3) positional specific lipases. The rate of hydrolysis of
the lipase toward a triglyceride (l,3-dipalmitoyl-2-oleoyl-glycerol) was 25-50 fold higher than
that toward the waxes (oleyl esters) and cholesteryl esters. Finally, it was conclusively shown
that the purified lipase could effectively release fatty acid residues from the triglycerides
isolated from wood.
The data and information obtained in this work have contributed to the understanding of the
physiological and biochemical features of sapstaining Ophiostoma species. The large amounts
of various lipids in wood, in particular TGs and FAs, are important carbon and energy sources
for the sapstaining fungi, which are capable of secreting extracellular lipases to degrade TGs.
The information implies that the growth of sapstaining fungi may be hindered by disrupting the
metabolic processes of lipid utilization, or by applying biological competitors which are more
efficient in assimilating lipids and other nutrients in wood. / Forestry, Faculty of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/6285 |
| Date | 11 1900 |
| Creators | Gao, Yong |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Format | 9397545 bytes, application/pdf |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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