Role of metal proteinases in the fruit-body formation ofHypsizygus marmoreus

We investigated the possible role of metal proteinase on the fruit-body formation ofHypsizygus marmoreus. The addition of a specific metal proteinase inhibitor, phosphoramidon, to the culture medium (10g/ml) completely inhibited fruit-body formation. Metal proteinase activity in both the medium and the mycelia of this fungus increased markedly during vegetative mycelial growth, and activity was maximal 25 days after inoculation. When phosphoramidon was added to the culture medium during vegetative mycelial growth, the metal proteinase activity in the mycelium decreased to 56% of the control (without inhibitor) level. Isoelectric focusing analysis showed that two kinds of metal proteinases with a pl of 7.7 and 8.4, respectively, were obtained from 29-day-old mycelia. Uptake of phosphoramidon into the mycelia was confirmed as the result of inhibition of thermolysin activity by the mycelial extracts. The degree of inhibitor uptake into mycelia was about 2.0% and was independent of the initial concentration of the inhibitor administered. The addition of peptone and amino acids to medium treated with phosphoramidon resulted in fruit-body dry weight yields that were about 50% that of the control.     

Characterization of monoclonal antibodies to woodderived β-1,4-xylanase of Postia placenta and their application to detection of incipient decay

Summary Murine monoclonal antibodies (mAbs) were produced to the extracellular -1,4-xy-lanase fraction of the brown-rot fungus, Postia placenta. The immunizing antigen was derived from decayed southern yellow pine wood blocks extracted in phosphate-buffered saline and Triton X-100, and fractionated by column chromatography. Five IgG monoclonal antibodies were selected for characterization by enzyme-linked immunosorbent assay, (ELISA) and western blot. Each mAb exhibited binding to two major protein bands on western blot at 32 kDa and 36 kDa. Characterization by chemical and enzymatic modification of the antigen determined whether mAbs were protein specific or specific for the carbohydrate portion of the glycoprotein. Affinity chromatography with the protein specific mAbs demonstrated specific binding to -1,4-xylanase and cross-reactivity with -1,3-glucanase, -1,4-glucanase, and -1,4-mannanase. These mAbs were also immunoreactive with polysaccharidases from liquid culture filtrates when tested by ELISA.The mAbs have been utilized to successfully detected P. placenta by ELISA in decayed southern yellow pine at a weight loss of 3.6%.This article was written and prepared by U.S. Government employees on official time, and it is therefore the public domain and not subject to copyright. The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin     

Transferase activity of GhCesA2 (putative cotton cellulose 4- β -glucosyltransferase) expressed in Pichia pastoris

GhCesA2 is a cotton (Gossypium hirsutum) homologue of the bacterial cellulose synthase gene that encodes cellulose 4--glucosyltransferase. The central catalytic region of GhCesA2 was expressed as a soluble protein in the methylotrophic yeastPichia pastoris. The molecular size of the recombinant protein was 100kDa, which decreased to 85 kDa after treatment with endoglycosidase H. The recombinant GhCesA2 catalyzed transfer of glucose from UDP-glucose into unknown products in the presence of an extract of cotton hypocotyls, but the products were not-1,4-glucan.     

Isolation of a β-1,3-glucan (laricinan) from compression wood of Larix laricina

Summary A -d-(13)-linked glucan has been isolated from compression wood of tamarack [Larix laricina (Du Roi) K. Koch]. The polysaccharide, which had []_D + 17.6°, consumed 0.17 mole of periodate per glucose residue and gave a methyl derivative which consisted of 115 2,4,6-tri- and 2,3,4,6-tetra-O-methyl-d-glucose residues in a ratio of 17.8:1. Partial acid hydrolysis yielded a series of -d-(13)-linked oligosaccharides, while enzymic hydrolysis with a -1,3-glucanase afforded glucose, laminaribiose, and laminaritriose. The glucan occurred in amounts varying from 2 to 4% in compression wood of this and other conifer species and was present in the wall of both tracheids and ray cells. It is proposed that this structural polysaccharide be designated as laricinan to distinguish it from callose and the various -1,3-glucans in lower plants.     

Effect of thawing regime on growth and mortality of frozen-stored Norway spruce container seedlings planted in cold and warm soil

One-year-old frozen-stored Norway spruce (Picea abies (L.) Karst.) container seedlings were planted in a controlled environment providing an air temperature of 22°C and soil temperature of 9±1 or 18±1°C. At planting the root plugs were either frozen or had been thawing for 4 days at 9°C. During a 5-week growing period, in both cold and warm soil the root growth and height growth were less in frozen-planted seedlings than in thawed seedlings. In addition, frozen-planting delayed bud burst and increased mortality. Soil temperature, however, had no effect on bud burst or mortality. Low soil temperature retarded root growth of seedlings thawed before planting but resulted in both retarded root growth and height growth if root plugs were frozen when planted. These results indicate that planting Norway spruce seedlings with frozen root plugs constitutes a considerable risk for successful forest regeneration at soil temperatures normally prevailing in Fennoscandia in spring or early summer especially if the soil is dry at the time of the planting.     

Effect of water potential on fruit body formation ofLentinula edodes in sawdust-based substrate

The influence of substrate water potential () on the growth and fruiting of three genotypes of shiitake (Lentinula edodes) was investigated. A slight reduction of (–0.5MPa) stimulated mycelial and colony growth on liquid, agar, and sawdust-based substrates.L. edodes has been found to grow well at a around –0.5 MPa, which corresponds to a moisture content around 55%. A small decrease in at the final vegetative growth phase had positive effects on flush quantity. The substrate was significantly affected by the interaction between genotypes and spawn run time. The of well-colonized mature substrate was –0.7MPa before and –4.OMPa after the fruiting. The rose again to –0.7MPa during rapid absorbance of water by soaking, and this rise was repeated during the second and third flushes. It is suggested that the water-holding capacity of a substrate is related to culture maturity. Excellent water-providing capacity (higher) is expected in the substrate of well-matured cultures with a high density of mycelial colonization.Part of this paper was presented at the 46th annual meeting of the Japan Wood Research Society, Kumamoto, April 1996     

Simple method for synthesizing phenolicβ-O-4 dilignols

A modified synthetic method for phenolic-O-4 lignin substructure model dimers was developed involving protection of the phenolic hydroxyl group of acetophenons with benzoyl chloride, bromination with 4-dimethylaminopyridiniumbromide perbromide, condensation with phenols in the presence of 18-crown-6-ether, condensation with paraformaldehyde, reduction with NaBH₄ and debenzoylation. This method results in shorter reaction times and increasing yields without the application of strict anhydrous and drastic conditions or chloric solvents. This alternative route could be applied to the-O-4 dilignol syntheses of four combinations of guaiacyl and syringyl derivatives.     

Erythro/threo ratio ofβ-O-4 structures as an important structural characteristic of lignin. I: Improvement of ozonation method for the quantitative analysis of lignin side-chain structure

Ozonation as a quantitative tool to analyze the stereo structures of arylglycerol--aryl ether linkages was examined using wood meal, milled wood lignin, and a lignin model compound, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol (veratrylglycerol--guaiacyl ether, VG). The procedure was improved. When mild postreduction was conducted for ozonation products, the total yield of erythronic and threonic acids from this model compound was 74%, which is 15% higher than the yield without postreduction. A decrease in the recovery of these two acids under prolonged ozonation treatment was successfully suppressed by postreduction. Theerythro/threo ratio of VG determined by the ozonation method with postreduction is in good agreement with the ratio determined by¹H-nuclear magnetic resonance. Excellent reproducibility of the yield was obtained by adopting a procedure that included trimethylsilylation of ammonium salts of ozonation products using a dimethylsulfoxide-hexamethyldisilazane-trimethylchlorosilane mixture and subjecting it to gas chromatography analysis. It was concluded that arylglycerol--aryl ether structures comprise at least 35% of the C₃-C₆ structure in birch wood meal, with anerythro/threo ratio of 2.8.This paper was presented at the 43rd Lignin Symposium, Fuchu, Japan, October 1998; and at the 10th ISWPC, Yokohama, Japan, June 1999     

Prevalence and incidence of the root-inhabiting fungi, Fusarium, Cylindrocarpon and Pythium, on container-grown Douglas-fir and spruce seedlings in British Columbia

Surveys were made at the end of the 1990 and 1991 growing seasons for root-inhabiting fungi in the genera Fusarium, Cylindrocarpon and Pythium from the roots of one year-old container-grown Douglas-fir and spruce seedlings grown under greenhouse conditions. In the 1990 survey of four nurseries, it was found that 61–97% of both Douglas-fir and spruce roots were colonized with Fusarium, Cylindrocarpon or Pythium. There were significantly (p0.05) more Douglas-fir roots than spruce roots colonized by Fusarium at all nurseries, however, there were significantly (p0.05) more spruce roots than Douglas-fir roots colonized by Cylindrocarpon and Pythium. Root colonization of Douglas-fir and spruce by the three fungal genera during 1991 varied from 0–82% at three nurseries, however, only at a south coastal nursery was there significantly (p0.05) more spruce than Douglas-fir roots colonized by Cylindrocarpon. Significantly more seedlings were infected in 1990 than in 1991. In 1991, there were few significant differences between Douglas-fir and spruce, in the percentage of seedlings with colonized roots and in the percentage of growth medium colonized by the fungi. However, there were significant differences between nurseries.     

Specific degradation ofβ-aryl ether linkage in synthetic lignin (dehydrogenative polymerizate) by bacterial enzymes ofSphingomonas paucimobilis SYK-6 produced in recombinantEscherichia coli

Sphingomonas paucimobilis SYK-6 produces unique and specific enzymes, such as-etherases,O-demethylases, and ring fission dioxygenases, for lignin degradation. Cleavage of arylglycerol--aryl ether linkage is the most important process in the lignin metabolic pathway ofS. paucimobilis SYK-6. We reported the genes (ligD, ligE, ligF) for enzymes that cleaved-aryl ether linkage of dimeric compounds in previous studies. In this study we synthesized the fluorescent high-molecular-weight lignin (UBE-DHP) by dehydrogenative polymerization. We investigated the-aryl ether cleavage ability of these enzymes produced in recombinantEscherichia coli. When UBE-DHP was incubated with LigF, 4-methylumbeliferone was released as a result of-aryl ether cleavage of O-methylumbelliferyl--hydroxypropiovanillone (compound III) incorporated in UBE-DHP. Here, we report that-etherase ofS. paucimobilis SYK-6 can be expressed inE. coli and is able to cleave the-aryl ether linkage in synthetic high-molecular-weight lignin.     

Chemical constituents ofInonotus obliquus II: a new triterpene, 21,24-cyclopentalanosta-3β,21,25-triol-8-ene from sclerotium

A new lanostane-type triterpene with a cyclopentanol partial structure in the side chain was isolated from the sclerotium of the wood rotting fungusInonotus obliquus along with four known compounds: lanosterol, inotodiol, trametenolic acid, and 3-hydroxy-8,24-dienlanosta-21,23-lactone. The new compound was determined to be 21,24-cyclopentalanosta-3,21,25-triol-8-ene by spectroscopic analyses.An outline of this study was presented at the 50th Annual Meeting of the Japan Wood Research Society, Kyoto     

Inhibition ofTrichoderma cellulase activity by a stilbene glucoside fromPicea glehnii bark

The stilbene glucoside isorhapontin (5,4-dihydroxy-3-methoxystilbene-3--D-glucoside) is the major constituent of the ethyl acetate extracts fromPicea glehnii bark. Isorhapontin inhibited the hydrolytic activity ofTrichoderma cellobiohydrolase I (CBH I) for both bacterial microcrystalline cellulose and the soluble cellooligosaccharide celloheptaitol. The inhibitory effect for celloheptaitol, however, was more drastic than that for bacterial microcrystalline cellulose. The hydrolytic activity of the CBH I core domain for celloheptaitol was also inhibited by isorhapontin to a similar extent, suggesting that the interaction between isorhapontin and the core domain of CBH I is the reason for this phenomenon. The inhibition of CBH I activity by isorhapontin showed mixed noncompetitive and uncompetitive types in a concentration of the inhibitor of less than 125M. TheK _i andK _i values were estimated to be 57.2 and 33.3M, respectively. Whereas isorhapontin strongly inhibited CBH I activity, its aglycone isorhapontigenin (3-methoxy-3,5,4-trihydroxystilbene) showed almost no inhibition. Consequently, both the stilbenic and the-glucosidic structures in isorhapontin are essential for the inhibitory effect on CBH I activity. Isorhapontin also inhibited the activity ofTrichoderma endoglucanase I for celloheptaitol, whereas almost no effect was observed for the activities of both endoglucanases II and III.An outline of this work was presented at the 49th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

Micropropagation of western hemlock (Tsuga heterophylla [Raf.] Sarg.) from embryonic explants

An in vitro propagation protocol is described for western hemlock, an important forestry species in Canada. For shoot bud induction, embryonic explants were placed initially on one-third strength Schenk and Hildebrandt medium containing 5 M N⁶-benzyladenine (BA) or 5 M BA in combination with either 5 M kinetin or 5 M 2-isopentenyl adenine for 14 days. The explants were transferred to basal medium without cytokinins for 3 weeks, and then to basal medium containing 0.05% activated charcoal. Elongating shoots were subcultured every 4 weeks on charcoal medium. Shoots, 10 mm in stem height, were rooted either in agar or sterilized peat/perlite (1:1). Up to 70% of the shoots formed roots when they were transferred to the latter, moistened with 1/2 strength Gresshoff and Doy medium containing 5 M -naphthaleneacetic acid. About 90% of the plantlets survived transfer to greenhouse conditions.     

Influence of cavity size, seedling growing density and fungicide applications on Keithia blight of western redcedar seedling growth and field performance

To determine the influence of cavity size, seedling density and fungicide application on severity of Keithia blight caused by the fungus Didymascella thujina (Durand) Maire, western redcedar (Thuja plicata Donn) seedlings were grown in styroblocks of 3 cavity sizes, thinned to 3 growing densities and sprayed several times monthly with mancozeb fungicide. After two growing seasons cavity size, seedling growing density and number of fungicide applications all significantly (p0.05) affected blight severity. Blight severity was inversely related to the number of mancozeb fungicide applications. The only significant two-way interaction was for cavity size and seedling growing density.Eighteen seedlings from each of the nine cavity sizes and seedling densities combinations were assessed for shoot height, root collar diameter and blight severity, prior to planting at both Holt Creek and Salmon Creek on Vancouver Island, British Columbia in March 1994. Growth was reassessed during October 1994. Seedling height and root collar diameter increased significantly (p0.05) with certain cavity size and seedling density combinations. Blight severity increased significantly (p0.05) for all nine treatments at both sites, however, between sites blight severity increased significantly (p0.05) only at the wetter, Salmon Creek site.     

Sapling architecture and growth in the co-occurring species Castanopsis cuspidata and Quercus glauca in a secondary forest in western Japan

This study quantitatively compared the sapling (height 62–289cm) architecture and growth of Castanopsis cuspidata and Quercus glauca, both of which are major components in the temperate zone of western Japan, under shaded light conditions in secondary forest. When the sapling architectures were compared at the same support mass (trunk + branch mass), C. cuspidata had a larger crown area but a smaller height gain than did Q. glauca owing to the allocation of more biomass to lateral branches in C. cuspidata. The above-ground relative growth rate (RGR) of C. cuspidata (0.442gg^–1 year^–1) was nearly twice that of Q. glauca (0.256gg^–1year^–1), primarily as a result of a greater total leaf area per above-ground biomass (LAR) in C. cuspidata (56cm²g^–1) as compared to Q. glauca (33cm²g^–1). Because it has a disadvantage in height gain, related to its allocation pattern of biomass, C. cuspidata realized the same height growth (RGR_H) as Q. glauca, despite the large biomass production. The great potential for photosynthesis in C. cuspidata, which results from its vigorous lateral spreading, is presumed to give it a long-term advantage over Q. glauca in the shaded forest understory. Q. glauca invests preferentially in trunk biomass, possibly giving it an advantage in sunny sites as opposed to a shaded forest understory.     

Effects of stilbenes from bark of <Emphasis Type="Italic">Picea glehnii</Emphasis> (Sieb. et Zucc) and their related compounds against feeding behaviour of <Emphasis Type="Italic">Reticulitermes speratus</Emphasis> (Kolbe)

The effects of stilbene glucosides and related compounds on termite feeding behavior were investigated using paper disc methods against the subterranean termite Reticulitermes speratus. The stilbene-rich fraction and isorhapontin (3-methoxy-3,4, 5-trihydroxystilbene-3--d-glucoside) from bark extracts of Picea glehnii showed avoidance by termites in choice tests. In the no-choice tests using compounds purified from the stilbene-rich fraction, the largest feeding deterrent effect was observed for piceid (3,4,5-trihydroxystilbene-3--d-glucoside), followed by isorhapontin, and astringin (3,3,4,5-tetrahydroxystilbene-3--d-glucoside), at the concentrations from 0.63 to 2.5µmol/disc. No change in activity was observed at retentions of more than 5.0µmol/disc. When the activities of isorhapontin and its aglycone derivative (isorhapontigenin: 3-methoxy-3,4,5-trihydroxystilbene) were compared with that of taxifolin (3,3,4,5,7-pentahydroxyflavanone) in the no-choice test, the stilbenes exhibited a larger antifeedant potential. Methylation of isorhapontigenin increased its termiticidal activity.Part of this study was presented at the 32nd Annual Meeting of the International Research Group on Wood Preservation, 2001     

On the distribution of the diastereomers of the structural elements in lignins: the steric course of reactions mimicking lignin biosynthesis

Summary Stereochemical studies on the formation of the diastereomers of arylglycerol--aryl ether structures during lignin biosynthesis have been carried out with model compounds. The addition of water to quinone methides of the -syringyl ether type gives arylglycerol -syringyl ethers with a predominance of the erythro isomer when the pH of the medium is low. Since erythro forms of arylglycerol -syringyl ethers are prevalent in hardwood lignins, this indicates that the pH of the medium in which lignin biosynthesis occurs is lower than has been assumed until now. Equilibration studies with non-phenolic model compounds of the arylglycerol-guaiacyl ether and -syringyl ether types under acidolysis conditions indicate that the erythro predominance observed in the syringyl ethers in lignins does not correspond to equilibrium conditions. A remarkable resistance to acidolysis is observed in the model compounds of etherified syringylglycerol -syringyl ether type.     

Glucanohydrolase enzyme activity in embryos of Scots,Corsican and lodgepole pines infected in vitro with Heterobasidion annosum

Embryos of Scots pine (Pinus sylvestris), Corsican pine (P. nigra var. maritima) and Lodgepole pine (P. contorta) were infected in vitro with Heterobasidion annosum and the activities of 1,3-β-glucanase, chitinase and β-glucosidase were monitored. Both the infected and control embryos of the three pine species showed the presence of the enzymes. Statistical analysis showed no significant increase (P > 0.05) in the levels of 1,3-β-glucanase in infected embryos for all the three pine species. However, there was a significant increase in levels of chitinase in infected embryos of P. contorta and P. nigra var. maritima and also in levels of β-glucosidase in infected embryos of all three of the pine species. The study has therefore revealed that constitutive levels of glucanohydrolase enzymes exist in the embryos of the three pine species and that the induction of chitinase and β-glucosidase was a factor in the response of the embryos to stimulation by H. annosum infection.     

Provenance variation in growth characters of four subtropical pine species planted in Mexico

In 1995 a species/provenance test was established in the state of Puebla, Mexico to assess and compare the growth of Pinus greggii var. australis, P. maximinoi, P. patula and P. tecunumanii, including 10, 5, 11 and 4 provenances of each species, respectively. Each provenance test lot consisted of seed from 7 to 10 individual trees collected in natural stands. Range-wide samples were included in the test for P. greggii var. australis and P. patula. Material of P. maximinoi and P. tecunumanii from Guatemala, Honduras and Nicaragua, as well as Mexico was established in the test. The test site is in a mountainous area at 1440m elevation, near 20°N latitude. At 5 years of age significant differences were seen at both the species and provenance levels in survival, total height, diameter and volume. The overall test survival was 93%. Variance among species was three time greater than among provenances, comprising 25–33% of the total variation. P. maximinoi had the greatest height, diameter and volume (62dm³) followed by P. tecunumanii (53dm³). P. greggii var. australis had an average volume of 43dm³ and P. patula produced 30dm³. Although P. maximinoi had the lowest survival rate, the San Jeronimo provenance was the most productive of all 30 provenances tested; 98% survival and 73dm³ volume. This was equivalent to 2.8cm annual diameter increment and 2m in height growth per year. Significant growth differences among provenances within species indicate that potential gain exists using provenance selection.     

Biological activity of extracts fromCupressus lusitanica cell culture

Ethyl acetate extracts ofCupressus lusitanica suspension cell cultures were examined for their biological activities (viz., tyrosinase inhibitory, antioxidant, and antimicrobial activities). The extract from elicitor-treated cells showed all of the biological activities, whereas the extract from cultures without elicitor was not bioactive to a discernible extent. The biological activities shown by the cell culture extracts were almost solely accounted for by-thuj aplicin contained in the extracts. These results suggest that the ethyl acetate extract ofC. lusitanica cells treated with the elicitor is a valuable bioactive source without isolation or purification of-thujaplicin.