Evaluation of Schizophyllum commune Fr. potential for biodegradation of lignin: A light microscopic analysis

Lignin biodegradation potential of Schizophyllum commune Fr. is studied by using sound wood blocks of Ailanthus excelsa, Azadirachta indica, Tectona grandis, Eucalyptus sp. and Leucaena leucocephala. Initially, in vitro wood decay test showed minor weight loss, but it became rapid after one month. After 120 days of incubation, weight loss was minimum in T. grandis (24.05%) whereas it was maximum in A. excelsa (34.44%). Treated test blocks were characterised by enlargement of pits on ray cell wall, formation of additional boreholes in rays, separation of fibres and cell wall thinning and formation of ‘U’-shape notches. Fungal hyphae moved through the xylem cell lumen, and intercellular spaces formed in response to separation of fibres. Hyphae traverse in adjacent cell through the cell wall pits or by making additional boreholes. In all the species studied, xylem fibres and parenchyma (axial and ray) cells were more susceptible while vessels were resistant to fungal attack. In advanced stage of decay, fibres and axial parenchyma lost their rigidity while vessel walls showed uneven thinning. In the tension wood, G-fibres remained unaffected initially but loosening and separation of gelatinous layer facilitated fungal action and showed similar pattern of cell wall deterioration. Among the wood of different species studied, Tectona was more resistant whereas Ailanthus was more susceptible to fungal attack.     

Radial dislocations in the fibre wall of Eucalyptus regnans trees of high growth stress

Summary Microscopical examination of the xylem of straight Eucalyptus regnans trees of high growth stress revealed the presence of radial dislocations in the secondary wall of many of the fibres. These features were characterized by a localized disruption in the microfibrillar orientation of the cellulose and were evident in both unlignified and lignified cell walls. Dislocations were not detected in trees having low peripheral growth stress. The origin of these features was suggested to lie in the expansion of the cell wall during the relaxation of longitudinal growth stresses following removal of the sample from the tree. A similar origin of the typically convoluted form of the gelatinous layer of tension wood fibres is discussed.The author expresses his appreciation to Dr. J. D. Boyd for helpful discussions.     

Temporally and spatially controlled death of parenchyma cells is involved in heartwood formation in pith regions of branches of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> var. <Emphasis Type="Italic">inermis</Emphasis>

Focussing on four types of parenchyma cell around pith regions of branches of Robinia pseudoacacia L. var. inermis, we examined the timing and role of cell death during heartwood formation. Large parenchyma cells that were located in the inner part of the pith died within a year. By contrast, other parenchyma cells died within 4 years, with the timing of cell death depending on the type of cell. Axial parenchyma cells of the xylem close to the pith died first. Then, small parenchyma cells died in the perimedullary zone in the outer part of the pith. Finally, ray parenchyma cells in the xylem close to the pith died. Variations in the autofluorescence of cell walls, which might have been due to deposition of heartwood substances, were observed first in xylem ray parenchyma cells and small parenchyma cells in the perimedullary zone. Our results indicate that the initiation of heartwood formation occurs within 4 years in pith regions of branches in Robinia pseudoacacia L. var. inermis. Moreover, it appears that not only xylem ray parenchyma cells but also small parenchyma cells in the perimedullary zone might be involved in the synthesis of heartwood substances.     

Distribution of lignin and lignin precursors in differentiating xylem of Japanese cypress and poplar

Lignin is an integral component of the cell wall of vascular plants. The mechanism of supply of lignin precursors from the cytosol into the cell wall of differentiating xylem has not yet been elucidated. The present study showed that a certain amount of coniferyl alcohol glucoside (coniferin) occurred in the differentiating xylem of Japanese cypress (Chamaecyparis obtusa), as previously reported in gymnosperms. Coniferin content peaked in the early stages of secondary wall formation and decreased during lignification. In contrast to gymnosperms, coniferin content was limited in the differentiating xylem of poplar (Populus sieboldii × Populus grandidentata). Moreover, coniferyl alcohol was not detected in all specimens. In the differentiating xylem of poplar, a higher amount of sinapyl alcohol occurred than glucoside (syringin). However, the phloem contained syringin and not sinapyl alcohol. The sinapyl alcohol content in the xylem peaked in the cells with ceasing cell wall formation, and decreased gradually towards the boundary of the annual ring, where the lignin content kept increasing. Sinapyl alcohol in the differentiating xylem of poplar may be used for the lignification of the xylem.     

Distribution of lignin interunit bonds in the differentiating xylem of compression and normal woods of Pinus thunbergii

The lignification process and lignin distribution at different stages of cell wall differentiation in the secondary xylem of compression and normal woods of Pinus thunbergii were investigated by thioacidolysis and subsequent desulfuration. We prepared 50-µm-thick, contiguous tangential sections of pine shoots, cut from the cambial zone through to mature xylem. In compression wood, uncondensed guaiacyl (G) and p-hydroxyphenyl (H) lignins were deposited simultaneously from early to late stages of lignification. The various types of G-G, G-H, and H-H dimers were detected in compression wood, and the ratio of G-H and H-H dimers to total dimers increased as lignification proceeded. In contrast, uncondensed and condensed H units were detected in trace amounts in normal wood. Significant differences in the relative distributions of lignin interunit linkages were not observed between compression and normal woods or between differentiating and mature xylems in either compression or normal woods.Part of this report was presented at the 10th International Symposium on Wood and Pulping Chemistry, Yokohama, June, 1999     

Lignin deposition during earlywood and latewood formation in Scots pine stems

Lignin deposition at consecutive secondary wall thickening stages of early and late xylem cells during annual ring wood formation in Scots pine (Pinus sylvestris L.) stems was studied. Lignin patterns, isolated by thioglycolic acid method, consisted of alcohol-soluble (LTGA-I) and alkali-soluble (LTGA-II) fractions. The sum of two fractions, being the total lignin content, gradually increased in the course of lignification. However, the increments of lignin amount at each development stage of early and late tracheids were different. The intensity of lignin deposition increased in the course of earlywood tracheid maturation and decreased toward the end of latewood cell differentiation. The deposition of two lignin fractions in each layer of forming wood also occurred oppositely. The increment of LTGA-I descended, whereas that of LTGA-II increased from the beginning to the end of early xylem lignification. In contrast, LTGA-I increment dropped, whereas LTGA-II rose during late xylem lignification. Gel permeation chromatography showed that the lignins, formed at the beginning of lignification, were more homogeneous and had higher molecular weight compared with the lignins at the end of cell differentiation. Besides, the content of cellulose, estimated as the residue after lignin isolation, and of cell wall substances, presented as cell wall cross-section areas, at consecutive maturation stages of early and late xylem cells have been found to be different. The data show that lignin deposition occurred in different conditions and with opposite dynamics during early and late xylem formation.     

Effects of applied gibberellins and uniconazole-P on gravitropism and xylem formation in horizontally positionedFraxinus mandshurica seedlings

The present study deals with the effects of gibberellins (GA₃ GA₄) and uniconazole-P, an inhibitor of gibberellin biosynthesis, on negative gravitropism and xylem formation in the stems of horizontally positioned, 2-year-oldFraxinus mandshurica Rupr. var.japonica Maxim. seedlings. Each growth regulator (100 g) dissolved in 5 l acetone (50%) was applied to the basal node of the current shoot on May 24, 1995. The same treatment was repeated five times weekly until June 28. Five seedlings were used for each treatment. The seedlings were positioned horizontally 24h after the first application on May 25. Within 5 weeks the horizontal stem of control and GA-treated seedlings exhibited negative gravitropism. In contrast, the application of uniconazole-P inhibited negative gravitropic stem bending. The application of GAs increased the number of gelatinous fibers having thickened cell walls on the upper side of stems. The uniconazole-P application decreased xylem cell formation but did not inhibit the formation of gelatinous fibers. These results indicate that not only the differentiation of gelatinous fibers but also xylem increment is important in the negative gravitropism of horizontally positionedF. mandshurica seedlings. These results also suggest that GAs may be involved in xylem cell :formation rather than the differentiation of gelatinous fibers in this species.Part of this research was reported at the 23rd annual meeting of the Plant Growth Regulation Society of America, Calgary, Canada, July 1996     

Immunolocalization of an anionic peroxidase in differentiating poplar xylem

Peroxidases are the major candidate enzymes involved in dehydrogenative polymerization of monolignols. Peroxidases have the signal sequence at their N-terminus and this suggests that they are transported to extracellular spaces or developing cell walls. In this study, we focused on an anionic peroxidase isozyme encoded by prxA3a, which seems to be related to lignification. To investigate the localization of peroxidase in differentiating xylem cells of poplar (Populus sieboldii × Populus grandidentata), anti-PRX3 antibody was raised against the anionic peroxidase. Western blotting and peroxidase activity inhibition assay showed specificity of the antibody. Labeling by anti-PRX3 antibody was localized in vessels and fibers during the secondary wall formation and was observed along the plasma membrane beside the microtubules. The labeling was not seen in the cell wall, where localization of peroxidases was expected during lignification. The peroxidase isozyme, which is suggested to be involved in monolignol polymerization, is localized on the plasma membrane and its localization might be regulated by microtubules.     

Formation and structure of reaction wood in Buxus microphylla var. insularis Nakai

Summary Anatomical differences in xylem between the upper and lower sides formed in the inclined stems of Buxus microphylla with different angular displacement from the vertical were examined microscopically. B. microphylla exhibited a pronounced growth promotion on the lower side of the inclined stems. Formation of tension wood (gelatinous fibers) was not observed. Xylem formed on the lower side showed some interesting features resembling the compression wood formed in gymnosperms. The reaction wood tracheids and vessels showed an excessive lignification in their secondary walls but lacked both helical cavities and an S₃ layer, features that were almost the same as those of primitive gymnosperms. These results indicate that B. microphylla has an ability to form compression wood, suggesting that in the genus Buxus a different mechanism in the conducting elements was developed in the phylogenetic evolution.The first author would like to express his sincere thanks to Dr. T. E. Timell, College of Environmental Science and Forestry, State University of New York, Syracuse, New York, for his invaluable suggestions in connection with this research     

Peridermium pini (Pers.) Lev. and the Resin-top Disease of Scots Pine: II. Lesion Anatomy

The stages in the formation of Peridermium pini lesions aredescribed. The hyphae of the parasite were septate, uninucleate,and intercellular, with unbranched haustoria penetrating allphloem cells and xylem parenchyma cells. Lesion extension occurredby apical growth of the hyphae along the outer edge of the functionalphloem. The hyphae stretched unbroken across the active cambium.Active centripetal and longitudinal growth of the mycelium occurredin the resin ducts of the xylem, the hyphae commonly reachingthe pith. Spermogonia were produced in late summer and fall,aecia appeared in spring. In lesion xylem, tracheids were shorter,rays taller, more numerous and wider, but ray cell height remainedunchanged. Fusiform rays increased in number and size and verticalresin ducts increased in number. Changes in xylem tissues didnot become evident until two to four years after invasion ofthe cambium by the mycelium. Invasion of the secondary phloemresulted in an increase in number and size of the longitudinalphloem parenchmya cells, while changes in phloem rays were similarto those in xylem rays.     

Liquid culture and transformation of hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

Hinoki cypress (Chamaecyparis obtusa) is one of the most important timber resource forest trees in Japan. Because seed production from a seed orchard of hinoki cypress is not constant every year, micropropagation from a limited amount of material is useful. Up to now, the conventional tissue culture method using solid medium has been used. Here a new method using liquid culture in tubes rotated vertically is described. Shoot primordium of hinoki cypress was inoculated in Campbell and Durzan’s (CD) liquid medium containing different cytokinins (6-benzylaminopurine (BAP), Zeatin, thidiazurone (TDZ)), and the container tubes were rotated vertically around the axis at 2 times / min. Culture room temperature was 25°C and light condition was 16 h photoperiod per day of fluorescent lamps. Zeatin at 1μM concentration was the best for maintaining the shoot primordium production and TDZ induced callus on the surface of the shoot primordia. After shoot primordium multiplication in the liquid culture, they were transplanted to agar medium for shoot elongation. A high concentration of agar (up to 16 g/L) or AVF (anti vitrification factor from Dr. Nairn, 1995) was effective to prevent vitrification of the shoots. Transformation of shoot primordium was done using particle bombardment with vectors containingβ-glucuronidase (GUS) gene or herbicide resistance gene (bar). Positive result for transient transformation was observed with the histo-chemical study for transformation with GUS. Integration of a useful herbicidebar gene into the shoot primordium culture system was also tried and stably transformed plants were obtained. This is the first report of stable transformation of Japanese conifer using practically useful gene. The generous supply of AVF-B from Dr. B.J. Nairn, Tasman Forestry, NZ is also appreciated.     

Anatomical and cellular responses of Pinus monticola stem tissues to invasion by Cronartium ribicola

White pine blister rust (Cronartium ribicola) causes extensive damage to white pines and their associated ecosystems across North America. The anatomical and cellular characteristics of C. ribicola colonization in Pinus monticola branch and stem tissues were studied as a basis for understanding host tree reactions that may be related to resistance. Samples examined showed typical fusiform swelling and some had produced aecia. The reaction of phloem and xylem tissues was compared with non-infected tissue using light and electron microscopy. Cortical parenchyma and phloem polyphenolic parenchyma cells underwent mitotic division, cell swelling, and ca sixfold greater accumulation of phenolic compounds in colonized vs. control stems. In the cortex and secondary phloem, haustoria were common in parenchymatous cells, and hyphae were abundant in the intercellular spaces, but cell death was rare, unless aecia had ruptured the stem cortex. Hyphae were also common in xylem rays, tracheids and between tracheids. Disease-induced changes in the cambial zone included development of cambium-derived xylem traumatic resin ducts. Results demonstrate that diverse host defence responses were activated in the bark of apparently susceptible trees, but lack of mechanical damage by C. ribicola to the phenol-containing host cells and the resin duct system allowed extensive colonization and development of aecia despite elicitation of these stem defences. Interactions between P. monticola and C. ribicola are discussed and compared with other conifer–fungus pathosystems.     

Role of periderm in resistance of Eucalyptus marginata roots against Phytophthora cinnamomi

Phytophthora cinnamomi Rands invaded phloem tissue preferentially in Eucalyptus marginata Sm. (Jarrah) roots of both primary and secondary structure. Rate of fungal growth in the “primary” roots was less than in roots with secondary thickening. In the main field study jarrah roots between 1.5–2.5 mm in diameter, were inoculated with P. cinnamomi at regular intervals, over 12 months and in most cases invasion of the phloem was halted within the first 8 weeks of infection. In the majority of roots which exhibited secondary structure, lesion shape reflected the pattern of periderm development. Some lesions were peripheral in the outer phloem as cylindrical periderms had formed close to the vascular cambium. Other lesions were limited to a sector of the roots; periderm had formed radially through the secondary phloem and a zone of suberization was often continuous through the cambial zone and xylem. Formation of periderm at the edge of lesions was a multi-step process with various changes occurring, including lignification and suberization of walls and accumulation of autofluorescent compounds in cell walls and vacuoles. The layers of suberized and lignified cells formed near lesion fronts, were necrophylactic periderms. Necrophylactic periderm formation generally preceded the establishment of periderm of the exophylactic type.     

The Delignification Pattern of Ailanthus excelsa Wood by Inonotus hispidus (Bull.: Fr.) P. Karst.

In vitro laboratory decay tests on Ailanthus excelsa Roxb. wood revealed that I. hispidus exhibits a combination of both white-rot and soft-rot patterns of wood decay. Early stages of wood decay showed dissolution of the middle lamella as well as defibration and localized delignification of fiber walls; vessels, axial, and ray parenchyma remained unaltered. Delignification commenced from the middle lamellae at the cell corners without any marked effect on the primary and secondary wall layers. In later stages of growth, the species produces typical soft-rot decay pattern by forming erosion channels through the S₂ layers of fiber walls, transverse bore holes in the cell walls, and erosion channels alongside/following the orientation of cellulose microfibrils. The rays showed signs of cell wall alterations only after the extensive damage to the fiber walls. After 120 days of incubation, the vessels also showed localized delignification, the erosion of pits, and separation from associated xylem elements. The extensive weight losses under natural and in vitro decayed wood as well as the very soft nature of severely degraded wood indicate that I. hispidus alters wood strength and stiffness.     

Determination of the distribution and reaction of polysaccharides in wood cell walls by the isotope tracer technique VIII: Selective radiolabeling of xylan in mature cell walls of magnolia (Magnolia kobus DC.) and visualization of its distribution by microautoradiography

To radiolabel xylan in mature cell walls selectively, magnolia (Magnolia kobus DC.) was administered withmyo-inositol-[2-³H] and allowed to metabolize for 1 month. The radiolabeled xylem tissue was then submitted to sulfuric acid hydrolysis and nitrobenzene oxidation. A large amount of radioactivity was found mainly in xylose, although slight activities were detected in glucose and in vanillin and syringaldehyde. The labeled tissue was submitted to a preparation of holocellulose followed by treatment with 24% potassium hydroxide (KOH). Radioactivity was distributed mainly in the KOH-soluble part of the holocellulose. These results indicate that most radioactivity was incorporated into xylan in the cell walls. The distribution of the incorporated radioactivity in the xylem tissue was visualized by microautoradiography. Radioactivities were distributed in the xylem more than 400 m from the cambium; and an inner layer of a secondary wall had formed at the labeled xylem. Consequently, selective radio-labeling of xylan was visualized in mature cell walls.Part of this report was presented at the 47th annual meeting of the Japan Wood Research Society, Kouchi, April 1997     

A study of lignification in loblolly pine tracheids by the SEM-EDXA technique

Summary The lignification process in different morphological regions of loblolly pine tracheids was studied by the SEM-EDXA technique. Prior to S₂ layer formation, lignification was initiated in the cell corner middle lamella and compound middle lamella regions. Subsequently a rapid lignin deposition was observed in both regions, whereas secondary wall lignification was a more gradual process and initiated when the middle lamella lignin concentration was approximately 50% of maximum. Within the secondary wall, the S₁ layer is lignified first. Then, lagging just behind cell wall formation, lignification of the S₂ layer is initiated adjacent to the S₁ layer and extends toward the lumen. Finally, the S₃ layer lignified. Upon completion of lignification, the cell walls had a higher concentration of lignin in both the S₁ and S₃ layers than in the S₂ layer.This Paper is an excerpt from the Ph.D. dissertation of Shiro Saka     

Heterogeneity in formation of lignin

Summary The formation of lignin in the cell wall of compression wood of Pinus thunbergii was examined by selective radio-labeling of specific structural units in the lignin and visualization of the label in the different morphological regions by microautoradiography. Deposition of lignin in the tracheid cell wall of compression wood occurred in the order: p-hydroxyphenyl, guaiacyl and syringyl lignin, which is the same order as observed in normal wood. However, the period of lignification in the compression wood was quite different from those of normal and opposite woods. The p-hydroxyphenyl units were deposited mainly in the early stage of cell wall formation in compound middle lamella in normal and opposite woods, while in compression wood, they were formed in both the compound middle lamella and the secondary wall. The most intensive lignification was observed during the formation of the S₂ layer, proceeding from the outer to inner S₂ layers for a long period in compression wood. In the normal or opposite woods, in contrast, the lignification became active after formation of S₃ had begun, then proceeded uniformly in the secondary wall and ended after a short period.A part of this report was originally presented at the 1989 International Symposium on Wood and Pulping Chemistry at Raleigh, NC, U.S.A.     

Resistance of Pinus contorta to the European race of Gremmeniella abietina

Pinus contorta seedlings, together with Pinus resinosa and Pinus banksiana seedlings, were planted adjacent to 25‐year‐old red pine trees infected by the European (EU) race of Gremmeniella abietina. Resistance to this race was assessed over 5 years. All P. resinosa seedlings were dead after that period while 65% of P. contorta and 86% of P. banksiana seedlings appeared resistant to the disease. The tip blight that occurred on P. contorta was slightly longer than that observed on P. banksiana. In microscopy, one, two, or even more suberized boundaries were seen to be initiated near the surface of the shoot at the base of healthy needles where they extended downward in the direction of the vascular cambium. Suberized boundaries occasionally crossed the xylem and joined together in the pith region to form continuous barriers around necrotic tissues. However, in most cases, these suberized barriers were not continuous across the shoot and compartmentalization was then completed by other barriers mainly constituted of parenchyma cells and xylem tracheids that accumulated phenolic compounds. Meristematic‐like cells were observed adjacent to the necrophylactic periderm. Tissue regeneration, restoration of cambial activities and formation of traumatic resin canals also seemed to be associated with the defence system of P. contorta against the EU race of G. abietina.     

Tracheid length in relation to seedling height in conifers

Summary Changes in tracheid length during the development of primary and secondary xylem are analysed in relation to the division and elongation of cambial initials, in first year seedlings ofPinus sylvestris andPicea sitchensis. During primary and early secondary growth tracheid length varies with leaf internode length, but this relationship soon becomes obscured during later secondary growth. Correlations between seedling height and tracheid length thus depend on variation in tracheid length with radial xylem increment before and after terminal bud production. Since the relation between shoot and tracheid length is a complex one, the clonseness of the correlation varies with environmental treatment, but overall, a doubling of shoot length increased tracheid length by about 10%.I am grateful to ProfessorS. D. Richardson andDr. G. K. Elliott for many helpful suggestions. This work was financed by a grant from the Natural Environment Research Council. Plants were grown from seed kindly supplied by the Forestry Commission Research Division (Scotland and North England).     

Xylem dysfunction in Yezo spruce (Picea jezoensis) after inoculation with the blue‐stain fungus Ceratocystis polonica

The blue‐stain fungus Ceratocystis polonica is pathogenic to Norway spruce (Picea abies) in Europe, as well as to Yezo spruce (Picea jezoensis) and Sachalin spruce (Picea glehnii) in Japan. The wilting mechanism in P. jezoensis saplings after inoculation with C. polonica was examined based on anatomical studies of the phloem and xylem of periodically harvested trees. In addition, the course of sap ascent in the trunks was traced by injection of acid fuchsin solution at harvest. As an initial external symptom, needle discolouration was observed. In dye conduction tests, xylem dysfunction in the xylem of inoculated trees became obvious. The dehydrated xylem area (dry zone) had extended more than 20 cm above the inoculation wounds, within 1 month after inoculation. When the sap flow to the branches had nearly stopped, the leaves began to discolour. Hyphae of C. polonica colonized the ray tissue around the inoculation wounds, but were absent at the front of the dry zones. Defence reactions occurred in ray parenchyma cells adjacent to the penetrating hyphae. It is suggested that secondary metabolites, which are formed by the ray cells and epithelial cells of resin canals, are involved in the obstruction of sap flow. Limited necrotic lesions of the phloem and cambium were not associated with foliar symptoms. It is proposed that the dry zone formation caused by C. polonica is the main mechanism leading to tree death.