The external morphological characterization ofTricholoma matsutake infection of hostPinus densiflora lateral roots

The external morphology ofTricholoma matsutake/Pinus densiflora mycorrhizal lateral roots, collected from a Shiro in Tottori Prefecture, western Japan, was described in detail. Four distinct stages within a progressive developmental continum were recognized and color illustrations of each stage were presented. This research was supported by a grant from the Bio-oriented Technology Research Advancement Institution (BRAIN).     

Isolation and partial characterization of a pectic polysaccharide from the fruit pulp of Spondias cytherea and its effect on peritoneal macrophage activation

The total carbohydrate content of the intact pulp of Spondias cytherea was 41%. Polysaccharides were obtained via hot aqueous extraction after defatting with organic solvents. The aqueous extract was treated with excess ethanol to form a precipitate, which was then solubilized in water. The material precipitated upon acidification when HCl was removed. The resulting supernatant fraction was submitted to freeze-thawing treatment yielding a soluble fraction (sFTS). This fraction had Ara, Rha, Gal and GalA in its structure as determined by GC-MS. 13C NMR analysis showed signals assigned to alpha-L-Araf, beta-D-Galp, alpha-D-GalpA and alpha-L-Rhap units, in addition to galacturonic acid units, which were present also as methyl ester. These results suggest a type I rhamnogalacturonan with arabinogalactan branches. Cell eliciting activity in a dose-depending pattern was observed in vitro on peritoneal macrophages treated with sFTS.     

Detectionin situ of endophyte withinTricholoma matsutake/Pinus densiflora mycorrhizal roots

Tricholoma matsutake/Pinus densiflora mycorrhizas andP. densiflora roots were collected from beneath matureT. matsutake fruit-bodies in a Shiro in central Japan and investigated for evidence of endophytic infection.Tricholoma matsutake infection was determined microscopically on cleared, bleached and stained mycorrhizas by the presence of both a thin, discontinuous mantle and highly branched, sparingly septate Hartig net mycelium within the root cortex. Endophytic infection of Matsutake mycorrhizas was characterized by intracortical sclerotia, simple septate mycelium within the cortex and vascular cylinder and intracellular spore masses within the root cortex. This research was supported by a grant from the Bio-oriented Technology Research Advancement Institution (BRAIN).     

Short-term effects of fire on soil properties inPinus densiflora stands

This study was performed to investigate a short time change (one week after fire) on soil properties due to the fire inPinus densiflora Sieb. et Zucc stands of the Kosung area in Kangwon Province in Korea. Twenty seven sampling plots [16 burned (8 low intensity fire, 8 high intensity fire) and 11 unburned plots] were chosen. Mineral soil samples from three depths (0–5, 5–15, and 15–25 cm) under the forest floor were collected. Forest fire in the area affected soil chemical properties. Soil pH, total nitrogen, available phosphorus, potassium, calcium, and magnesium in the surface soil (0–5 cm) of the burned area compared with the unburned area increased, but there was no marked change in the subsurface soil (5–25 cm). Organic matter, total nitrogen, available phosphorus, and exchangeable cations in the surface soil were generally lower in the high than in the low intensity fire areas. This indicates that these nutrients on the high intensity fire may be volatilized. The results suggest that change in soil chemical properties in the area was restricted mainly to the surface soil and was different between the high and the low intensity fire types.     

Influence of osmotic pressure on somatic embryo maturation in Pinus densiflora

The effect of an osmoticum, polyethylene glycol (PEG), on somatic embryo production was examined using embryogenic cells of Pinus densiflora. In the basal medium containing 30 μM abscisic acid and 6% maltose, the quality of the embryos formed was poor even though somatic embryos were produced. The addition of PEG with molecular weight of 4000 or 8000 significantly enhanced the development of both the quality and quantity of somatic embryos. Furthermore, higher levels of a constant osmotic pressure with PEG 8000 in a range from about 300 to 450 mmol/kg could remarkably enhance the morphogenesis of somatic embryos and their number of embryos produced. A higher stable osmotic pressure with an appropriate molecular weight of PEG is a key factor for the production of good quality somatic embryos in P. densiflora.     

Morphological changes in the cytoskeleton, nuclei, and vacuoles during cell death of short-lived ray tracheids in the conifer Pinus densiflora

Morphological changes in the cytoskeleton, nuclei, and vacuoles were monitored during the cell death of short-lived ray tracheids in the conifer Pinus densiflora. After formation of the dentate thickenings that occurred at the final stage of formation of cell walls, organelles started to disappear in differentiating ray tracheids. First, the microtubules and vacuoles disappeared. Then actin filaments disappeared in the differentiating ray tracheids adjacent to ray tracheids that lacked nuclei, and, finally, the nuclei disappeared. These features indicate that cell death in ray tracheids might differ from the programmed cell death of tracheary elements that has been studied in vitro in the Zinnia culture system. This study was presented at the 57th Annual Meeting of the Japan Wood Research Society, August 8–10, 2007, Hiroshima, Japan     

Promotion of lateral female strobili production inPinus densiflora by cytokinin application at a specific stage

BAP (N ⁶-benzylaminopurine) was sprayed 5, 10 and 15 times at four different periods to promote flowering of a grafted Japanese red pine (Pinus densiflora) clone. Four stages were I, August 4 to September 1; II, September 6 to October 4; III, October 5 to November 14 and IV, November 30 to December 28, in 1994. Concentration of BAP was 250 mg/l. In May, 1995, no apical female strobili were observed at the top of the new shoots in any treatment. However, BAP application at stage III (floral differentiation period) produced lateral female and bisexual strobili at the lower part of new shoots. The numbers of lateral female and bisexual strobili were 87 out of 147 to 234 out of 162 new shoots. Frequency of BAP application did not affect the numbers. Marked decrease in number of male strobili induced by BAP application at stages I and II was observed. In contrast, the number of new shoots was increased by the BAP application at the above stages. Treatment at stage III and IV did not affect significantly the number of male strobili and new shoots. These results indicate that BAP application at stage III specifically induces the development of lateral female and bisexual strobili in Japanese red pine.     

Lateral female strobili production in a Japanese red pine (Pinus densiflora Sieb. et Zucc.) clone by exogenous cytokinin application

Application ofN ⁶-benzylaminopurine (BAP) and gibberellin (GA₃) to a grafted Japanese red pine (Pinus densiflora Sieb. et Zucc.) clone promoted flowering. BAP (250 mg/l) was sprayed five times to the top of branches in July or September 1992. GA₃ (100 or 500 mg/l) was applied five times to the whole plants by spray treatment during the above period, or GA₃ solution of the same concentration was injected once into a small incision made in the main stem of clone trees at the beginning of July or the middle of September 1992. In May 1993, no apical female strobili were observed at the top of the new shoots in any treatment. However, lateral female and bisexual strobili were produced in the lower part of new shoots by BAP application in September, regardless of GA₃ application. BAP applied in September without GA₃ application was sufficient to produce lateral female and bisexual strobili. In such treatment, production of female and bisexual strobili was 47 and 25, and they occurred in 16 out of 68 new shoots, 11 out of 15 treated branches and 5 out of 5 treated trees. The average number of female and bisexual strobili per new shoot was 1.06. BAP treatment either in July or in September significantly decreased (p<0.05) the production of male strobili. These results indicate that BAP application induces the development of lateral female strobili in Japanese red pine when applied during the period of floral differentiation. A part of this paper was presented at the 105th annual meeting of the Japanese Forestry Society.     

Survival ofPinus densiflora andP. thunbergii forests in Ibaraki Prefecture severely damaged byBursaphelenchus xylophilus

The total area ofPinus densiflora andP. thunbergii forests in Ibaraki Prefecture in 1978 was 65,200 ha, which decreased to 30,300 ha by 1985 mainly due to pine wilt mortality caused byBursaphelenchus xylophilus. This damage has also continued thereafter. To estimate the survivability of pine trees in Ibaraki Prefecture, pine tree mortality has been studied in eight experimental forests for over 20 years, and ground surveys throughout the Prefecture were also conducted in 1995. Survival in the experimental forests corresponded well to the results of ground surveys. Pine forests remained as pure stands if control measures were undertaken or if they were located in cool areas. In warm areas where no control was undertaken, most of the pine forests disappeared and only a few pine trees remained in mixed forests, while on dry soils no mature or old pine trees survived. Since surviving pine forests are often cut for wood utilization,P. densiflora andP. thunbergii may decrease in area to become rare species in the future unless controls are applied and/or reforestation with resistant pines is carried out. A part of this paper was orally presented at the 108th Annual Meeting of the Japanese Forestry Society (1997).     

The role of litter and humus layer as a mortality factor of seedlings of Japanese red pine,Pinus densiflora Sieb. et Zucc.

The process of decrease in seedlings of Japanese red pine (Pinus densiflora Sieb.et Zucc.) and the effect of litter and humus layers on the mortality were studied in their stand and adjacent bare land. After the rainy season, only small number of individuals died in the bare land but more seedlings died in the forest. The number of seedlings killed by drought was greater in the forest than in the bare land. The population of naturally occurring current year seedlings was larger in the forest than in the bare land, but that of 1- to 5-year-old seedlings was smaller. Severe summer drought exceeding the permanent wilting point was observed only in the forest F-H horizon where greater part of seedling roots distributed, this may be one of major mortality factors. Inhibitory substances to seedling growth in fresh fallen pine needles were also suggested.     

A new C-methylated flavonoid glycoside from Pinus densiflora

A new C-methyl flavonol glycoside, 5,7,8,4'-tetrahydroxy-3-methoxy-6-methylflavone 8-O-beta-D-glucopyranoside (1), has been isolated from the needles of Pinus densiflora, together with kaempferol 3-O-beta-(6"-acetyl)-galactopyranoside.     

Only two weeks are required forTricholoma matsutake to differentiate ectomycorrhizal Hartig net structures in roots ofPinus densiflora seedlings cultivated on artificial substrate

There has been conflicting debate over many years regarding the trophic status ofTricholoma matsutake (Ito et Imai) Sing., and further investigations are necessary to better understandT. matsutake physiology, particularly carbon nutrition, during ectomycorrhizal symbiosis. For this purpose, we developed a technique to rapidly synthesizein vitro ectomycorrhizas betweenPinus densiflora Sieb. et Zucc. andT. matsutake on artificial substrate (vermiculite: perlite: peat: beech sawdust; 5:5:1:1.), without added sugar in the nutrient solution. Only 1 week was required before the first rudimentary Hartig net ‘palmetti’ could be observed in roots. Well-developed Hartig net structures appeared in taproots after 2 weeks and in lateral roots after 3 weeks. Such rapid root infection may be attributed to the quality of the substrate and the inoculum used. This research was supported by a grant from the Bio-oriented Technology Research Advancement Institution (BRAIN).     

Comparison of organic matter dynamics in soil between Japanese cedar (Cryptomeria japonica) forest and adjacent Japanese red pine (Pinus densiflora) forest established on flatland

In order to clarify the effects of tree species on organic matter dynamics in soil, we investigated the amount of forest floor material, leaf litter decomposition rate, soil chemical characteristics, soil respiration rate and cellulose decomposition rate in a Japanese cedar forest (cedar plot) and an adjacent Japanese red pine forest (pine plot) established on a flatland. The amount of forest floor material in the cedar plot was 34.5 Mg ha⁻¹ which was greater than that in the pine plot. Because the leaf litter decomposition rate was higher in the pine plot than in the cedar plot, it is likely that the difference in the amount of forest floor material between the plots is caused by the difference in the leaf litter decomposition rate. The C concentrations of soil in the cedar plot were 1.2–2.1 times higher than those in the pine plot. Soil pH(H₂O)s in the cedar plot were significantly higher than those in the pine plot. The soil respiration rates and the rates of mineralized C in the cedar plot byin vitro incubation were higher than those in the pine plot. From this result, it is assumed that soil organic matter in the cedar plot was decomposed relatively faster compared with the pine plot. Furthermore, microbial activities, which were reflected as cellulose decomposition rates in the cedar plot, were higher than those in the pine plot. A part of this paper was presented at the 109th Annual Meeting of the Japanese Forestry Society (1998).     

A comparison of needle retention on Japanese black pine and Japanese red pine

Needle retention (number of needle sets), needle density, height increment and radial increment were surveyed on seven Japanese black pines (Pinus thunbergii Parl.) and seven Japanese red pines (Pinus densiflora Sieb. et Zucc.) growing on the same site. Number of needle sets on branches with respect to whorl position was estimated visually. In 1999, maximum summer needle retention was observed on the fifth and sixth whorl from the top, with values of 3.4 needle sets for the black pines and 2.4 needle sets for the red pines, respectively. The needle trace method (NTM) was used to determine needle retention and needle density along the main stems retrospectively for the years 1968–1998. The long-term mean summer needle retention along the main stem was 3.7 needle sets for the black pines and 2.2 needle sets for the red pines. In both pine species, the number of needle sets varied from year to year. However, the long-term budget between newly born and annually shed needle sets was in equilibrium. The long-term average of needle density per cm of stem shoot was 9.4 needle pairs for the black pines and 7.4 needle pairs for the red pines. The results showed clear intra-specific and inter-specific similarities in needle retention, height increment and radial increment trends. The values for number of needle sets, height increment and radial increment positively increased with favorable growing conditions, whereas the value of needle density had an opposite tendency.     

Bark thickness of host pine trees and female body size affecting ovipositional success ofSirex nitobei matsumura (hymenoptera: Siricidae)

We examined the effect of bark thickness of host trees on the ovipositional success ofSirex nitobei in relation to female body size by analyzing the relationships between female body-weight and ovipositor length and between bark thickness ofP. densiflora andP. thunbergii trees and oviposition proportion (the proportion of eggs laid to the potential female lifetime fecundity) of the woodwasp. The ovipositor length ranged from 6 to 14 mm and was correlated positively with the fresh body-weight of females. The bark thickness of host trees had no significant correlation with the oviposition proportion of large females of more than 100 mg f.wt, whereas it was correlated negatively with that of small females of less than 100 mg f.wt. Thus,P. densiflora, which has thinner bark as a whole thanP. thunbergii, may be more suitable for oviposition, especially for smaller females. Research Fellow of the Japan Society for the Promotion of Science. This study was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture (No. 80003292).     

Physiological process of the symptom development and resistance mechanism in pine wilt disease

Pathophysiological changes during the symptom development of pine wilt disease are reconsidered from recent investigations. The symptom development is divided into two stages: the early and the advanced stages. In the early stage, small number of nematodes migrate in cortex, then in xylem of the stem, and induce denaturation and necrosis of parenchyma cells. These changes in parenchyma are regarded as defense reactions of pines which result in terpene synthesis in xylem cells and embolism in tracheids. Such changes in the early stage can be induced in both susceptible and resistant pine species by either virulent or avirulent isolates of pinewood nematode (Bursaphelenchus xylophilus), or byB. mucronatus. No change occur in physiological status of leaves, and nematode reproduction is suppressed during this stage. Pine trees can survive if symptom does not progress from this stage. The symptoms of the advanced stage usually occur only in susceptible pines infected by virulent nematode isolates. At the beginning of the advanced stage, enhanced ethylene production by stem which coincides with cambial destruction occurs, and results in embolism of the outermost xylem in the portion. The embolism causes decrease in leaf water potential and cessation of photosynthesis. After cessation of photosynthesis, symptoms develop drastically with a burst of nematode population. There seems to be some unknown mechanism which suppress nematode reproduction and invasion to the cambial zone. This mechanism is thought to be photosynthesis-dependent, so that in photosynthesis-decrased conditions, even avirulent nematodes can multiply and invade cambium to induce tree death. Water stress in hot and dry summer should accelerates symptom development from the early to the advanced stage through such decrease of photosynthesis-dependent “cambial resistance”.     

Effect of electric impulse on sporocarp formation of ectomycorrhizal fungusLaccaria laccata in Japanese red pine plantation

We examined the effect of electric impulses on sporocarp formations of ectomycorrhizal fungus in a Japanese red pine (Pinus densiflora) plantation.Laccaria laccata was the dominant species in this study plantation. Spatial distribution concentrated in the electric impulse treatment zone, and recorded a markedly larger number of sporocarps compared with untreated control plots. The effect of electric impulses was also recognized onL. laccata fruiting with a mycorrhizal sapling in a pot. Sporocarp production ofL. laccata was strongly promoted by the electric impulse in experiments bothin vivo natural plantation stands andin vitro pot saplings. Our work indicates the possibility of enhancement of sporocarp production of the mycorrhizal fungi with electric impulses in the nursery and plantation. This work was partly supported by a grant from the Kyushu Electric Power Company.     

Oviposition and larval performance of Monochamus alternatus (Coleoptera: Cerambycidae) on the Japanese cedar Cryptomeria japonica

To determine whether Monochamus alternatus can use the Japanese cedar (Cryptomeria japonica) as a host tree, experiments were conducted in a laboratory using three insect populations of different localities. Adult females chose Pinus densiflora bolts as oviposition substrate when supplied with cedar and pine bolts simultaneously, whereas some females from one locality oviposited on cedar bolts when supplied with them exclusively. Seventy-three percent of 40 eggs hatched in cedar bolts. When the first instar larvae were inoculated on cedar bolts, the development was stunted greatly and all died during the larval stage. Two of 20 larvae that were inoculated on cedar bolts at the third instar entered the diapause and one larva developed into an adult female, which produced viable eggs but was much smaller than those obtained from pine bolts. The results did not exclude the possibility that M. alternatus can use recently killed C. japonica trees as a host.     

Community-based management of rural pine forests in a suburban village of Hiroshima prefecture, western Japan

Community-based management of a rural pine forest in a small suburban community, was examined and assessed. In particular, the study focused on theMatsutake project, which is an initiative seeking to help in the maintenance of a communal pine forest via the cultivation of theMatsutake mushroom by a local seniors’ group. From an ecological perspective, the maintenance work is found to be effective in the conservation and regeneration of the pine forest ecosystem, including its species diversity, especially in the herb layer. From a sociological perspective, theMatsutake project presented a valuable opportunity to strengthen connections not only within the seniors’ group, but also between senior citizens and other generations. However, subsidies for the project have tended to be gradually reduced, because it is difficult for non-involved community members to appreciate the benefits of the project. For the project to be sustainable, more widespread participation of the community is essential. A framework for wider analysis of local participatory forest management is also deemed necessary.     

Statistical interferometric investigation of nano-scale root growth: effects of short-term ozone exposure on ectomycorrhizal pine (<Emphasis Type="Italic">Pinus densiflora</Emphasis>) seedlings

This study presents the effects of short-term ozone exposure on the nano-scale growth behavior of the fine roots of Pinus densiflora (Japanese red pine) seedlings. Root elongation measurements were obtained in nanometers for very short (sub-second) time intervals by using the optical interference method called statistical interferometry, developed by the authors. Three categories of P. densiflora seedlings were investigated; two categories were infected with ectomycorrhiza of Pisolithus sp. (Ps) and Cenococcum geophilum (Cg), while the third was without any fungal infection. In experiments, two points on a root with a separation of 3 mm were illuminated by laser beams and the elongation was measured continuously by analyzing speckle patterns successively taken by a CCD camera. The ectomycorrhizal fungi-infected and uninfected seedlings were exposed to ozone at concentrations of 120 and 240 ppb for periods of 1, 3, or 5 h in separate treatments. The root elongations of P. densiflora seedlings were measured before and immediately after the each ozone treatment and then the root elongation rates (RER) were determined for growth-measurement periods of 5.5 s and 9.5 min. From the measurements obtained for 9.5 min, we found that the RERs of uninfected and Cg-infected seedlings were reduced by 42 and 18%, respectively, after 5 h of exposure to 120 ppb ozone compared with that before exposure, while the reduction in RER of Ps-infected seedlings was not significant. When the concentration of ozone was increased to 240 ppb, the RERs of Ps-infected and Cg-infected seedlings were reduced by 32 and 44%, respectively, after exposure for 5 h, while the reduction in RER of uninfected seedlings was 59%. These observations prove that the non-mycorrhizal seedling roots are more sensitive to ozone stress. From this study, we found that the RERs of both mycorrhizal and non-mycorrhizal seedlings apparently fluctuated throughout the measurements, even within a few minutes.