Efficient plant regeneration of Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis

This report describes the efficient plant regeneration of Chamaecyparis obtusa Sieb et Zucc. via somatic embryogenesis. Embryogenic cultures were initiated from megagametophytes containing immature zygotic embryos. Embryogenic cultures were maintained and proliferated by 2–3-week interval subcultures in medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. High maturation frequencies of cotyledonary embryos were obtained on maturation medium containing maltose, polyethylene glycol, activated charcoal, and abscisic acid. Somatic embryos germinated readily after transfer to plant growth regulator-free medium. Growth of regenerated emblings has been monitored in a greenhouse.     

Resinous stem canker development during the growing season of Chamaecyparis obtusa (Hinoki cypress) inoculated with pathogenic fungus Cistella japonica

Seasonal development of resinous stem canker was determined in Chamaecyparis obtusa trees wound‐inoculated with Cistella japonica in January 1996. Samples for anatomical analysis were harvested on 20 May, 9 July and 6 August in the second year of inoculation, and the sections were observed under light microscope. Resin exudation was most abundant in the second year, decreasing in the third. All new resin exudation in the second year was recognized at the May observation. New traumatic resin canal formation was observed in the August samples, and an expansion of necrotic lesions was observed mainly in summer samples in the inoculated trees and in naturally affected trees. Cistella japonica was re‐isolated from all bark lesions or inoculated wounds examined. These results suggest that the activity of Ci. japonica in the tree affects the seasonal development of resinous stem canker during the growing season of the trees.     

Soil and the foliage nutrient status following soil amendment applications in a Japanese cypress (Chamaecyparis obtusa Endlicher) plantation

The aim of this study was to evaluate the response of soil amendment applications on soil and the foliage nutrient status of a Japanese cypress (Chamaecyparis obtusa Endlicher) plantation established following clear-cutting in a pine-wilt-disease (PWD)-disturbed forest. We established four soil amendment treatments [(compound fertilizer (CF), compound fertilizer + biochar (CFB), compound fertilizer + sawdust (CFS) and a non-treated control treatment] in an 8-year-old Japanese cypress plantation. Soil organic carbon (C) and total nitrogen (N) were not significantly different (P > 0.05) between the soil amendment treatments and the control treatments, whereas extractable phosphorus (P), NH₄⁺, K⁺, and Mg²⁺ concentrations were significantly affected by the addition of biochar in CF. The mean soil CO₂ efflux rates during the study period were the highest in CFB (0.79 g CO₂ m^?2 h^?1), followed by CFS (0.71 g CO₂ m^?2 h^?1), CF (0.62 g CO₂ m^?2 h^?1), and the control (0.46 g CO₂ m^?2 h^?1) treatments. Foliar N and P concentrations were significantly higher in the CFB than in the control treatments. The results suggest that the addition of biochar in CF can enhance extractable soil nutrients and foliar N and P conditions of Japanese cypress established in a PWD-disturbed forest.     

Mitigation of aluminum toxicity by calcium and magnesium in Japanese cedar (Cryptomeria japonica)

We investigated the short-term and long-term mitigation of Al toxicity by Ca and Mg in pot trials of Japanese cedar (Cryptomeria japonica D. Don). We found that in the initial stages of treatment, Al toxicity at high Al concentration (5mM) was mitigated by Ca and Mg through the stimulation of antioxidant enzyme activities in needles. However, growth reduction occurred after 11 months exposure to Al despite the coexistence of Ca and Mg. Growth reduction was related to Al³⁺ activity in solution rather than the concentration of Ca and Mg. Therefore, when considering the influence of soil acidification on Al toxicity in forest ecosystems, it is important to consider not only the potential for mitigation of Al toxicity by base cations, but also the potential for factors in the soil solution to change the chemical form of Al.     

In vitro screening of angiotensin I-converting enzyme inhibitors from Japanese cedar (Cryptomeria japonica)

Screening and isolation of angiotensin I-converting enzyme (ACE) inhibitors from Japanese cedar (Cryptomeria japonica) based on the in vitro ACE inhibitory assay were attempted. The ethanol extract from outer bark showed the highest inhibitory activity (IC₅₀ is 16g/ml) among 24 extracts prepared from roots, leaves, heartwood, sapwood, inner bark, and outer bark by successive extraction with four solvents. The fractionation of the outer bark ethanol extract followed by the bioassay resulted in the isolation of two strong ACE inhibitors, catechin and dimeric procyanidin B3. The bioassay of three flavan-3-ols including (+)-catechin and six flavones revealed that most of these compounds have high ACE inhibitory activity. The results suggest that the phenolic hydroxyl group at the C7 position and heterocyclic oxygen atom of these compounds are important for expressing the inhibitory activity.     

Drought-dependent responses of photosynthesis,transpiration and water use efficiency of Japanese cypress and Japanese red pine seedlings

This study was conducted to investigate the potential for modifying drought tolerance of Japanese cypress (Chamaecyparis obtusa Endl.) and Japanese red pine (Pinus densiflora Sieb. et Zucc.). Three-year-old seedlings were controlled for five-months at three different soil water potentials ({ie73-1}). Japanese cypress exposed to high {ie73-2} was able to maintain higher photosynthesis (Ph_n), transpiration (Tr) and stomatal conductance to H₂O (gH₂O) in comparison to low {ie73-3} pretreatments, however, there was no significant difference in Ph_n for Japanese red pine. Soil water potential at the threshold from the maximum to limited Ph_n was higher in high {ie73-4} pretreatments than in low {ie73-5} pretreatments. Net photosynthesis, Tr and gH₂O decreased more rapidly in high {ie73-6} pretreatments than in low {ie73-7} pretreatments. Transpiration decreased more significantly than Ph_n, thus, resulted in increased water use efficiency. All these factors are likely to result in significant improvements in the drought tolerance. Japanese red pine seems more drought-tolerant than Japanese cypress. Japanese cypress is suitable to soil of −0.05 MPa water potential, and Japanese red pine is suitable to −0.16 MPa and even dryer soils.     

Estimation of the fine root biomass in a Japanese cedar (Cryptomeria japonica) plantation using minirhizotrons

We estimated fine root biomass in a Japanese cedar (Cryptomeria japonica) plantation using a min-irhizotron technique. Since data obtained from minirhizo-trons are limited to the length and diameter of fine roots observed on minirhizotron tubes, data conversion is necessary to determine the fine root biomass per unit soil volume or unit stand area. We first examined the regression between diameter squared and weight per unit length of fine roots in soil core samples, and calculated the fine root biomass on minirhizotron tubes from their length and diameter. Then we determined conversion factors based on the ratio of the fine root biomass in soil core samples to that on minirhizotron tubes. We examined calculation methods, using a single conversion factor for total fine root biomass in the soil for depths of 0–40cm (Cal1), or using four conversion factors for fine roots in the soil at 10-cm intervals (Cal2). Cal1 overestimated fine root biomass in the lower soil or underestimated that in the upper soil, while fine root biomass calculated using Cal2 better matched that in soil core samples. These results suggest that minirhizotron data should be converted separately for different soil depths to better estimate fine root biomass.     

Spatial distribution of soil carbon and nitrogen storage and forest productivity in a watershed planted to Japanese cedar (Cryptomeria japonica D. Don)

Digital terrain modeling was used to evaluate landscape-level spatial variation of soil C and N storage and site productivity in Japanese cedar (Cryptomeria japonica D. Don) stands. Soil C and N storage were measured in samples from surface soils (0–25 cm depth) of 29 Japanese cedar stands in the 205-ha Myougodani watershed, Toyama Prefecture. The site index (C. japonica tree height at age 40 years) was used as a measure of forest productivity. Seven terrain attributes (elevation, slope gradient, aspect, profile curvature, plan curvature, openness, and wetness index) were calculated from a digital elevation model. Soil C and N storage were negatively correlated with slope gradient and positively correlated with openness. Variation in the site index was closely related to the wetness index. The prediction models using terrain attributes as explanatory variables explained 50% of the variability in soil C storage, 53% of the variability in soil N storage, and 75% of the variability in site index. This result demonstrated that this technique is useful for estimating the spatial distribution of soil properties and productivity in forest landscapes. On the other hand, there was no correlation between site index and soil C and N storage. Use of the prediction models in a geographic information system revealed that the spatial distribution of forest productivity differed considerably from those of soil C and N storage.     

Responses of antioxidant enzymes in the needles of hinoki cypress (Chamaecyparis obtusa) seedlings to nutrient solutions containing various calcium/aluminum ratios

An attempt was made to examine the possible connection between the various ratios of calcium/aluminum (Ca/Al) in the nutrient solution of plant cultures and the active oxygen scavenging system of hinoki cypress (Chamaecyparis obtusa) seedlings. The hinoki cypress seedlings were transferred to nutrient solutions containing 5 mM AlCl₃ together with various concentrations of Ca(NO₃)₂ in pots containing glass beads and Teflon tips. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) in the needles were estimated at several stages during the 7-day treatment. The samples treated with the lower Ca/Al solutions show the highest SOD activities. The activities of APX and GR, both of them involved in the ascorbate-glutathione cycle, showed the same tendency with decrease to higher Ca/Al ratio. These results indicate that rhizospheric Ca might compete with Al and ameliorate Al toxicity on and in the roots, the Al stress is not transformed to the needles after a few days, and the ascorbate-glutathione cycle in the hinoki cypress needles might fluctuate and be suppressed by the rhizospheric Al stress during the 7 days. This work was supported in part by funding from the Japan Science & Technology Corporation, the CREST program 1996–2001, and the Center for Forest Decline Studies.     

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).     

Dependence of the aboveground respiration of hinoki cypress (Chamaecyparis obtusa) on tree size

Nighttime respiration was measured at monthly intervals over one year on the aboveground parts of five sample trees in an 8-year-old hinoki cypress (Chamaecyparis obtusa (Sieb. et Zucc.) Endl.) stand, by an enclosed standing-tree method. The respiration rate rose rapidly from early spring to a maximum in June, and decreased abruptly in July and then gradually toward autumn and winter. The seasonal change in the respiration rate was synchronized with stem volume increment rather than with monthly mean air temperature. The respiration rate, r, of individual trees increased with increasing tree dimensions, such as stem volume, v(S), and stem girth at the base of the live crown, G(B). The dependence of respiration rate on tree size was successfully represented by a power function. The r - v(S) dependence was rather stronger than the r - G(B) (2) dependence, especially toward the end of the growing season (from July to September). The observed respiration rate was almost the same as the respiration rate corrected for the monthly mean air temperature. The annual respiration of individual trees was directly proportional to their phytomass or to its increment. Although the annual respiration of individual trees decreased proportionally to the square root of the leaf mass, it decreased abruptly in the range close to the smallest sample tree. Combining the monthly relationship between respiration rate and stem volume with the tree size distribution in the stand, the stand aboveground annual respiration was estimated to be 20.4 Mg CO(2) ha(-1) year(-1) (= 12.5 Mg dry mass ha(-1) year(-1)) for an aboveground biomass of 17.4 Mg ha(-1) with an annual increment of 6.51 Mg ha(-1) year(-1), i.e., the stand aboveground annual respiration amounted to the equivalent of 72% of the biomass or to almost twice the biomass increment.     

Growth recovery of young hinoki (Chamaecyparis obtusa) subsequent to late weeding

The effects of late weeding treatment on the growth of young hinoki (Chamaecyparis obtusa) during the sixth growing season after planting were examined. Furthermore, the contribution toward hinoki growth recovery of crown productivity (NAR) and biomass allocation to crown (CAR) were determined. In the late weeding plot, no decline in height growth was observed subsequent to weeding, and growth in diameter at breast height (DBH) and crown projection area (CPA) began to recover subsequent to weeding; however, DBH and CPA were reduced and experienced a 1- to 1.5-year growth delay compared to values associated with the weeding plot at the end of the second year subsequent to weeding. Relative growth rate (RGR) and NAR in the late weeding plot recovered and possessed similar values to those of the weeding plot in the second year subsequent to weeding. CAR values of the late weeding plot were similar to those of the weeding plot both before and after weeding. These results suggested that the recovery of NAR rather than that of CAR was primarily responsible for the recovery of RGR. The present study demonstrated that hinoki were able to quickly acclimate to an environment dramatically altered by late weeding and recover growth rate within a short period of time. The present study also showed that delays in crown expansion associated with late weeding may have impeded subsequent matter production. Therefore, it was concluded that late weeding treatments should be employed only when the associated delays in growth are taken into account.     

Effect of alcohol-based organosolv treatment combined with short-time ball milling on the enzymatic hydrolysis of Japanese cypress (Chamaecyparis obtusa)

Japanese cypress (Chamaecyparis obtusa) is a recalcitrance softwood widely used as building material in Japan. A combined technique of alcohol-based organosolv treatment and ball milling (BM) of this softwood for the production of fermentable sugars from lignocellulose was investigated. Ethanol, ethylene glycol, water, and their mixtures were used as solvents, and acetic acid was used as a proton donor. After solvolysis, a residual sample was subjected to short-time BM, and the entire residue was evaluated by saccharification with Acremonium cellulase. Short-time BM after alcohol-based organosolv treatment greatly improved the enzymatic digestibility and decreased the required severity of organosolv treatment. Moreover, alcohol-based organosolv treatment increased the efficiency and reduced the time required for BM despite small quantity of removed lignin. It was found that the combination of alcohol-based organosolv treatment in mild condition and short-time BM had a synergistic effect on the enzymatic digestibility of Japanese cypress.     

Within- and between-site variations in leaf longevity in hinoki cypress (Chamaecyparis obtusa) plantations in southwestern Japan

We investigated mean leaf retention time in order to elucidate the factors affecting regional and local variations in stand-level leaf longevity in hinoki cypress (Chamaecyparis obtusa) plantations. Our study sites consisted of six stands at a low elevation (320–370 m a.s.l.) and 12 stands at a high elevation (850–970 m a.s.l.) in southwestern Japan. We also used published data on leaf longevity in stands at various elevations to clarify the regional-scale variations in leaf longevity and their relationships to environmental factors. At the regional scale, leaf longevity increased with increasing elevation and with decreasing air temperature, growing season length, and Kira’s warmth index across sites. Similar relationships were obtained for the variation in leaf mass. At the local scale, leaf longevity did not show a clear relationship with topographic position, soil water content, or soil C/N ratio. Contrary to our expectation, leaf longevity was negatively correlated with the leaf C/N ratio at both study sites, although the significance level was marginal. This indicates greater leaf longevity with better leaf nutrient status. Our results suggest that responses of leaf longevity in hinoki cypress stands to environmental factors would be more prominent at the regional scale than at the local scale, although large variation was detected at the local scale. Air temperature and growing season length appear to be the main drivers of the variation in leaf longevity at the regional scale, whereas the causal factors are unclear at the local scale.     

Maintenance and growth respiration of the aboveground parts of young field-grown hinoki cypress (Chamaecyparis obtusa)

Aboveground respiration of five 8-year-old trees of field-grown hinoki cypress (Chamaecyparis obtusa (Sieb. et Zucc.) Endl.) was nondestructively measured at monthly intervals over 1 year with an enclosed standing tree method. The relationship between monthly specific respiration rate and monthly mean relative growth rate at the individual tree level was described by a linear equation. During the dormant season, respiration was used mainly for maintenance purposes, whereas during the growing season, more than 40% of the respiration was used for growth purposes, i.e., 60 to 70% in May. We conclude that annual maintenance and growth respiration of a tree are directly proportional to the aboveground phytomass and its annual increment, respectively. The maintenance coefficient was estimated to be 0.504 +/- 0.039 (SE) kg kg(-1) year(-1), indicating that the amount respired for maintaining already existing phytomass was equivalent to about half of the existing phytomass. The growth coefficient was estimated to be 0.772 +/- 0.043 (SE) kg kg(-1), indicating that the amount respired for constructing new phytomass was equivalent to about three-fourths of the annual phytomass increment. The annual stand maintenance and growth respiration were, respectively, 8.8 Mg ha(-1) year(-1) for an aboveground biomass of 17.4 Mg ha(-1) and 5.0 Mg ha(-1) year(-1) for an annual stand aboveground biomass increment of 6.5 Mg ha(-1) year(-1). About two-thirds of the total respiration was used to maintain already existing biomass, and about one-third was used to construct new biomass.     

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.