Temporal changes in nitrogen acquisition of Japanese black pine （Pinus thunbergii） associated with black locust （Robinia pseudoacacia）

The alien woody legume, black locust(Robinia pseudoacacia), has invaded Japanese black pine(Pinus thunbergii) forests located in Japan's coastal plain and hill regions where gaps are formed in pine forests after nematode infestation. Nitrogen fixation by legumes accelerates N cycling in forest ecosystems. We studied temporal change in the annual tree-ring resolution N stable isotope composition(δ15N, a per mil deviation of δ15N/14 N ratio, relative to atmospheric N2δ15N=0‰) at two natural locations of Japanese black pine forest with black locust that differed in the time since black locust establishment(Shohnai in northeast and Kita-Kyushu in southwest Japan). Analyzed tree-rings covered the period from 1990/1992 to 2009. N acquisition by Japanese black pine from black locust N input to the soil was evidenced by temporal shifting of N stable isotope composition on the annual pine tree rings. With progressive development of the forest stand, δ15N values of earlier tree-rings δ15N of-5‰) from black pine associated with black locust shifted towards values similar to those of black locust δ15N values nearly to-1‰), which suggests acquisition of N by N2 fixation(Shohnai site). In contrast, in a forest where black locust had settled for two or three generations, in a black pine stand(Kita-Kyushu site), longer periods of N enrichment in the soil were reflected in the elevated tree-ring δ15N values of newly established black pine trees. Based on tree-ring δ15N data from the Shohnai site, we determined that about 10 years after black locust establishment, soil N had already been enriched by black locust N, this, in turn, contributed to N fertilization of surrounding trees in mixed stands.     

Wind profiles in a coastal forest of Japanese black pine (Pinus thunbergii Parl.) with different thinning intensities

Simultaneous measurements of windspeed were made outside and inside a pine (Pinus thunbergii Parl.) coastal forest with different thinning intensities. Synchronously, optical stratification porosity (OSP), which is defined as vertical distribution of the proportion of sky hemisphere not obscured by tree elements inside a forest stand, was estimated using hemispherical silhouettes in each treatment area. Based on the observations, the frequency distribution of velocity, turbulence intensity, wind profile, and the corresponding relationships of these respective parameters with the vertical forest structure (OSP) were examined and compared among the treatment areas. A normal distribution of wind velocity was observed at the lower zone of the forest stand in all treatment areas. The turbulence intensity, the highest value of which was found near the canopy, changed greatly with height, wind velocity, and treatment, and decreased with windspeed and increased with the thinning intensity. It was found that the exponential relationship between windspeed and height could be used to describe the wind profiles within the canopy of the coastal forest. The results demonstrate that the attenuation coefficient of the wind profile corresponds to the grade of thinning intensities. The wind patterns in a coastal forest with different thinning intensities are related to the vertical forest structure, particularly, wind profiles within the canopy are closely correlated with the distribution of OSP. The results indicate that wind profiles can be estimated simply based on the measurement of OSP with a very high coefficient of determination. Reciprocally, the estimation of OSP can also be obtained from the measurement of wind profile. Portions of this paper were presented at the 111st Annual Meeting of the Japanese Forestry Society. Appreciation is due to Professor Masashi Yamamoto, Mr. Kenji Sakioka, Mr. Takahiro Yoshida, Mr. Hirotaka Yamazaki, Mr. Yasunori Hasegawa and the members of the Sabo Division of the Faculty of Agriculture of Niigata University for their help in collecting the field data and in constructing the observation towers.     

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.     

Retardation of pine wilt disease symptom development in Japanese black pine seedlings exposed to simulated acid rain and inoculated withBursaphelenchus xylophilus

Three- or 4-year-old Japanese black pine seedlings were exposed to simulated acid rain (SAR) at pH 3 for two months, then inoculated with a virulent isolate (S 10) of pinewood nematodes. The experiments were repeated three times in 1996–1998. The exposure to SAR killed no seedlings, and retarded the development of disease symptoms in the seedlings inoculated with nematodes. In the experiments in 1996 and 1997, however, cessation of resin exudation, and primary decrease in the xylem conductivity occurred earlier in the seedlings exposed to SAR than in those exposed to tap water as a control. These results imply that acid rain at pH 3 influenced an increase in some resistance of Japanese black pine seedlings to pinewood nematode, and that this improved resistance could potentially overcome damage caused by acid rain. The exposure to SAR did not have any significant effect on the water relations of the seedlings itself, suggesting that retardation of the symptom development after the exposure to SAR could not be attributed to the improvement in the water relations. This work was supported in part by a Grants-in-Aid for Scientific Research from the Japan Forest Technical Association (JAFTA).     

日本黑松和赤松精英树半同胞家系生长及遗传参数分析

通过对泰山竹林寺景区引种27年生的日本黑松和赤松精英树半同胞家系生长特性和遗传参数的研究发现：（1）黑松生长量略优于赤松,它们的平均胸径分别为21．83cm和20．84cm,平均材积分别为0．264m^3和0．262m^3。从遗传特性上来看,黑松和赤松的胸径的家系h^2分别为0．7668和0．4404。（2）从家系间比较可知：黑松大岛6^#、黑松柳井3^#、当地黑松、黑松柳井2^#生长指标居于前4位,材积分别为0．4725m^3、0．3526m^3、0．3179m^3和0．3144m^3。赤松都浓3^#、赤松都浓2^#、赤松大岛1^#居于前列,材积分别为0．429m^3、0．343m^3和0．365m^3。     

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).     

Somatic Embryogenesis and Plantlet Regeneration in Callus Cultures Derived from Mature Zygotic Embryos of Slash Pine

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Biomass distribution in 40-year-old trees of Japanese black pine

Measurements were carried out to survey the quantity of above- and below-ground biomass and its distribution of five Japanese black pines (Pinus thunbergii Parl.) growing on a sandy soil. The roots, divided into diameter groups, were surveyed using two methods—soil coring and excavation. Average dry weight of total biomass of the trees was 176,185 g. Roots represented 13.2%, below-ground stump 6.5%, stem 70.4% and branches with needles 9.9% of total biomass. Roots made up about two thirds and stump one third of below-ground biomass. Total length of below-ground biomass (except roots with diameter < 0.1 cm) was 479.1 m/tree. Roots with diameter of 0.1–0.2 cm represented only 0.7% of below-ground biomass, however as much as 49.9% of their total length. Roots with diameter over 10.0 cm constituted as much as 21.6% of below-ground biomass, however were only 0.3% of its total length. Root systems had well developed tap roots to maximal depth of 231 cm. The results indicated that mass and length of roots with diameter 0.5–2.0 cm had a close correlation with branch mass. Mass and length of roots with diameter 2.0–10.0 cm closely correlated to stem mass. Stem mass, root mass and root length closely correlated to DBH. A rather low correlation was found between DBH and mass of branches and below-ground stump. DBH was a suitable variable for predicting total biomass.     

Somatic embryogenesis and plant regeneration inAcanthopanax sciadophylloides

Somatic embryos ofAcanthopanax sciadophylloides Franch. et Sav. were differentiated from both zygotic and somatic embryos and calli, and plants were regenerated from these somatic embryos. A zygotic embryo, enclosed within a small portion of the endosperm, was incubated on Murashige and Skoog (MS) media supplemented with various combinations (range 0–10.0 mg/l) of 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D). After 4 months, swelling of the zygotic embryos and callus formation was observed. When the swollen embryos were transferred to MS medium supplemented with 0.5 mg/l of 2,4-D, somatic embryos were formed in one to two months. After subculture on the same medium, new embryos were differentiated from various parts of the older somatic embryos. The calli were cultured on medium supplemented with 2.0 mg/l of 2,4-D and BAP for three weeks. Proliferated calli were transferred to medium supplemented with 1.0 mg/l of 2,4-D and BAP. Somatic embryos were differentiated from the calli within one to two months. Somatic embryos were germinated on half-strength MS medium without plant growth regulators and the plantlets were grown in soil. A part of this paper was presented at the 106th Annual Meeting of the Japanese Forestry Society (1995) & First Asia-Pacific Symposium on Forest Tree Genetic Improvement (Beijing).     

Promotion of the population growth of pinewood nematode in 4-month-old Japanese black pine seedlings by pretreatment with simulated acid rain

Simulated acid rain (SAR) at three pH levels (pH 4, 3, 2) was applied to only the top or both the top and roots of 4-month-old Japanese black pine seedlings repeatedly for two months. Then the seedlings were inoculated with a virulent isolate (S10) of pinewood nematode,Bursaphelenchus xylophilus. The exposure to SAR at any level did not kill the seedlings. When inoculated with nematodes, however, the nematode population build-up was significantly higher in the seedlings pretreated with SAR at any level than in the control at the 7th and 17th day after inoculation, and subsequent disease symptom development was also significantly accelerated by the exposure to SAR at pH 2 and 3. This result indicates that even acid rain at pH 4 has the potential for promoting population growth of pinewood nematodes in 4-month-old Japanese black pine seedlings.     

Ectomycorrhizal development in a Pinus thunbergii stand in relation to location on a slope and effect on tree mortality from pine wilt disease

The relationship between ectomycorrhizal development and mortality from pine wilt disease was studied in an artificial Pinus thunbergii Parl. stand on a slope. The development of ectomycorrhizae and the survival of the trees showed the same tendency, which suggests a correlation between mycorrhizal development and resistance to pine wilt disease. The development of pine roots and mycorrhizae was greater in the upper part of the slope. The ratio of mycorrhizae to the total of mycorrhizae and fine taproots was also higher in the upper part of the slope. Tree mortality was clearly biased and more trees survived in the upper part of the slope than in the middle and the lower parts. There was no significant difference between the upper and the lower part of the slope in the number of feeding wounds made by the pine sawyer beetle, which demonstrates the opportunity of infection with this disease. There was no clear correlation between the development of mycorrhizae and the composition of the soil substrate such as total carbon, nitrogen, and phosphorus. The abundant mycorrhizae in the upper part of the slope, which mitigate drought stress, may also have decreased the rate of tree mortality.     

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.     

Biomass of fine and small roots in two Japanese black pine stands of different ages

The biomass and the spatial distribution of fine and small roots were studied in two Japanese black pine (Pinus thunbergii Parl.) stands growing on a sandy soil. More biomass of fine and small roots was found in the 17-year-old than in the 40-year-old stand. There were 62 g m⁻² of fine roots and 56 g m⁻² of small roots in the older stand, which represented mean values of 608 g for fine and 552 g for small roots per tree, respectively. In the younger stand, a total of 85 g m⁻² of fine roots and 66 g m⁻² of small roots were determined, representing a mean of 238 g for fine and 186 g for small roots per tree, respectively. Fine and small root biomasses decreased linearly with a soil depth of 0–50 cm in the older stand. In the younger stand, the fine and small roots developed only up to a depth of 30 cm. Horizontal distributions (with regard to distance from a tree) of both root groups were homogeneous. A positive correlation in the amount of biomass of fine and small roots per m² relative to tree size was found. Fine and small root biomasses increased consistently from April to July in both stands. The results also indicated earlier growth activity of the fine roots than small roots at the beginning of the growing season. The seasonal increases in fine and small root biomasses were slightly higher in the younger stand than the older stand.     

Individual variations in monoterpenes released from Cryptomeria japonica and Pinus thunbergii needles

Variations in the compositions of low boiling point (LBP) monoterpenes in needle samples from 99 sugi (Cryptomeria japonica) and 100 kuromatsu (Pinus thunbergii) trees were investigated using a headspace technique. Considerable variations in the proportions of monoterpenes were revealed in both species. In sugi, the proportions of sabinene and α-pinene in the total LBP monoterpenes, ranging from 8.8% to 73.3% and from 14.5% to 73.3%, respectively, showed enormous variations among nine monoterpenes. The proportions of 3-carene and limonene, ranging from 0.1% to 29.5% and from 0.2% to 20.4%, respectively, also showed very specific variations. In kuromatsu, the proportions of β-pinene and α-pinene in the total LBP monoterpenes, ranging from 26.5% to 66.3% and from 18.7% to 46.9%, respectively, showed considerable variations among ten monoterpenes. The proportions of myrcene and 1,8-cineole, ranging from 0.9% to 18.5% and from 0.8% to 12.3%, respectively, also showed specific variations. Part of this article was presented at the 50th Annual Meeting of the Japan Wood Research Society, Kyoto, April, 2000, and the 13th Annual Meeting of the Chugoku Shikoku Branch of the Japan Wood Research Society, Okayama, September 2001     

Different development of pine wilt disease in artificially infectedPinus thunbergii seedlings potted together with different tree species

To evaluate the effect of adjacent tree species on the susceptibility of Japanese black pine (Pinus thunbergii) to pine wilt disease, an inoculation experiment was conducted usingP. thunbergii seedlings potted with seedlings of six tree species,i. e. Alnus sieboldiana, Eurya japonica, Lespedeza bicolor formacutifolia, Pinus thumbergii, Robinia pseudo-acacia andSarothamus scoparius. About ten months after planting, they were inoculated with the pinewood nematode (Bursaphelenchus xylophilus) in early July 1992. After that, the proportion of pine seedlings with completely discolored foliage increased more quickly when the seedlings were potted withR. pseudo-acacia, S. scoparius orA. sieboldiana than when potted withP. thunbergii, L. bicolor orE. japonica. At the end of the study period, 17 weeks after inoculation, it reached 90.6%, 90.0%, 87.5%, 72.7%, 63.3%, and 59.4% when the pine seedlings were potted withR. pseudo-acacia, S. scoparius, A. sieboldiana, P. thunbergii, L. bicolor andE. japonica, respectively. This indicated that the susceptibility ofP. thunbergii seedlings to pine wilt disease was influence by the species of adjacent trees.     

Pathogenicity of the pine wood nematode at different developmental stages

The ability of the pine wood nematode,Bursaphelenchus xylophilus, a pathogen that causes pine wilt disease, to kill cortical cells of Japanese black pine,Pinus thunbergii, during early development of the disease was conjectured to be a function of nematode developmental stage. A tangential segment of bark was removed from a 2-cm-long current-year stem. The cortex-exposed segments with cut cortical resin canals were designated as + RC-segments and those without them as − RC-segments. When a nematode population containing many older juveniles and adults (NL) was inoculated onto the cut surface, the − RC-segments were still alive 4 d after inoculation, as were non-inoculated control segments. When cortex-exposed segments were inoculated with either a nematode population containing many younger nematodes (NS) or with nematodes isolated from inoculated pine cuttings that also contained many younger juveniles, most tissue cells in − RC-segments died 4 d after inoculation, suggesting that younger juveniles killed pine cells directly, in contrast with older juveniles and adults. When nematodes were inoculated onto + RC-segments in which they could easily enter resin canals, both NL and NS killed the segment tissues. This suggests that NL is pathogenic to pine cells while living in resin canals. Such differences in the pathogenicity of NL and NS to pine parenchymatous cells were also demonstrated in a pathogenicity assay system using bark peelings, which allowed an estimate of direct attack on the cambial cells by nematodes. Based on these results, we hypothesize that younger juveniles are pathogenic to pine parenchymatous cells, while adults and older juveniles are not pathogenic. This work was supported in part by Grants-in-Aid for Scientific Research (No.01440012 and 06454088) and for Young Scientists (to K.I.) from Ministry of Education, Science, Culture, and Sports of Japan, and by a grant from PROBRAIN.     

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.     

First-year freeze hardiness of pure species and hybrid taxa of Pinus elliottii (Engelman) and Pinus caribaea (Morelet)

Six pine taxa were compared for freeze hardiness following 2 whole plant freeze tests carried out in early and late winter 1993–94. Pure species included slash pine (Pinus elliottii var. elliottii Engelman) (PEE), Caribbean pine (Pinus caribaea Morelet) var. bahamensis (PCB), and var. hondurensis (PCH). The same six slash pine parents served as the female parents of the pure slash pine taxon and were control pollinated to create the F₁ hybrids of slash pine × Caribbean pine var. bahamensis (F1B), the F₁ of slash pine × Caribbean pine var. hondurensis (F1H), and the backcross of slash pine × (slash pine × var. hondurensis (B1H). Four traits related to adaptation (bud set, foliage/stem damage, survival) were compared. Significant differences existing among the three species at all temperatures for both freeze tests. The 3 hybrid taxa were intermediate to parental taxa for all measured traits and only at extreme temperatures was there an indication of hybrid vigor. However, inspection of foliage damage curves to estimate temperatures at which 50 percent damage occurs indicated that the hybrids deviated from the mid parent value toward the more freeze susceptible parent. Discriminant functions which included foliage damage and survival traits provided the best segregation of taxa groupings and the combining of canonical variable 1 for these discriminant functions placed hybrids intermediate to parental taxa. Parent × taxa interaction was significant, suggesting that within these taxa, family to family variation occurred, but different maternal parents were associated with the freeze hardly families in different taxa.This is Florida Agricultural Experiment Station Journal Series No. R-04852 of the Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, 32611     

火炬松体细胞胚胎发生和干化体细胞的氧化物酶活性(英文)

培养于附加2,4-D、BA和KT的愈伤组织诱导培养基上的火炬松成熟合子胚在培养3-9周后形成白色、半透明、有光泽的粘性愈伤组织。这类愈伤组织形成于成熟合子胚的子叶,但当用NAA或者IBA代替愈伤组织诱导培养基中的2,4-D时,它的诱导频率明显降低。这种粘性愈伤组织在分化培养基上形成体细胞胚。体细胞胚经过去50μm ABA和8.5%PEG600处理后成为耐干化胚。扫描电镜观察表明,萌发处理36小时后,耐干化胚恢复到干化处理之前的状态且大小和形态正常,而不耐干化胚不能恢复到干化处理之前的状态且表面撕裂。过氧化物酶活性的分析结果表明,耐干化胚有更高的过氧化物酶活性。耐干化胚的高过氧化物酶活性可能与催化H2O2的分解和保护体细胞胚免受氧化的伤害有关。     

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).