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.     

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.     

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.     

Leaf water relations in <Emphasis Type="Italic">Pinus densiflora</Emphasis> Sieb. et Zucc. on different soil moisture conditions

To elucidate the differences in the leaf water relations of Pinus densiflora Sieb. et Zucc. growing in different soil moisture conditions, we examined the pressure-volume curve and the diurnal changes in the stomatal conductance, the transpiration rate, and the leaf water potential. The leaf water relations were compared using field-grown 40-year-old pine trees growing on the upper and lower parts of a slope. We also compared the leaf water relations of potted 4-year-old saplings growing at pF 4.2 and pF 1.8 soil moisture levels for almost 1 year. The values of the ratio of symplasmic water at turgor loss point to symplasmic water at saturated point (V_p/V_o) and bulk modulus of elasticity () of both the adult trees on the upper part of the slope and the potted saplings growing on pF 4.2 soil moisture were higher than those values of both the adult trees on the lower part of the slope and the potted saplings growing on pF 1.8 soil moisture, respectively. The field-grown adult tree and the potted saplings growing under long-term water stress tended to reduce their stomatal conductance in response to the acute soil drying. It is suggested that P. densiflora growing under long-term water stress rapidly closed its stomata in response to soil drying and avoided losing water, and could also rapidly absorb water with reducing water loss because of the decrease in the leaf pressure potential derived from the high values.     

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).     

Response of Japanese red pine to inoculation with a blue stain fungus,Ceratocystis piceae

To confirm the pathogenicity of a blue stain fungus,Ceratocystis piceae (Münch) Bakshi to the Japanese red pine (Pinus densiflora Sieb. et Zucc.), the responses of healthy young pine trees and stressed trees which were girdled by the half-circumferential girdling technique were investigated by the fungal inoculation test. Although neither of the pine trees inoculated withC. piceae in the non-girdled treatment nor the controls died, mortality of the trees girdled and inoculated withC. piceae was 28.6%. In the pine trees inoculated withC. piceae, the mean area of the necrotic lesion of the sapwood was significantly larger than that of the controls, and the mean of the water pressure potential of the xylem decreased, regardless of the girdling treatment. TheC. piceae was reisolated from the wood pieces near the inoculation points on the inoculated trees, but not from the controls. These results suggest that under strongly stressed conditions, the Japanese red pine trees might have been killed by heavy infestations ofC. piceae carried by bark beetles. A part of this paper was presented at the 103rd Annual Meeting of the Japanese Forestry Society (1992).     

赤松天然更新规律及人工促进更新技术的研究

本文对赤松天然更新的规律进行了调查，并对人工促进更新技术进行了研究。结果表明，赤松初果期为１２ａ，盛果期为５１ａ，公最大更新幅度为４０ｍ；更新幼苗幼树的７０％以上集中分布在距母树９ｍ或树高１．２倍以内。人工促进效果以拖拉机耙和人工耧地好；只要耧掉草皮，移栽７－８ａ幼树，３－４ａ就可达到更新指标；单位面积上培育母树的数量随时间长而减少。     

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.     

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     

Reduction of frost heaving of Norway spruce and Scots pine seedlings by planting in mounds or in humus

This study was carried out in northern Sweden to determine the effects of frost heaving on the establishment of Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings in relation to planting methods. For this purpose, one year old containerised seedlings were planted on two sites and on two dates: during the spring (early planting) and during the fall (late planting). In each case, two planting depths (normal and deep planting) and four planting sites (in mineral soil in the depressions, in the scalp/trench area, on the top of the mound and in the untreated humus layer) were used. On each site, 50 seedlings were planted for each treatment. Frost heaving was observed and measured during two years. The amount of heaving was highest in the hole and almost insignificant on the top of the mound and in the humus layer. Planting depth influenced the degree of heaving only for Scots pine planted in the hole and was not related to the planting time.     

红松仁蛋白的分离、纯化及抗氧化作用研究

研究了松仁蛋白分离、纯化、一级结构及部分蛋白清除·OH和O-2·的活性,探讨松仁蛋白的功能机理。松仁蛋白经过硫酸铵盐析初步分级,对收集量最大的分离蛋白P80通过Sephadex G-75凝胶过滤柱和DEAE 52离子交换层析,收集得到P80分离纯化的组分D,对组分D进行基质激光解析质谱分析,结果显示组分D与北美白松松仁清蛋白Albumin 4具有较高的匹配分数,匹配分数为99,分子质量为18 647 u,等电点为5.35,序列覆盖率为41%,松仁蛋白组分D可能为与Albumin 4蛋白同类的一种松仁清蛋白或其他蛋白,称其为HSR-Protein 1。体外抗氧化实验表明松仁蛋白具有清除·OH和O-2·的能力。在质量浓度为800 mg/L时,松仁蛋白组分D、松仁蛋白组分P80和松仁粗蛋白对·OH的清除率分别为93.12%、90.89%和89.12%,对O-2·的清除率分别为64.19%、56.45%和35.19%,松仁蛋白组分D对·OH和O-2·的清除能力最强。相同浓度下,纯度越高的松仁蛋白清除·OH和O-2·能力越强。     

Thinning method and intensity influence long-term mortality trends in a red pine forest

Tree mortality shapes forest development, but rising mortality can represent lost production or an adverse response to changing environmental conditions. Thinning represents a strategy for reducing mortality rates, but different thinning techniques and intensities could have varying impacts depending on how they alter stand structure. We analyzed trends in stand structure, relative density, stand-scale mortality, climate, and correlations between mortality and climate over 46 years of thinning treatments in a red pine forest in Northern Minnesota, USA to examine how thinning techniques that remove trees of different crown classes interact with growing stock manipulation to impact patterns of tree mortality. Relative density in unharvested plots increased during the first 25 years of the study to around 80%, then began to plateau, but was lower (12–62%) in thinned stands. Mortality in unharvested plots claimed 2.5 times more stems yr⁻¹ and 8.6 times as large a proportion of annual biomass increment during the last 21 years of the study compared to the first 25 years, but showed few temporal trends in thinned stands. Mortality in thinning treatments was generally lower than in controls, particularly during the last 21 years of the study when mortality averaged about 0.1% of stems yr⁻¹ and 4% of biomass increment across thinning treatments, but 0.8% of stems yr⁻¹ and 49% of biomass increment in unharvested plots. Treatments that combined thinning from above with low growing stock levels represented an exception, where mortality exceeded biomass production after initial thinning. Mortality averaged less than 0.1% of stems yr⁻¹ and less than 1% of annual biomass production in stands thinned from below. These trends suggest thinning from below minimizes mortality across a wide range of growing stock levels while thinning from above to low growing stock levels can result in dramatic short-term increases in mortality. Moderate to high growing stock levels (21–34 m² ha⁻¹) may offer greater flexibility for limiting mortality across a range of thinning methods. Mean and maximum annual and growing season temperatures rose by 0.6–1.8 °C during the study, and temperature variables were positively correlated with mortality in unharvested plots. Mortality increases in unharvested plots, however, were consistent with self-thinning principles and probably not driven by rising temperatures. These results suggest interactions between thinning method and intensity influence mortality reductions associated with thinning, and demonstrate the need for broader consideration of developmental processes as potential explanations for increased tree mortality rates in recent decades.     

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.     

Growth responses of red pine seedlings to the chemical bioregulator,DCPTA

The chemical bioregulator, 2-(3, 4-dichlorophenoxy)-triethylamine (DCPTA), which has markedly stimulated photosynthesis, carbon allocation, and above- and below-ground growth of several agricultural crops, caused small growth effects on red pine (Pinus resinosa L.) germinants and one and two-year-old seedlings, mainly to foliage and stems. At low DCPTA concentrations, i.e. 10 ppm, growth tended to be promoted, whereas at 100 ppm growth appeared to be suppressed. Continued testing of the bioregulator's effect on growth and physiology of other tree species is recommended.     

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.     

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.     

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.     

The effect of late season damage on the apical bud development of Scots pine seedlings

The effect of Lygus rugulipennis Popp. feeding and artificial damage on the apical bud of Scots pine (Pinus sylvestris L.) late in the growing season was studied on two-year-old seedlings. Lygus feeding in late July and August caused inhibition of bud formation in the subsequent year's shoots, which led to loss of apical dominance and formation of interfascicular buds. Mechanical damage caused by piercing the apical bud with a needle in July, August and October produced scars and malformations on subsequent year's shoots and buds, but did not inhibit bud formation. Damage to the apical meristem could not be detected visually on the dormant bud before shoot elongation.     

Somatic embryo production and plant regeneration of Japanese black pine (Pinus thunbergii)

Somatic embryogenesis in Pinus thunbergii was initiated from megagametophytes containing immature zygotic embryos. Embryogenic cultures were maintained and proliferated in medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. High maturation frequencies of somatic embryos were obtained on maturation media containing maltose, activated charcoal, abscisic acid, and polyethylene glycol as osmotic agent. The best result among the cell lines tested was achieved with the cell line T-205-3. More than 900 somatic embryos per petri dish, on average, were obtained after about 8 weeks of culture on maturation medium. Sixty percent of somatic embryos tested germinated after transfer to plant growth regulator-free medium and then 85% of them converted into plantlets.     

The role of germination microsite in the establishment of sugar pine and Jeffrey pine seedlings

Mature shrubs can provide microhabitats that are beneficial to tree seedling growth and development. Sugar pine trees (Pinus lambertiana) grow in a narrow zone on the eastern slope of the Carson Range in extreme western Nevada, whereas Jeffrey pine (Pinus jeffreyi) is the dominant tree species in the region, an area extensively disturbed by wild fire. This study compares seedling establishment of sugar pine and Jeffrey pine relative to mature shrubs. In the fall of 2002 (cohort 1) and 2003 (cohort 2), 13,600 seeds of both species were planted in wire mesh enclosures, at three sites, under a variety of microhabitat treatments: under shade and in the open, under two species of shrub cover, and with and without plant litter. Seedlings were monitored for survival through two growing seasons. Even though more sugar pine seedlings emerged, more Jeffrey pine seedlings survived, and Jeffrey pine was the more drought tolerant species, better suited for the xeric climate found in the Carson Range. Litter slightly hindered seedling emergence but had no effect on survival and there was no significant species × litter interaction. Supplemental water facilitated survival in all treatments with highest survival in shade treatments. Sugar pine seedlings showed a significant increase in survival over Jeffrey pine seedlings with the addition of water, particularly in open treatments and more of both species survived under manzanita shrubs with water. The highest seedling mortality occurred when shrub canopy was removed, and seedlings experienced the effect of full sun and competition for soil water. For either species, microhabitat is a significant factor in determining success or failure in rehabilitation efforts after disturbance.