Identification of Armillaria species in Japan using PCR-RFLP analysis of rDNA intergenic spacer region and comparisons of Armillaria species in the world

Armillaria species from Japan were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the intergenic spacer region-1 (IGS-1) of ribosomal DNA (rDNA). Eleven different digestion patterns by restriction endonuclease Alu I were found among 70 isolates of seven Armillaria species in Japan. Isolates within Armillaria nabsnona, A. ostoyae, A. cepistipes, and Japanese biological species E showed the same Alu I digestion patterns. Five Alu I patterns were detected for A. gallica, three patterns for A. mellea, and two patterns for A. tabescens. Seven Armillaria species in Japan were clearly distinguished by using the profiles obtained when PCR products were digested with Alu I, Msp I, and Hae III restriction enzymes. There was considerable variability of Alu I restriction sites within the IGS-1 between the isolates of five Armillaria species, A. gallica, A. nabsnona, A. cepistipes, A. mellea, and A. tabescens, in Japan and those of their European and North American counterparts.     

RFLPs in internal transcribed spacers of ribosomal DNA from three species ofTilletia

DNA polymorphisms were evaluated for the eight strains representingTilletia controversa Kuhn,T. laevis Kuhn andT. tritici (Bjerk.) Winter using polymorase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. DNA regions coding for the internal transcribed spacers (ITS) were amplified and digested with five four-base restriction enzymes (Msp I,Hinf I,Taq I,Hha I andHae III). No differences were shown in the PCR-RFLP patterns among the strains tested. This indicatedT. controversa, T. laevis andT. tritici are very closely related. This study was supported in part by systematic Mycology & Liohenology Laboratory of Sciance Institute of China     

Pre- and post-dispersal damage to the acorns of two oak species (Quercus serrata Thunb. andQ. mongolica Fisher) in a species-rich deciduous forest

A comparative study of pre- and post-dispersal damage to the acrons of two oaks,Quercus serrata Thunb. andQ. mongolica Fischer, was done over 3 years in a species-rich deciduous forest in central Japan. The numbers of acorns bored into by moth larvae and byKobuzo rectirostris (Roelofs) was low and relatively uniform each year for both oak species, but the numbers bored into byCurculio spp. varied from year to year. The proportions of acorns attacked were independent of the density of acorns produced for both oak species. The proportions of the dispersed acorns of both oak species on the forest floor that disappeared between autumn and spring were less than 27%, and were lower for species in the stand that belong to family Fagaceae. However, by the next summer, most dispersed sound acorns had been infested by moths that specialized on post-dispersal acorns.     

Effects of crown release on basal area growth rates of some broad-leaved tree species with different shade-tolerance

We compare three or four years effects of crown release on stem growth rates among four deciduous broad-leaved tree species,Fagus crenata, Quercus crispula, Magnolia obovata andAcer mono, in an even-aged coppice forest, central Japan. The crown release significantly improved relative growth rates for basal-area (RGR_BA) of the four species. However the effects of neighboring gap area (GAPA) on the RGR_BA differed among the species. The effects were significant forQ. crispula andM. obovata, indicating that growth rates of these two species increase with intensity of the crown release. In contrast, the RGR_BA ofF. crenata andA. mono were not correlated with the GAPA, suggesting that their growth rates are unrelated to the intensity. We considered that such differences among species were closely related to their photosynthetic characteristics of light use; less-tolerant species (Q. crispula andM. obovata) exhibit greater growth rates relative to the intensity of crown release than tolerant species (F. crenata andA. mono). Based on these results, we proposed implications for thinning practices in mixed forests of species with different shade-tolerance.     

Screening of extracts of Japanese woods for melanin biosynthesis inhibition

The effects of 120 methanol extracts prepared from bark and heartwood of 69 types of Japanese wood on the melanin production of B16 melanoma cells were examined. The melanin content of B16 melanoma cells was determined spectrophotometrically at 405nm. The extracts were also examined for their effects on cell viability. We found that the methanol extracts of Fagus crenata (buna, wood, 100μg/ml), Sapium sebiferum (Nankinhaze, wood, bark, 10μg/ml), and Zelkova serrata (keyaki, wood, 10μg/ml) greatly inhibited the melanin production of B16 melanoma cells without significant cytotoxicity. However, these extracts did not inhibit tyrosinase activity at the concentration of 100μg/ml. These findings indicate that the depigmenting mechanism of these extracts involves the suppression of some pigmenting signals in stimulating melanogenesis rather than the inhibition of tyrosinase activity. Part of this study was presented at the 53rd Annual Meeting of the Japan Wood Research Society, Fukuoka, Japan, March 2003     

Factors affecting the dynamics of the population of Fagus crenata in the Takakuma Mountains, the southern limit of its distribution area

The refugee population of Fagus crenata in the Takakuma Mountains, Kyushu, Japan, represents the southern limit of this species' distribution area. Because this population is the most exposed to the global warming effect, records of this population are likely to provide useful information on the response of this species to global warming. The aim of this study was to record the present status of this valuable population, enabling judgment of its sustainability. The density of successive F. crenata trees and saplings was low regardless of the coverage of dwarf bamboo. Moreover, the proportion of empty nuts observed was considerably high, suggesting to be the likely major factor limiting recruitment of saplings or successors in this population. Radial growth of F. crenata was shown to have been decreasing for the past 50 years, and there was a significantly negative correlation with the warmth index. The decreasing growth rate and poor regenerative ability caused by the high proportion of empty nuts suggest the possibility that this population will degrade further in the future, possibly being replaced by shrubs and small tree species.     

Predicting the spatial distribution of major species composition in secondary hardwood forests on Mt. Gozu,central Japan,based on environmental factors

Our goal was to predict the spatial distribution of canopy species composition in secondary deciduous hardwood forests at a fine spatial resolution, based on climatic and topographical factors using a geographic information system. We studied secondary forests on Mt. Gozu, Niigata Prefecture, central Japan. Canopy species composition was investigated in 100 sample plots within the study area. A digital elevation model (DEM) was created, and topographical, hydrological, and light factors were calculated using the DEM. Climatic factors were interpolated by kriging. The five major species used as response variables were Fagus crenata, Quercus serrata, Quercus crispula, Magnolia obovata, and Castanea crenata. We prepared three possible explanatory variable sets: climatic variables only, both climatic and topographic variables, and topographic variables only. Multivariate regression trees were derived, and the accuracy of predicting the major species composition was tested. The multivariate regression trees derived from the climatic variable set and from the climatic and topographic variable set had better accuracy than the regression tree derived from the topographic variable set. In the regression tree generated by the climatic and topographic variable set, the warmth index was the principal explanatory variable in classifying forest types, followed by topographic factors. This regression tree would be preferable to the other two regression trees for the prediction of canopy species composition.     

Photosynthesis and biomass allocation of beech (Fagus crenata) and dwarf-bamboo (Sasa kurilensis) in response to contrasting light regimes in a Japan Sea-Type beech forest

Regeneration of beech (Fagus crenata) forests depends on the formation of canopy gaps. However, in Japan Sea-type beech forests, a dwarf bamboo (Sasa kurilensis) conspicuously occupies sunny gaps. Therefore,F. crenata seedlings must escape the severe interference ofS. kurilensis in the gaps and persist beneath a closed canopy of the beech forest. We hypothesized that the growth ofF. crenata seedlings in the understory would be favored by their being more plastic thanS. kurilensis in photosynthetic and morphological traits, which would support the matter production ofF. crenata seedlings in a wide range of light availabilities. To examine this hypothesis, the photosynthetic-light response of individual leaves and the biomass allocation in aboveground parts (i.e., the culm/foliage ratio) were surveyed at sites with contrasting light availabilities in a Japan Sea-type beech forest in central Japan. InF. crenata, photosynthetic light utilization efficiency at relatively low light was greater, and the dark respiration rate was smaller in the leaves of seedlings (10 cm in height) beneath the closed canopy than in the leaves of saplings at the sunny forest edge. The culm/foliage (C/F) ratio of theF. crenata seedlings at the shady site was small, suggesting effective matter-production beneath the beech canopy. On the other hand,S. kurilensis both in the gap and beneath the beech canopy showed low plasticity in photosynthesis and the culm/foliage ratio. Because the shoot density ofS. kurilensis was smaller beneath the beech canopy than in the gap, the light availability at the bottom of theS. kurilensis layer was greater beneath the beech canopy. These results suggest that the photosynthetic productivity of theF. crenata seedlings would be enough for the seedlings to survive in the understory with a low density ofS. kurilensis shoots beneath the closed beech canopy.     

Effects of altitudinal gradient on species composition of naturally regenerated trees in <Emphasis Type="Italic">Larix kaempferi</Emphasis> plantations in central Japan

Species composition and community structure of naturally regenerated trees in Larix kaempferi plantations and natural forests were compared in relation to altitudinal gradient. Fifty-nine L. kaempferi plantations and 26 natural forest stands including old-growth and secondary forests were selected from 1300 to 2000 m a.s.l. in Mt Kushigata of central Japan. Vegetation plots (10 × 10 m) were established in each stand (85 plots in total). Species composition differed significantly between forest types, particularly on high altitude plots. Some species were significantly biased to natural forests, while no species were biased to plantations. Although some species that responded significantly to altitude were common to both forest types (e.g., Acer ukurunduese, Castanea crenata, and Fraxinus lanuginose f. serrata), some differed between forest types (e.g., Fagus japonica, Prunus maximowiczii, and P. nipponica). Thus, plantation management altered species responses to altitude. Altitude should be considered when planning and carrying out forest ecological restoration in plantations, because species composition of naturally regenerated trees has already been changed by past plantation management. In particular, attention should be paid to ecological restoration at high altitude.     

Pre- and post-dispersal damage to the nuts of two beech species (Fagus crenata blume andF. japonica Maxim.) that masted simultaneously at the same site

A study of pre- and post-dispersal damage to the nuts of two beeches,Fagus crenata Blume andF. japonica Maxim., which masted simultaneously, was performed on a single site in Japan. Most insect damage to the nuts before dispersal resulted from boring by lepidopteran larvae, and the level of attack was markedly higher onF. crenata than onF. japonica. Early nut growth ofF. crenata allowed these borers to act as the first attackers on the nuts. Conversely, late nut growth ofF. japonica delayed the attack of borers, and foliage feeders acted as the first attackers instead, chewing on nuts from the outside. The low level of borer attacks on the nuts ofF. japonica in mast years was thought to have been caused by foliage feeders decreasing the amount of food resource available for later attack by borers in lean years. This could then lead to a subsequent depression of the population of borers in mast years. The proportion of nuts damaged by moth larvae was independent of the density of nuts for both beech species. Caching by vertebrates was thought to be the most important factor in post-dispersal damage because the density of nuts of both beech species on the forest floor in March was half the level measured using seed traps in the previous year. However, post-germination damage caused by a fungus was also an important factor. The survival rate of the nuts (including current-year seedlings) in the year after dispersal was independent of the density of dispersed sound nuts in the previous year for both beech species, with the exception of one set of observations (forF. japonica in July). This research was supported in part by a grant from the Japanese Ministry of Agriculture, Forestry and Fisheries.     

Effect of pressure on organic acid production from Japanese beech treated in supercritical water

The effect of pressure as described by density was studied on organic acid production from Japanese beech (Fagus crenata) treated in supercritical water. At a reaction temperature of 380°C, the maximum yield of organic acids was 35% at a pressure of 30 MPa (density of water: 0.53 g/ml) for 1 min in a batch-type system. Furthermore, the yield of organic acids decreased with increasing reaction pressure. It was also found that fragmented products from sugars such as methylglyoxal and glycolaldehyde could be more easily converted to organic acids than dehydrated products such as furfural and 5-hydroxymethyl furfural. This result suggests that organic acids can be mainly derived from fragmented products. Part of this article was presented at the 55th Annual Meeting of the Japan Wood Research Society (2005), and at the 17th Annual Meeting of Japan Institute of Energy (2007)     

Wood identification of JapaneseCyclobalanopsis species (Fagaceae) based on DNA polymorphism of the intergenic spacer betweentrnT andtrnL 5′ exon

DNA was extracted from wood samples of six representativeCyclobalanopsis species (Fagaceae) growing in Japan that cannot be distinguished from one another by conventional microscopy. A part of the intergenic spacer region betweentrnT andtrnT 5 exon was amplified and sequenced. The sequences obtained from wood samples were grouped into three DNA types by a single nucleotide polymorphism as reported previously in leaf samples: I (Quercus acuta, Q. sessilifolia, Q. salicina), II (Q. myrsinaefolia, Q. glauca), and III (Q. gilva). Thus,Q. gilva can be distinguished from the otherQuercus species, and the others are separated in two subgroups based on DNA polymorphism. The present findings support the possibility of wood identification based on DNA polymorphism.     

Comparison of beetle samples captured by traps with those of flowers in a broad-leaved forest

Beetle samples were compared between catches by traps and those on flowers in a Japanese beech (Fagus crenata Blume) forest, in Minakami, Gunma Prefecture, Japan. White plastic traps (Sankei Chemical Co., Ltd.) equipped with benzyl acetate were set out for a month from July 23 to August 23, 1993, and all beetles captured were collected every six days. During six days from August 10 to August 16, beetles were captured with traps and on wild flowers (Aruncus sylvester Kostel andAngelica polyclada Franch) at three hour intervals. Over the month, the traps captured 1,677 individuals of 73 species of 20 families, which included 16 (89%) species captured on flowers in the six-day period. Over the six days, the traps captured 327 individuals of 21 species of 12 families, compared with 685 individuals of 18 species of 5 families on the flowers; all of the families and nine (50%) species of beetles taken on the flowers were also captured by the traps; all of the species with more than five individuals captured on the flowers were also trapped. This suggests that capture by traps might sufficiently cover the beetle fauna on flowers. The number of species and individuals, as well as their changing patterns, however, varied more irregularly during the six-day period with the traps than with those on the flowers. A part of this paper were presented at the 46th meeting of the Kanto Branch of the Japanese Forestry Society (1994) These English titles are tentative translation by the authors from the originals     

Effects of previous forest types and site conditions on species composition and abundance of naturally regenerated trees in young <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> plantations in northern Japan

Naturally regenerated trees in young Cryptomeria japonica plantations were investigated in 141 quadrats of 10 × 50 m within a watershed of 1,000 ha, and factors affecting their composition and abundance were examined. The species composition of naturally regenerated trees was classified into four types. Dominant species were Swida controversa, Magnolia hypoleuca and Pterocarya rhoifolia in Type A1, Betula maximowicziana, M. hypoleuca, Quercus crispula and Castanea crenata in Type A2, Q. serrata and C. crenata in Type B1, and Pinus densiflora in Type B2. The results of path analysis showed significant influences of previous forest type and geology among the factors that correlated with the species composition of naturally regenerated trees; previous forest type in particular showed a higher absolute path coefficient value. Species composition types of naturally regenerated trees corresponded to the previous forest types: Types (A1 and A2) corresponded to the natural forests composed of Thujopsis dolabrata var. hondai, Fagus crenata, Aesculus turbinata, etc., Type B1 corresponded to the secondary Q. crispula and Q. serrata forest, and Type B2 corresponded to the secondary P. densiflora forest, respectively. The abundance of naturally regenerated trees was strongly affected by geology; i.e., plantations on soft-sedimentary dacitic tuff contained great amounts of colonizing P. densiflora trees.     

Reaction behavior of wood in an ionic liquid, 1-ethyl-3-methylimidazolium chloride

Reaction of Japanese beech (Fagus crenata) in an ionic liquid, 1-ethyl-3-methylimidazolium chloride ([C2mim][Cl]), which can dissolve cellulose, was investigated. Although both lignin and polysaccharides such as cellulose and hemicelluloses can be liquefied at a treatment temperature of around 100°C, the liquefaction of polysaccharides mainly occurs at the beginning of the treatment with [C2mim][Cl]. Cellulose crystallinity in the wood was gradually broken down as the treatment continued. The solubilized polymers were depolymerized to low molecular weight compounds. The results indicate that [C2mim][Cl] is an effective solvent and reagent for the liquefaction of wood components and subsequent depolymerization of them. Part of this report was presented at the 58th Annual Meeting of the Japan Wood Research Society, Tsukuba, April 2008     

Liquefaction of beech wood in various supercritical alcohols

The liquefaction of Japanese beech (Fagus crenata Blume) was studied with various straight-chain alcohols in subcritical and supercritical states using a batch-type reaction vessel to obtain liquid fuel from lignocellulosics. Under the reaction condition of 270°C, beech wood was liquefied to some extent in all alcohols with about 50%–65% insoluble residue left after treatment for 30min. Under the condition of 350°C, however, more than 90% of wood was decomposed and liquefied in all alcohols. Alcohols with longer alkyl chains liquefied lignocellulosics in shorter reaction times. Because many kinds of alcohols, such as methanol and ethanol, can be produced from biomass, 100% biomass-based liquid fuel can be prepared by supercritical alcohol technology when using such bioalcohols. This study was presented in part at the 55th Annual Meeting of the Japan Wood Research Society, Kyoto, Japan, March 16–18, 2005     

Blue stain fungi associated withTomicus piniperda (Coleoptera: Scolytidae) on Japanese red pine

A number of various species of blue-stain fungi were isolated fromTomicus piniperda adults at various stages of development, as well as from the galleries, pupal chambers and sapwood underneath galleries on Japanese red pine. This study was an attempt to identify the species, composition of blue-stain fungi associated withT. piniperda, the frequency of occurrence of the fungi, and their role in the sapwood-staining of Japanese red pine in Tsukuba City, central Japan. Among the seven species of blue-stain fungi isolated, an undescribed species ofOphiostoma together withO. minus were the dominant species and closely associated withT. piniperda. These two species occurred on newly emerging adults more frequently than the overwintered adults.Hormonema dematioides was also associated with the beetle, however, its frequency of occurrence from the emerged new adults was very low. Although the two other species,O. ips andGraphium sp. were also isolated from emerged beetles, the frequency of these fungi from gallery systems suggested that they were accidentally carried byT. piniperda. Leptographium wingfieldii, known to be associated with the beetle in Europe, was also isolated at a very low frequency and the fungus seemed not to be closely associated with the beetle.Ophiostoma sp. andO. minus appear to be the most important causes of blue-stain of Japanese red pine sapwood after infestation byT. piniperda.     

Risk assessment of ozone impact on <Emphasis Type="Italic">Fagus crenata</Emphasis> in Japan: consideration of atmospheric nitrogen deposition

Tropospheric ozone (O₃) is considered to be the air pollutant relating to the decline of Fagus crenata forest in Japan. In the present study, we assessed a risk of O₃ impact on the growth of F. crenata in Japan, giving consideration to the effects associated with atmospheric nitrogen (N) deposition based on the experimental study, national monitoring data for oxidant concentration and atmospheric N deposition, and a national vegetation survey. The average and maximum O₃-induced relative growth reduction (RG_red) of F. crenata across Japan were estimated to be 3.2 and 9.7%, respectively. Current levels of atmospheric N deposition were found to significantly affect the sensitivity of F. crenata to O₃. When the N deposition was assumed as zero, the estimated average and maximum RG_red were 2.3% and 5.7%, respectively. The inclusion of atmospheric N deposition data thus increased the estimated values for average and maximum RG_red (by 38% and 71%, respectively). Our results demonstrate that a change in the sensitivity to O₃ associated with atmospheric N deposition is an important consideration in the risk assessment of O₃ impact on the growth of F. crenata in Japan.     

Spatial distribution pattern of attack of the oak borer,Platypus quercivorus (Murayama) (Coleoptera: Platypodidae), and scolytid ambrosia beetles (Coleoptera: Scolytidae) on fresh logs

The spatial distribution patterns of the attack on fresh logs ofPasania edulis was studied for the oak borer,Platypus quercivorus (Murayama), and two species of Scolytid ambrosia beetles,Xylosandrus crassiusculus (Motschulsky) andXyleborus attenuatus Blanford, in 1994 and 1995. On the logs where onlyP. quercivorus attacked, the entry holes were distributed uniformly when attack intensity was low. However, the distribution pattern became more aggregated with the increase in attack intensity. On logs where bothP. quercivorus and the two Scolytid species attacked, there was a negative association between the spatial distribution of the entry holes ofP. quercivorus and that of scolytids. Simultaneous attack of two scolytids also increased the degree of aggregation of the entry holes ofP. quercivorus. The entry holes of scolytids were distributed in groups irrespective of the attack intensity of scolytids andP. quercivorus. These results suggest an asymmetrical interspecific relationship betweenP. quercivorus and scolytids. Concentration of the entry holes ofP. quercivorus in a small area may cause a considerable decline in the reproductive success in the galleries constructed there.     

“Candidatus Phytoplasma solani” associated with Eucalyptus witches’ broom in Iran

During summer of 2015, Eucalyptus camaldulensis plants showing witches’ broom, little leaf and general yellowing of the foliage were observed in west of Fars and Khozestan province of Iran. DNA from samples of 22 symptomatic and two asymptomatic trees was extracted and subjected to molecular analyses. Nested‐PCR test using R16F2n/R16R2 primers confirmed phytoplasma presence in 63% of symptomatic Eucalyptus plants. Sequence analysis along with virtual RFLP of the 16S ribosomal DNA allowed to classify three Eucalyptus witches’ broom strains into the “stolbur” (“Candidatus phytoplasma solani”) 16SrXII‐A subgroup. Comparison of the secA and secY gene sequences with sequences deposited in GenBank confirmed the phytoplasma identity. Real and virtual RFLPs of the amplified secY gene using HaeIII, MseI and RsaI restriction enzymes showed profiles indistinguishable from each other. This is the first study reporting E. camaldulensis as a new host species for “Ca. P. solani.”