Japanese Chestnut Research Articles

Fertilization and In Vitro Growth of Ovules in Japanese Chestnut (Castanea crenata Sieb. et Zucc.)

Ovules were removed from ovaries from mid-July to early August and cultured in vitro. As a result, all ovules which were sampled and cultured by July 19 in the central female flowers and by July 22 in the lateral female flowers died before early September. However, in ovules sampled and cultured on and after July 22 and 26 in the central and lateral female flowers, respectively, nearly one ovule per ovary survived and developed on the culture medium until mid-September. The development and enlargement of the embryos within the ovules were also observed. Differences in the weight between the largest ovule per ovary and the other ovules began to become clear after July 22 in the central and lateral female flowers. The differences increased rapidly on and after July 29. The longest pollen tubes which penetrated into the ovaries reached the nucelli on July 19-22 and July 22-26 in the central and lateral female flowers, respectively. The pollen tubes which reached the zone where the nucelli were located were observed on July 15 and 19 in the central and lateral female flowers; their numbers increased rapidly during July 15-19 and July 19-22, respectively. Subsequently, their numbers gradually decreased. In the central female flowers, the ovules with a single pollen tube per ovary numbered 5, 6 and 5 on July 22, 26 and 29, respectively. A pollen tube, which penetrated into the micropyle, reached the nucellus in only one ovule in the ovary during July 22-29. The pollen tubes which had penetrated into the micropyles of the other ovules stopped growing at the same length, about half of the length of the entire micropyle, on July 29 in particular. It was suggested that when one ovule had already been fertilized, the other ovules, into which pollen tubes had penetrated, could not be fertilized owing to the cessation of the pollen tube growth.

Identification of Fragrances from Chestnut Blossom by Gas Chromatography-Ion Trap Mass Spectrometry

In far-eastern Asia including Korea, Japan, and China, chestnuts are an important source of food. The scientific name for the Korean chestnut tree that widely distributed in Korean peninsular is Castanea crenata var dulcis Siebold et Zuccarini (Fagaceae) or Castanea kusakuri. The Japanese chestnut is classified as Castanea crenata, the Chinese chestnut as Castanea mollissima or Castanea bungeana Blume, the European chestnut as Castanea sativa, the American chestnut as Castanea dentata and some American species the chinquapin as Castanea pumila. It was commonly found on mountains, hills, and slopes in gravelly or rocky, well-drained glacial soils. The flowers of the Korean chestnut appeared in June, producing a spectacular display of creamy-yellow blossoms and were strongly scented. The flowers were similar to those of the Japanese chestnut. Chestnut honey was produced in this season. Interestingly, it has been believed that chestnut blossoms emit strong sperm-like scents for attracting mate. The edible nuts ripened and dropped from September. The bark and wood were rich in tannic acid which provided tannin for use in the tanning of leather. To date, however, chemical composition of fragrances emanated from chestnut blossom has never been studied. The main purpose of this study is to characterize the fragrance composition emanated from chestnut blossom. The screening of new compounds and combination formulas for cosmetics, aroma therapy and many other applications is the final goal. In this study, solid-phase trapping-solvent extraction (SPTE) was used to collect fragrances emitted from chestnut blossom. Then, fragrant compounds were analysed by GC-MS n with electron impact (EI) ionization and methane chemical ionization (CI) modes for the crosschecking. For the first time, fragrance constituents of chestnut blossom have been reported here.

'Candidatus Phytoplasma castaneae', a novel phytoplasma taxon associated with chestnut witches' broom disease.

In Korea, Japanese chestnut trees (Castanea crenata Sieb. and Zucc.) showing symptoms indicative of witches' broom disease, including abnormally small leaves and yellowing of young leaves, were examined. Since the symptoms were suggestive of a phytoplasma infection, tissues were assayed for phytoplasmas by PCR analysis using a pair of universal primers that amplify a 1.4-kbp phytoplasma 16S rDNA fragment. The phytoplasma-specific fragment was amplified from diseased plants, but not from healthy plants, indicating that a phytoplasma was the causal agent of the chestnut witches' broom (CnWB) disease. The phylogenetic relationship of the CnWB phytoplasma to other phytoplasmas was examined by sequence analysis of the 16S rDNA. A phylogenetic analysis of 16S rDNA sequences of the phytoplasmas placed the CnWB phytoplasma within a distinct subgroup in the phytoplasma clade of the class Mollicutes. The phylogenetic tree indicated that the CnWB phytoplasma is related most closely to coconut phytoplasmas and suggested that they share a common ancestor. The unique properties of the CnWB phytoplasma sequences clearly establish that it represents a novel taxon, 'Candidatus Phytoplasma castaneae'.

Effects of burying depth and seed size on seedling establishment of Japanese chestnuts, Castanea crenata

The extent to which caching depth and nut size affect seedling establishment of Japanese chestnuts, Castanea crenata Sieb. et Zucc. , were investigated by burying the nuts of three different size-classes at different depths (0 cm on litter; 0, 5, 10, 20 and 30 cm beneath litter) in a nursery. Seedling emergence, seedling mass and height were usually reduced by deep burial, particularly for small nuts. With increasing burial depth and decreasing nut size, the biomass allocation to stems (particularly below ground stems) increased, while allocation to roots (part below cotyledons including hypocotyl) decreased. Such morphological changes would amortize the limited seed reserve to facilitate emergence. Seedling emergence, seedling mass and height were greatest at 5 cm burial depth, but no seedlings emerged when nuts were sown on litter, suggesting that animal caching in shallow burial (scatter hoarding) favors chestnuts regeneration. At the optimal burial depth (5 cm), there was little difference in the seedling height among nut size-classes which showed that nut size has little effect on their competitive abilities with neighboring herbs in the early successional habitats where the chestnuts usually regenerate. The fact that most of the larder- or nest-hoarded nuts ( depth≧10 cm) were consumed by wood mice ( Apodemus spp.) in the fields suggests that large nuts would not always have distinct advantages over small nuts.

Impact of site‐induced mouse caching and transport behaviour on regeneration in Castanea crenata

Abstract. Dispersal and retrieval site selection by mice, transport distance, cache depth, and emergence and survival of seedlings of Castanea crenata (Japanese chestnut) were investigated by a magnet‐locating experiment in two habitat conditions (gap vs. forest understorey). Magnets were inserted into nuts (n= 450) and the nuts placed in the edge of forest gaps. Although wood mice (Apodemus speciosus and A. argenteus) initially buried nuts singly in shallow surface caches near the nut source, by the following spring these cached nuts were retrieved and re‐cached in larger, deeper caches farther from the source, particularly in forest understories, probably to reduce the threat of pilferage. All the nuts cached in the forest understories were consumed, but 4 seedlings emerged in gaps, apparently because of lower foraging activity in the gaps by the mice. Seed size was not correlated with cache depth or cache site selection. With increasing seed size, transport distance increased, particularly in gaps, possibly due to a greater potential energy gain (relative to handling cost to the cacher), or to attempts to prevent density‐ or mass‐dependent loss of caches by other foragers. Variable seed dispersal behaviour based on variation in seed size may influence the chances of colonization and distribution of the light‐demanding Castanea trees in mosaic landscapes and may play an important role in community organization and dynamics.

Life history and spatial distribution of Oriental beetle (Coleoptera: Scarabaeidae) in golf courses in Korea.

Larval and adult activity of the oriental beetle Exomala orientalis (Waterhouse), a pest of turfgrass in Korea, was investigated at four golf clubs in Pusan, Korea, from 1995 to 1999. Adult emergence was first observed on the greens in late May with peak activity occurring 2 wk later. During the day, E. orientalis adults were most active between 1800 and 2200 hours. First instars were found mostly in early July, second instars mostly in late July, and third instars from August to April. The density of larvae in fixed plots decreased steadily from the time of egg laying to pupation: 667/m3 on 26 July, 267/m3 on 29 August, and 122/m3 on 2 October 1997. All the observed E. orientalis completed one generation per year. Adult females were observed feeding on flowers of a late-blooming variety of Japanese chestnut (Castanea crenata Sieb & Zucc). E. orientalis larval densities were higher in greens with Japanese chestnut nearby, and where magpie, Pica pica sericea (Gould), feeding was observed. More E. orientalis adults emerged from the right, left, and back of greens than from the front or middle. The intensity of emergence was inversely proportional to the amount of golfer traffic on various parts of the green. Counting emergence holes may be a way that golf course superintendents can predict which greens and tees are most likely to be damaged from E. orientalis larvae without destructive sampling.

The Relationship Between Cultural Practices and Arbuscular Mycorrhizal(AM) Activity in Orchards Under Different Management Systems.

It has been demonstrated that soil disturbance, excessive fertilization and the use of some agrochemicals affect the survival and function of arbuscular mycorrhizal (AM) fungi. To clarify the effect of different cultural practices on mycorrhizal activity in orchards, spore population dynamics, soil and plant mineral content and mycorrhizal infection were surveyed monthly from May to October, 2000 in kiwifruit, Japanese chestnut, Iyo tangor, satsuma mandarin, Japanese persimmon, apple, peach, Japanese plum and grape plantings, subjected to different management regimes. In intensively managed orchards, levels of major elements in the soil were generally high, owing to periodic fertilization ; consequently, the content of these elements in the leaves was within the recommended range for normal growth and function. High levels of AM infection were maintained throughout the season in the apple orchard that received no fertilizer or fungicide. The infection levels on roots of kiwifruit vines decreased in July and August after three fungicide sprays in June ; it also decreased in Iyo tangor in July following four fungicide applications in May and June. These orchards had no under-story cover plants. However, the infection level of peach trees under sod culture remained relatively high even after five fungicide sprays from May through August. Thus, population density of AM spores in the soils seems to be low in the clean cultivated orchards, subjected to frequent fungicide sprays, whereas it was high under sod culture. We conclude that excessive fertilization, frequent fungicide sprays and clean culture have an adverse effect on AM spore populations and tree infection in orchards.

Impact of site-induced mouse caching and transport behaviour on regeneration in Castanea crenata

Abstract Dispersal and retrieval site selection by mice, transport distance, cache depth, and emergence and survival of seedlings of Castanea crenata (Japanese chestnut) were investigated by a magnet-locating experiment in two habitat conditions (gap vs. forest understorey). Magnets were inserted into nuts (n = 450) and the nuts placed in the edge of forest gaps. Although wood mice (Apodemus speciosus and A. argenteus) initially buried nuts singly in shallow surface caches near the nut source, by the following spring these cached nuts were retrieved and re-cached in larger, deeper caches farther from the source, particularly in forest understories, probably to reduce the threat of pilferage. All the nuts cached in the forest understories were consumed, but 4 seedlings emerged in gaps, apparently because of lower foraging activity in the gaps by the mice. Seed size was not correlated with cache depth or cache site selection. With increasing seed size, transport distance increased, particularly in gaps, pos...

Pollen Tube Growth and Fertilization in Japanese Chestnut. (Castanea crenata Sieb. et Zucc.).

Pollen tube growth and the location of ovules within ovaries in Japanese chestnut (Castanea crenata Sieb. et Zucc.) were investigated in relation to fertilization. Pollen tubes were first observed on 18 July in the upper part of ovaries in both central and lateral female (pistillate) flowers ; 2 days later, 36 and 11 pollen tubes were counted in the former and the latter, respectively. The number of pollen tubes in central female flowers began to decrease gradually from 26 July, and decreased rapidly after 28 July. In lateral female flowers, the number of pollen tubes also decreased gradually after a maximum of 16 on 24 July. The distances from the top of ovaries to the nucelli in central female flowers ranged 3-6mm on 18-24 July and 5-7mm after 26 July. In lateral female flowers, the distances increased to 3-5mm, 4-6mm, and 5-7mm on 18-20 July, 22 July, and afterward, respectively. The time of fertilization was estimated to be 26-28 July in central female flowers based on the observation of the pollen tube within the micropyle, and 24-26 July in lateral female flowers based on the onset of nucellar degeneration. On 26 and 28 July, the numbers of pollen tubes reaching the zone where the nucelli are located in central female flowers were 2 and 9, respectively ; in lateral female flowers, the frequency of pollen tubes on 24 and 26 July were 1 and 11, respectively, indicating a rapid increase in the number of pollen tubes approaching the ovules at fertilization. The bur weight increased rapidly from 18 July, when the pollen tubes first penetrated into the ovaries ; the burs were covered with spire scales and stinging hairs that began to develop. At fertilization, 26-28 July, the stinging hairs covered all styles ; by the time the embryo appeared on 1 August, they covered the entire surface of the burs in central and lateral female flowers.

Complete Amino Acid Sequence of Japanese Chestnut Agglutinin

The complete amino acid sequence of Japanese chestnut (Castanea crenata Sieb. et Zucc.) agglutinin (CCA) was determined. Analysis by SIMS of the acidic peptide obtained by pepsin digestion revealed that the N-terminal amino acid sequence should be Acetyl-Met-Glu-Glu. Prior to sequence analysis, redetermination of cysteine residues indicated the presence of one cysteine residue per subunit. The complete sequence was determined by endoproteinase Arg-C and Achromobacter protease I digestion, and CNBr cleavage. CCA consists of 309 amino acid residues with a high content of glycine (16.5 mol%) and one cysteine residue. The calculated molecular mass was 33,387 Da including the N-terminal acetyl group. C-terminal sequence analysis of intact CCA gave only one sequence, HMEYF, indicating that no heterogeneous CCA formed by posttranslational cleavage at the C-terminal region, as occurs in some legume lectins. Analysis of the sequence of CCA itself revealed that CCA could be divided into two structural domains, the N-domain and the C-domain, almost at the center. These domains share about 35% identical residues, so CCA has a repeat sequence. Also, both domains show a homology to jacalin-related lectins with 27–38% identity. These results suggest that the structure of CCA resembles two molecules of jacalin-related lectin.

Chestnut Production in the Northwestern United States

The chestnut (Castanea Mill.) industry in the northwestern United States is in its relative infancy, with most orchards being less than 10 years of age. Currently there are an estimated 300 acres (121 ha) in Oregon and Washington. California has about 500 acres (202 ha) in chestnuts. Current worldwide production is over 500,000 tons (435,600 t). China is the leading producer with 40%, followed by Korea at 15%. Italy, Turkey and Japan grow 10% each, while France, Greece and Spain grow 4% each. The United States, Chile, Argentina, New Zealand and Australia each grow less than 1%. The value of chestnuts imported into the United States is estimated to be $10 to 15 million annually. Domestic producers hope to displace some of the imported chestnuts in the marketplace. The leading variety being grown in the western United States is `Colossal,' a hybrid between european chestnut (C. sativa Mill.) and japanese chestnut (C. crenata Gillet). `Dunstan' hybrids are chestnut blight (Cryphonectria parasitica Murr.) resistant, and were bred in Florida using chinese chestnut (C. mollisima Blume) and american chestnut (C. dentata Marsh. Borkh.) parentage. Prices received by chestnut producers in the northwestern United States have ranged from $1.20 to $7.00/lb ($2.64 to $15.40/kg). The marketing of chestnuts has been through brokers into wholesale markets, farmers markets, mail order and direct sales through catalogues and World Wide Web sites.

Purification and characterization of a mannose/glucose-specific lectin from Castanea crenata

A hemagglutinin was purified from the cotyledons of Japanese chestnut (Castanea crenata Sieb. et Zucc.) by affinity chromatography on asialo-fetuin Sepharose 4B column followed by anion-exchange and gel permeation chromatography. The hemagglutinating activity of Castenea crenate agglutinin (CCA) was strong for sialidase-treated human erythrocytes, but was inhibited by mannose, glucose, and their derivatives as well as by glycoproteins having an N-linked complex carbohydrate type. The apparent M r of intact CCA was determined to be ca 257,000 by gel filtration using a Superose 12 column. In SDS-PAGE, under reducing and non-reducing conditions, CCA migrated as a single band of M r 37,000. Therefore, the intact CCA may be composed of six or eight identical subunits without disulfide bonds. In addition, CCA showed strong mitogenic activity similar to other lectins. The N-terminal amino acid of CCA may be blocked since no amino acid was detected by direct sequence analysis. Amino acid analysis showed that CCA was rich in glycine, but did not contain cysteine residues. Some properties of CCA were similar to mannose/glucose-specific legume lectins, but our data suggest that the molecular structure of CCA is different.

Seasonal variation of peroxidase activity in chestnut trees

Peroxidase (E.C.1.11.1.7) activity in twigs of American ( Castanea dentata), Chinese ( C. mollissima), and Japanese ( C. crenata) chestnut was determined monthly over an eight month period. The total amount of peroxidase activity was highest in December and April for most trees. An inhibitor of peroxidase activity was present in the extracts which could be removed by gel filtration. Extracts from healthy twigs had consistent major bands on IEF gels. However, a new band was detected in bark of American chestnut after wounding or infection with chestnut blight fungus ( Cryphonectria parasitica).

The Role of Phenolic Substances in the Adhesion between the Pellicle and Kernel of Japanese Chestnut under Experimental Conditions.

The adhesion process between the pellicle and kernel under heat (62°C) and hypobaric (4 mm Hg) conditions was analyzed in the immature Japanese chestnut, which is easy to peel. The adhesion process under the experimental conditon was similar to that occurring in nature. The peeling time of the nuts heated for 10 hr was about 5 times longer than that of the nuts subjected to hypobaric treatments. Monophenolic substances in the water extracts from pellicle, composed mainly of (+)-catechin and gallic acid identified by GC and GC-MS, diffused to the outer cells of the kernel and the interface between the pellicle and kernel during the first 2 hr of both treatments. There the low molecular weight phenolic substances, including (+)-catechin, polymerized as noted by the shift of their peaks after 2-5 hr of heating. With continued heating, the content of phenolic substances in the alcohol insoluble solids (AIS) fraction of the pellicle increased rapidly and the pellicle tightly adhered to the kernel. Therefore, we postulate that the polyphenolic substances are the adhesive between pellicle and kernel. However, under hypobaric conditions, cell breakdown and migration of the phenolic substances in the tissues occurred during the initial 0-2 hr and ceased. Thus, little or no polyphenolic substances accumulated between the pellicle and kernel to cause their adhesion after 10 hr of treatment.

Partial Purification, Estimated Chemical Structure, and the Adhesive Property of Castahesion between the Pellicle and Kernel of Chestnut.

An adhesive substance, H-2, isolated by HPLC is present in the pellicle of the Japanese chestnut which makes it difficult to peel. The adhesive force of H-2 was measured by applying it between two wooden test pieces and its hemagglutination activity using in sheep and rabbit erythrocytes. The hemagglutination of H-2 was not inhibited by sorbitol, glucose, sucrose, glucosamine, Nacetyl-glucosamine or (+)-catechin ; its was accelerated by divalent cations, Ca2+, Mg2+, and Mn2+. The pyrolysis-gas chromatogram pattern of H-2 was similar to that of (+)-catechin. The IR spectrum had peaks of 3450 cm-1 (OH) and 1605, 1520 cm-1 (aromatic ring). The molecular weight of H-2, determined by HPLC, was approximately 1500. These findings indicate that H-2 is a polymer containing (+)-catechin. This adhesive substance between the pellicle and kernel of the chestnut is called Castahesion.

Genetic Characterization of Asian Chestnut Varieties Assessed by AFLP.

The genetic diversity of 24 Asian chestnut varieties, including 14 Japanese chestnuts (Castanea crenata) originating in Japan or the Korean Peninsula, 7 Chinese chestnuts (C. mollissima) and 3 other Castanea species, was characterized by AFLP (amplified fragment length polymorphism) using 9 primer combinations. About 60% of the amplified fragments (165/271) were polymorphic bands which were observed on both intra-species and inter-species. All varieties were identified from the differences of more than 14 bands and a lot of variety-specific AFLP fragments could be obtained. A phenogram of chestnut varieties was constructed based on polymorphisms. According to the phcnogram, all C. crenata cultivars originating in Japan were grouped into the same cluster with Shibaguri varieties which were considered the native species. In contrast, the genetic diversity of C. crenata cultivars from the Korean Peninsula was fairly extensive compared to that of C. crenata in Japan. These results suggest the possibility that cultivated chestnut in Japan might have been derived from Shibaguri or the possibility that both might have originated from the same ancestor. In addition, the genetic diversity among C. mollissima and the genetic relationship between C. crenata and C. mollissima were discussed.

Fast pattern recognition using a neurocomputer

This paper presents a new pattern recognition system based on moment invariants using a neurocomputer. The new pattern recognition system consists of a CCD video camera, an image processing system named FDM, a monitor, two stand lights, an NEC PC-9801 microcomputer and a RICOH RN-2000 neurocomputer; these two different types of computers can be considered to constitute an artificial brain. Experimental studies to recognize five dynamic patterns of Japanese chestnuts were performed. From the studies, a high speed of both learning and recognition has been achieved compared with the former pattern recognition system based on the software of artificial neural networks developed by us.

Effects of gall formation by Dryocosmus kuriphilus Yasumatsu (Hym., Cynipidae) on the growth of chestnut trees

AbstractEffects of gall formation by Dryocosmus kuriphilus on the growth of the Japanese chestnut tree (Castanea crenaia) were investigated from 1988 to 1989 using 15 wild trees. Mean leaf area on galled shoots currently expanded was about a half that on ungalled shoots. Galled shoots were lower than ungalled shoots both in biomass ratio of leaf/shoot and in mean number of winter buds per shoot. Bud expansion on galled shoots was less successful. Total biomass of current shoots diminished under heavy infestation with D. kuriphilus. Thus, D. kuriphilus not only retarded the development of photosynthetic organs but also reduced the biomass production of host trees in the next year. This may be a causal mechanism of biomass decline in gall‐infested trees.

Four New C-Glycosidic Ellagitannins, Castacrenins D-G, from Japanese Chestnut Wood (Castanea crenata SIEB. et ZUCC.).

In the course of studies on polyphenol metabolism in wood, four new C-glycosidic ellagitannins named castacrenins D-G were isolated from the wood of the Japanese chestnut tree (Castanea crenata). Castacrenins D and F are related to vescalagin and have a gallic acid and an ellagic acid moiety, respectively. Castacrenins E and G are oxidative metabolites of castacrenins D and F, respectively, in which a pyrogallol ring of each C-glycosidic ellagitannin unit is replaced by a cyclopentenone ring.

クリ果実における渋皮組織の発達と剥皮に関する形態学的研究

The developmental processes and the ease of peeling the pellicle in Japanese and Chinese chestnuts were investigated with light phase and scanning and transmission electron microscopes.During the early growth stage of Japanese chestnut, tannin accumulated in pellicle cells as scattered granules in vacuoles, aggregates along tonoplasts, electron-dense vacuoles, vacuoles with high density of tannin, etc. At harvest, however, most of the pellicle cells were filled with tannin. The tannin cells, which had high electron-dense vacuoles, were located mainly in the middle and outer layers of pellicle, whereas those adjacent to the cotyledons accumulated less tannin.Various aspects related to the ease of peeling the pellicle, i. e. the adhesive force between pellicle and cotyledon were examined but aspects specific to the variety were not recognized.In Japanese chestnut varieties, whose pellicles are difficult to peel, tannin accumulated in most of the cells of pellicle tissues; the cells in contact with the cotyledon and those in the middle layers of pellicle were crushed during the growth of fruits. The low electron density matrix and the electron-dense structure of network developed between the pellicle and the epidermis of the cotyledon. The network structure is probably made of tannins which penetrates into the epidermal cells of the cotyledon.In Chinese chestnut varieties, whose pellicle peels easily, the pellicle cells accumulate little tannin; those in contact with the cotyledon degrade slowly. No penetration of electron-dense structure of network into epidermal cells of the cotyledon occurs.These finding suggest that the adhesive force between the pellicle cells and the cotyledon depends, not only on tannin accumulation in the pellicle cells, but also on the degradation of the cells contacting the cotyledon.