Development of 11 EST-SSRs for Japanese white birch, Betula platyphylla var. japonica and their transferability to related species

Simple sequence repeat (SSR) markers were developed for Japanese white birch, Betula platyphylla var. japonica, using previously designed primer pairs derived from expressed sequence tags (ESTs). Out of 98 unpublished primer pairs, 35 yielded clear PCR amplification products, 11 of which revealed polymorphism in eight individuals sampled across the species’ range. The number of alleles detected and the expected heterozygosity ranged from 1 to 10 and 0.000 to 0.857, respectively, when these 11 loci were examined in 24 individuals from a single B. platyphylla var. japonica population. In cross-species transferability tests most of the 11 loci were also polymorphic in three other Betula species examined, but not B. maximowicziana. We have now developed a total of 25 polymorphic EST-SSRs for the genus Betula (including 14 we previously developed), which are likely to be highly useful in studies of various aspects of population genetics, including hybridization dynamics, in the genus.     

In vitro ectomycorrhizal specificity between the Asian red pine Pinus densiflora and Tricholoma matsutake and allied species from worldwide Pinaceae and Fagaceae forests

Tricholoma matsutake produces commercially valuable, yet uncultivable, mushrooms (matsutake) in association with pines in the Far East and Scandinavia and with both pines and oaks in the foothills of Tibet. Other matsutake mushrooms, such as Tricholoma anatolicum from the Mediterranean regions and Tricholoma magnivelare and Tricholoma sp. from the North Pacific Coast area of Canada and North America as well as Mexico, respectively, are associated with pines or oaks in their natural habitats. Tricholoma bakamatsutake and Tricholoma fulvocastaneum from Asia produce moderately valuable matsutake mushrooms and are solely associated with Fagaceae in nature. In this study, we demonstrate for the first time that matsutake mushrooms from Scandinavia, Mediterranean regions, North America, and Tibet form ectomycorrhizae with Pinus densiflora similar to the Far East T. matsutake. In general, worldwide T. matsutake and the symbionts of Pinaceae colonize the rhizospheres of P. densiflora as well as T. matsutake isolated from the host plant. However, T. fulvocastaneum and T. bakamatsutake formed a discontinuous Hartig net and no Hartig net, respectively, and colonized to a lesser extent as compared to T. matsutake. The data suggest that conifer-associated matsutake mushrooms in their native habitat will associate symbiotically with the Asian red pine.     

Antioxidant and anticancer activity of extract from Betula platyphylla var. japonica

The antioxidant and anticancer properties of a medicinal plant, Betula platyphylla var. japonica were investigated. The total methanol extract of B. platyphylla var. japonica had protective effects against hydrogen peroxide (H2O2) in the Chinese hamster lung fibroblast (V79-4) cell line and induced apoptotic cell death in human promyelocytic leukemia (HL-60) cells, a cancer cell line. B. platyphylla var. japonica extract significantly increased cell viability against H2O2. The extract also showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 2.4 microg/ml) and lipid peroxidation inhibitory activity (IC50 below 4.0 microg/ml). Furthermore, B. platyphylla var. japonica extract reduced the number of V79-4 cells arrested in G2/M in response to H2O2 treatment and increased the activities of several cellular antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. Treatment with B. platyphylla var. japonica extract induced cytotoxicity and apoptosis in HL-60 cells, as shown by nucleosomal DNA fragmentation, increases in the subdiploid cell population, and fluorescence microscopy. B. platyphylla var. japonica extract gradually increased the expression of pro-apoptotic Bax and led to the activation of caspase-3 and cleavage of PARP. These findings suggest that B. platyphylla var. japonica exhibits potential antioxidant and anticancer properties.     

Floral sex allocation at individual and branch levels in Betula platyphylla var. japonica (Betulaceae), a tall, wind-pollinated monoecious tree species

Floral sex allocation at the individual and first-order branch levels and the relation between these levels were examined in Betula platyphylla var. japonica, a wind-pollinated monoecious tree. Floral sex allocation at the individual level varied with resource availability in a pattern similar to that predicted by the Masaka and Takada model (Journal of Theoretical Biology 240: 114-125). Thus, individual trees with few reproductive resources produced only female or male inflorescences, whereas individuals with many resources rarely had a high male ratio (i.e., number of male inflorescences/total number of inflorescences). Furthermore, the number of male inflorescences tended to reach an upper limit, whereas the number of female inflorescences increased monotonically with increasing reproductive investment. The patterns of floral sex allocation at the first-order branch level were analogous to those at the individual level. Thus, each first-order branch of B. platyphylla var. japonica behaves like an individual, and the floral sex allocation of a given branch does not necessarily represent the individual tree. The effect of the individual-level floral sex ratio on branch-level floral sex allocation indicates that branch behavior is controlled by the individual.     

Root endophyte symbiosis in vitro between the ectomycorrhizal basidiomycete Tricholoma matsutake and the arbuscular mycorrhizal plant Prunus speciosa

We previously reported that Tricholoma matsutake and Tricholoma fulvocastaneum, ectomycorrhizal basidiomycetes that associate with Pinaceae and Fagaceae, respectively, in the Northern Hemisphere, could interact in vitro as a root endophyte of somatic plants of Cedrela odorata (Meliaceae), which naturally harbors arbuscular mycorrhizal fungi in South America, to form a characteristic rhizospheric colony or “shiro”. We questioned whether this phenomenon could have occurred because of plant–microbe interactions between geographically separated species that never encounter one another in nature. In the present study, we document that these fungi formed root endophyte interactions and shiro within 140 days of inoculation with somatic plants of Prunus speciosa (=Cerasus speciosa, Rosaceae), a wild cherry tree that naturally harbors arbuscular mycorrhizal fungi in Japan. Compared with C. odorata, infected P. speciosa plants had less mycelial sheath surrounding the exodermis, and the older the roots, especially main roots, the more hyphae penetrated. In addition, a large number of juvenile roots were not associated with hyphae. We concluded that such root endophyte interactions were not events isolated to the interactions between exotic plants and microbes but could occur generally in vitro. Our pure culture system with a somatic plant allowed these fungi to express symbiosis-related phenotypes that varied with the plant host; these traits are innately programmed but suppressed in nature and could be useful in genetic analyses of plant–fungal symbiosis.     

Reciprocal distribution of two congeneric trees, Betula platyphylla var. japonica and Betula maximowicziana, in a landscape dominated by anthropogenic disturbances in northeastern Japan

Aim Information has been compiled on disturbance regimes and the life‐history characteristics of Betula platyphylla var. japonica (Miq.) Hara and B. maximowicziana Regel to investigate the impact of humans on the present distribution patterns of these two congeneric tree species. Location The study area is in the central region of the northern Kitakami Mountain Range, located in the northeast of Honshu Island, Japan. Methods First, the present distributions of B. maximowicziana and B. platyphylla var. japonica were mapped at the landscape scale. To examine the factors affecting the distribution patterns, topographic features and past land use were taken into account. Second, life‐history traits of both species were clarified at various growth stages either by fieldwork or through a literature search. Previous studies have provided some information on seed production, seed dispersal, seedling dynamics, sprouting, and growth. In the present study, field observations and experiments were conducted regarding seed‐bank formation, size and age at reproduction, and the life span of canopy trees. Results Betula platyphylla var. japonica and B. maximowicziana were spatially segregated in the studied landscape (14,000 ha). The distribution of each species was correlated more strongly with land use at the beginning of the twentieth century than with site environmental factors such as altitude or slope angle. Betula platyphylla var. japonica was distributed more frequently on former grasslands, whereas B. maximowicziana was almost exclusively limited to past old‐growth forests. As typical pioneers, the two species showed similar life‐history traits but differed in several critical points. Betula platyphylla var. japonica has a vigorous sprouting ability, which might increase its resistance to burning and logging, whereas B. maximowicziana forms a persistent seed bank in the soil, indicating an advantage in regenerating in disturbances found in forest communities. Other critical differences were detected in age‐related characteristics such as minimum age of reproduction and life span. Main conclusions The earlier reproductive maturity and the shorter life span of B. platyphylla var. japonica indicate a shorter population cycle than that for B. maximowicziana. The latter would be excluded from grasslands that were burned frequently, as a result of the longer time span before initial reproduction and its poor sprouting ability. In contrast, B. platyphylla var. japonica would be excluded from old‐growth forests, where disturbance was infrequent, owing to its shorter life span and lack of a persistent seed bank.     

Tricholoma matsutake Y1 strain associated with Pinus densiflora shows a gradient of in vitro ectomycorrhizal specificity with Pinaceae and oak hosts

Tricholoma matsutake produces commercially valuable yet uncultivable matsutake mushrooms during an ectomycorrhizal association with coniferous trees. In the Far East, most matsutake are harvested in managed Pinus densiflora forests. To determine whether T. matsutake has host plant specificity, we synthesized mycorrhiza in vitro between T. matsutake Y1 that originated from a P. densiflora forest and various Pinaceae and oak hosts. The strain Y1 formed a continuous Hartig net, a sign of ectomycorrhization, in the lateral roots of Pinus sylvestris, Pinus koraiensis, Pinus parviflora var. pentaphylla, Picea glehnii, Picea abies, and Tsuga diversifolia seedlings in vitro, which resembled those formed with the natural host Pinus densiflora. The strain conferred a discontinuous Hartig net with Pinus thunbergii, Picea yezoensis, Abies veitchii, and Larix kaempferi. However, no such development by this strain was observed on the roots of Quercus serrata, unlike T. bakamatsutake B1, a false matsutake that is symbiotic with oak trees. The data suggest that T. matsutake can be associated with diverse conifers but may establish ectomycorrhizal relationships only with specific host plant species.     

Trichoderma songyi sp. nov., a new species associated with the pine mushroom (Tricholoma matsutake)

A new species, Trichoderma songyi, was found to be associated with the pine mushroom (Tricholoma matsutake) in Korea. This species was isolated from three different substrates: Tricholoma matsutake basidiomata, as well as roots of Pinus densiflora and soil in the fairy ring. Based on its molecular and phenotypic characteristics, we demonstrate that Trichoderma songyi is unique and distinguishable from closely related species. We performed phylogenetic analyses based on two molecular markers, the genes for both translation elongation factor 1-alpha and the second largest subunit of RNA polymerase II. Phylogenetic analyses showed that Trichoderma songyi is closely related to Trichoderma koningii aggregate and Trichoderma caerulescens. Morphologically, Trichoderma songyi can be distinguished from these closely related taxa by its growth rates, colony morphology on PDA in darkness, and coconut-like odour. Due to the economic importance of the pine mushroom, the relationship between Trichoderma songyi and Tricholoma matsutake should be studied further.     

Quantities and types of ectomycorrhizal and endophytic fungi associated with Betula platyphylla var. japonica seedlings during the initial stage of establishment of vegetation after disturbance

Ectomycorrhizal and endophytic fungi of Betula platyphylla Sukatchev var. japonica Hara seedlings were investigated by bioassay using soils from sites where the surface layer had been removed by destructive disturbances. Soil samples were taken from sites A, B, C and D, where 1, 2–3, 4–5, and 7–8 years, respectively had passed since disturbance. Naturally regenerated B. platyphylla var. japonica seedlings grew at sites C and D, but not at sites A or B. The percentages of ectomycorrhizal formation in seedlings were significantly lower in the soils from site A (4%) and site B (13%), compared to those in the soils from site C (53%) and site D (37%). The numbers of ectomycorrhizal morphologic types in sites A, B, C, and D were eight, five, one, and seven, respectively. The same dominant type of ectomycorrhiza was found in sites C and D, and this type was different from those in sites A and B. The frequencies of colonization of seedling roots by endophytic fungi, especially Mycelium radicis atrovirens Melin (MRA) in soils from sites A and B were 31 and 33%, respectively; these frequencies were significantly higher than those for site C (0%) and site D (2%). During the initial stage of establishment of vegetation following disturbance, the quantities and types of ectomycorrhizal fungi in the field that have the potential to associate with B. platyphylla var. japonica might rapidly change after invasion of the host plant. Ectomycorrhizal fungi seemed to compete with endophytic MRA fungi for colonization of the roots of B. platyphylla var. japonica seedlings.     

大兴安岭森林草原过渡带白桦及主要草本植物生态位关系的研究

以白桦、日阴菅及其它主要草本植物的个体数量为指标,分析了各植物种群在土壤有机质,速效P和pH值3个资源维上的生态位宽度和生态位重叠及其在不同海拔条件下的变化规律。日阴菅生态位宽度随海拔升高而增大,其余植物种类在有机质资源维上的生态位宽度,大都是以中等海拔（800m)的样带最宽,而在速效P资源维上,又以中等海拔的样带为最窄,由于高海拔及相应低气温在某种程度上限制了植物种群在土壤有机质和速效P的利用,主要植物种对之间在这两个资源维上的生态位重叠以高海拔（950m)样带为最小,在土壤pH值资源维上,溪荪与其它主要植物种间的生态位重叠皆以低海拔（650m)样带为最小,可能是其特殊的环境组合迫使溪荪发生了生态位移动,大多数种对在土壤有机质,速效P和pH值3个资源维上都以海拔800m的样带生态位重叠最大。     

Tricholoma matsutake in a natural Pinus densiflora forest: correspondence between above- and below-ground genets, association with multiple host trees and alteration of existing ectomycorrhizal communities

Tricholoma matsutake (matsutake) is an ectomycorrhizal (ECM) fungus that produces economically important mushrooms in Japan. Here, we use microsatellite markers to identify genets of matsutake sporocarps and below-ground ECM tips, as well as associated host genotypes of Pinus densiflora. We also studied ECM fungal community structure inside, beneath and outside the matsutake fairy rings, using morphological and internal transcribed spacer (ITS) polymorphism analysis. Based on sporocarp samples, one to four genets were found within each fairy ring, and no genetic differentiation among six sites was detected. Matsutake ECM tips were only found beneath fairy rings and corresponded with the genotypes of the above-ground sporocarps. We detected nine below-ground matsutake genets, all of which colonized multiple pine trees (three to seven trees per genet). The ECM fungal community beneath fairy rings was species-poor and significantly differed from those inside and outside the fairy rings. We conclude that matsutake genets occasionally establish from basidiospores and expand on the root systems of multiple host trees. Although matsutake mycelia suppress other ECM fungi during expansion, most of them may recover after the passage of the fairy rings.     

Divergent Selection and Local Adaptation in Disjunct Populations of an Endangered Conifer,Keteleeria davidiana var. formosana (Pinaceae)

The present study investigated the genetic diversity, population structure, F _ST outliers, and extent and pattern of linkage disequilibrium in five populations of Keteleeria davidiana var. formosana, which is listed as a critically endangered species by the Council of Agriculture, Taiwan. Twelve amplified fragment length polymorphism primer pairs generated a total of 465 markers, of which 83.74% on average were polymorphic across populations, with a mean Nei’s genetic diversity of 0.233 and a low level of genetic differentiation (approximately 6%) based on the total dataset. Linkage disequilibrium and HICKORY analyses suggested recent population bottlenecks and inbreeding in K. davidiana var. formosana. Both STRUCTURE and BAPS observed extensive admixture of individual genotypes among populations based on the total dataset in various clustering scenarios, which probably resulted from incomplete lineage sorting of ancestral variation rather than a high rate of recent gene flow. Our results based on outlier analysis revealed generally high levels of genetic differentiation and suggest that divergent selection arising from environmental variation has been driven by differences in temperature, precipitation, and humidity. Identification of ecologically associated outliers among environmentally disparate populations further support divergent selection and potential local adaptation.     

Erysiphe havrylenkoana and E. prunastri var. japonica: a new species and a new variety of Erysiphe sect. Uncinula (Erysiphaceae, Ascomycota)

A new species and a new variety of Erysiphe sect. Uncinula are described and illustrated. Erysiphe havrylenkoana is a new species found on Nothofagus alpina and N. obliqua in Argentina, and E. prunastri var. japonica is a new variety collected on Prunus maximowiczii in Japan. Erysiphe havrylenkoana differs from E. nothofagi, E. patagoniaca and E. magellanica in having unique helicoid appendages, brownish from the base to the beginning of helicoid part, spirally twisted at the center and straight or not spiral at the upper part with uncinate to circinate tip. The phylogenetic analyses of rDNA sequences clearly showed that the spiral pattern of helicoid appendages is an important morphological character to delimit species among Nothofagus powdery mildews. Erysiphe prunastri var. japonica is distinct from E. prunastri var. prunastri parasitizing hitherto Prunus species from East-Central Asia to Europe by having mainly eight asci per chasmothecium and smooth, rarely septate appendages.     

Competitive interactions between Microdochium nivale var. majus, M. nivale var. nivale and Fusarium culmorum in planta and in vitro

Microdochium nivale var. majus and var. nivale are economically important fungal pathogens of cereal seedlings, stem bases and ears, as is the toxigenic species Fusarium culmorum. Competition experiments on seedlings support an earlier report of differential host preference between the varieties of M. nivale on wheat and rye seedlings at 15 degrees C, but showed that it does not extend across a broad range of temperatures. The studies showed that, although interaction is disadvantageous to the less virulent pathogen, it does not confer an advantage to the more virulent pathogen. In mixed inoculum experiments on wheat seedlings at 15 degrees C and 20 degrees C, F. culmorum suppressed the growth of both varieties of M. nivale. However, if M. nivale var. majus became established on the seedlings, it was able to co-suppress colonization of wheat seedlings by F. culmorum. In contrast M. nivale var. nivale did not suppress F. culmorum significantly. The growth of M. nivale var. majus and F. culmorum was also co-suppressed in liquid culture. Significantly, the accumulation of deoxynivalenol mycotoxin was also reduced in the mixed in vitro culture compared with axenic culture of F. culmorum. However, in vitro interaction studies on solidified media were of only limited use in predicting the outcome of competitions in planta.     

Inheritance of caffeine and heteroside contents in an interspecific cross between a cultivated coffee species Coffea liberica var dewevrei and a wild species caffeine-free C. pseudozanguebariae

 Coffee species originating from Africa, in particular the two major cultivated species C. arabica and C. canephora, usually contain caffeine in their beans, whereas almost all Malagasy coffee species are caffeine-free. However, one wild coffee species C. pseudozanguebariae, collected near the coast in south Kenya, is also caffeine-free. Beans of this species contain a specific heteroside diterpene (hereinafter referred to simply as heteroside) and give a bitter coffee beverage. We have investigated the inheritance of the caffeine and heteroside contents in the first and second generations of an interspecific cross between C. pseudozanguebariae and C. liberica var. dewevrei, for which the caffeine content is about 1% dmb (dry matter basis). The caffeine content of F₁ hybrids (0.2% dmb) was lower than the parental average (0.47% dmb). Caffeine and heteroside contents appeared to be under polygenic control with a strong genetic effect. Nevertheless, one major gene with two alleles seemed to be involved in the control of both compounds. Absence of caffeine was apparently controlled by one recessive gene. Heteroside content seemed to be controlled by one co-dominant gene, heterozygotes being intermediate between the two different groups of homozygotes. Received: 15 September 1997 / Accepted: 6 October 1997     

Isolation and characterization of 19 microsatellite markers in a tropical and warm subtropical birch, Betula alnoides Buch.-Ham. ex D. Don

Betula alnoides is an ecologically and economically important species in the tropics and warm subtropics. Nineteen polymorphic microsatellite markers were isolated from this species, which displayed three to 12 alleles per locus. The observed heterozygosities ranged from 0.100 to 0.905, and the expected heterozygosities from 0.510 to 0.893. These markers would be useful tools in genetic resource assessment, molecular marker-assistant breeding, parentage analysis and genetic diversity studies for this species.     

Allocation,within and between organs,and the dynamics of root length changes in two birch species

Spatial and temporal dynamics of biomass allocation within and between organs were investigated in seedlings of two birch species of contrasting successional status. Seedlings of Betula alleghaniensis Britt (yellow birch) and B. populifolia Marsh (gray birch) were grown for 6 weeks at two nutrient levels in rectangular plexiglass containers to allow non-destructive estimates of root growth, production and loss. Leaf area and production were simultaneously monitored. Yellow birch responded more to nutrient level than gray birch in terms of total biomass, shoot biomass, leaf area and root length. Yellow birch also flexibly altered within-organ allocation (specific leaf area, specific root length and specific soil amount). In contrast, gray birch altered between-organ allocation patterns (root length:leaf area and soil amount:leaf area ratios) more than yellow birch in response to nutrient level. Yellow birch showed greater overall root density changes within a very compact root system, while gray birch showed localized root density changes as concentric bands of new root production spread through the soil. Species differ critically in their responses of standing root length and root production and loss rates to nutrient supply. Early successional species such as gray birch are hypothesized to exhibit higher plasticity in varied environments than later successional species such as yellow birch. Our results suggest that different patterns of allocation, within and between plant organs, do not necessarily follow the same trajectories. To characterize thoroughly the nature of functional flexibility through ontogeny, within- and between-organ patterns of allocation must be accounted for.