Development of microsatellite markers in white birch (Betula platyphylla var. japonica)

Betula platyphylla var. japonica is a typical pioneer tree species in the secondary succession in northern Japan. We describe the cloning and characterization of 13 polymorphic, codominant microsatellite loci isolated from this species. These polymorphic loci had 2–8 alleles per locus and a range of expected heterozygosities from 0.050 to 0.808.     

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 Out of 98 unpublished primer pairs, 35 yielded clear PCR amplification products, 11 of which revealed polymorphism in eight individuals sampled across the species’ range.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.     

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.     

Mesophyll conductance in leaves of Japanese white birch (Betula platyphylla var. japonica) seedlings grown under elevated CO2 concentration and low N availability

To test the hypothesis that mesophyll conductance (g_m) would be reduced by leaf starch accumulation in plants grown under elevated CO₂ concentration [CO₂], we investigated g_m in seedlings of Japanese white birch grown under ambient and elevated [CO₂] with an adequate and limited nitrogen supply using simultaneous gas exchange and chlorophyll fluorescence measurements. Both elevated [CO₂] and limited nitrogen supply decreased area‐based leaf N accompanied with a decrease in the maximum rate of Rubisco carboxylation (V_c,max) on a CO₂ concentration at chloroplast stroma (C_c) basis. Conversely, only seedlings grown at elevated [CO₂] under limited nitrogen supply had significantly higher leaf starch content with significantly lower g_m among the treatment combinations. Based on a leaf anatomical analysis using microscopic photographs, however, there were no significant difference in the area of chloroplast surfaces facing intercellular space per unit leaf area among treatment combinations. Thicker cell walls were suggested in plants grown under limited N by increases in leaf mass per area subtracting non‐structural carbohydrates. These results suggest that starch accumulation and/or thicker cell walls in the leaves grown at elevated [CO₂] under limited N supply might hinder CO₂ diffusion in chloroplasts and cell walls, which would be an additional cause of photosynthetic downregulation as well as a reduction in Rubisco activity related to the reduced leaf N under elevated [CO₂].     

In vitro shiro formation between the ectomycorrhizal basidiomycete Tricholoma matsutake and Cedrela herrerae in the Mahogany family (Meliaceae)

Tricholoma matsutake is an ectomycorrhizal basidiomycete that associates with Pinaceae plants, forming a rhizospheric mycelial aggregate called “shiro” from which the prized “matsutake” mushrooms form. Here we document that T. matsutake associates in vitro with Andean Cedrela herrerae (Meliaceae) via root endophyte interactions and efficiently forms shiro. C. herrerae produces many branches, leaves, and lateral roots in association with T. matsutake, unlike C. odorata, which grows in the tropics and produces few leaves and branches in association with the symbiont. This symbiosis may be a unique approach to culturing matsutake as well as to cultivating endangered plant species in vitro.     

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.     

Effect of ambient ozone at the somma of Lake Mashu on growth and leaf gas exchange in Betula ermanii and Betula platyphylla var. japonica

We examined the effects of ambient ozone, at the somma of Lake Mashu in northern Japan, on the growth and photosynthetic traits of two common birch species in Japan (mountain birch and white birch). Seedlings of the two birch species were grown in open-top chambers and were exposed to charcoal-filtered ambient air (CF) or non-filtered ambient air (NF) at the somma of Lake Mashu during the growing season in 2009. For the mountain birch, ambient ozone significantly increased the ratio of aboveground dry mass to belowground dry mass (T/R ratio), although no difference in the whole-plant biomass was observed between the treatments. For the white birch, in contrast, ozone exposure at ambient level did not decrease in growth and photosynthesis. These results suggest that ambient O₃ at the somma of Lake Mashu may shift the allocation of biomass to above-ground rather than below-ground in the mountain birch.     

Elevated CO2 and limited nitrogen nutrition can restrict excitation energy dissipation in photosystem II of Japanese white birch (Betula platyphylla var. japonica) leaves

Elevated atmospheric CO₂ concentration [CO₂] and different levels of nitrogen (N) nutrition can influence the amount of excess excitation energy in photosystem (PS) II and related photosynthetic properties. The interactive effect of two [CO₂] levels (ambient: 360 µM M⁻¹ and elevated: 720 µM M⁻¹) and two N levels (high: 700 mg N plant⁻¹ and low: 100 mg N plant⁻¹) on these properties was examined in seedlings of Japanese white birch (Betula platyphylla var. japonica) using simultaneous measurements of gas exchange and chlorophyll fluorescence. Photosynthetic acclimation to elevated [CO₂], as indicated by a decline in carboxylation efficiency (CE), was observed in plants grown at elevated [CO₂] especially under low N. Elevated [CO₂] resulted in a decrease in area-based leaf N content (N_area) irrespective of N treatment. The adverse effect of elevated [CO₂] and low N on CE may have been exacerbated by a greater accumulation of leaf sugar and starch contents in these plants leading to a lower electron transport rate (ETR). While these plants also showed higher non-photochemical quenching (Nq_P) that could offset the reduction in energy dissipation through ETR to some extent, they still have a higher risk of photoinhibition from excessive excitation energy in PSII as indicated by a decrease in photochemical quenching (q_P). However, chronic photoinhibition was not observed in plant grown at elevated [CO₂] and low N because they showed no difference in F_v/F_m (the maximum photochemical efficiency of PSII) from those grown at ambient [CO₂] and low N after an overnight dark adaptation. High levels of Nq_P in plants grown at elevated [CO₂] and low N reflect a near saturation of thermal energy dissipation. This impaired capacity of photoprotection would render these plants more vulnerable to photoinhibition in the event of additional environmental stresses such as drought, low or high temperature.     

Effects of isolates of ectomycorrhizal fungi and endophytic Mycelium radicis atrovirens that were dominant in soil from disturbed sites on growth of Betula platyphylla var. japonica seedlings

Effects of ectomycorrhizal fungi and endophytic Mycelium radicis atrovirens Melin (MRA) on growth of Betula platyphylla var. japonica seedlings were investigated under aseptic culture conditions. Three isolates of ectomycorrhizal fungi and two isolates of MRA were used. One MRA isolate was Phialocephala fortinii. Previous field work revealed that these isolates were dominant on the roots of B. platyphylla var. japonica seedlings grown in a mineral subsoil that had been exposed by the removal of surface soil. After a 100-day incubation, the growth of the seedlings was significantly enhanced by the colonization of these ectomycorrhizal fungal isolates as compared with uninoculated seedlings. In contrast, the growth of seedlings was retarded by the colonization of the MRA isolates. The growth of seedlings that were co-inoculated with ectomycorrhizal fungi and MRA was similar to that of uninoculated seedlings in most cases. These results suggest that ectomycorrhizal fungi have a beneficial effect on the growth of B. platyphylla var. japonica seedlings and that they suppress the deleterious effect of MRA. Thus, these ectomycorrhizal fungi probably have an important role in establishing B. platyphylla var. japonica seedlings during the initial stage of re-vegetation following site disturbance by the removal of surface soil.     

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.     

长白山阔叶红松林和杨桦次生林土壤有机碳氮的协同积累特征

次生演替是森林土壤有机碳、氮库变化的重要驱动因素.本研究以长白山原始阔叶红松林和杨桦次生林为例,通过成对样地途径,研究了森林土壤有机碳、氮的数量分布及其协同积累特征,探讨了次生演替导致的温带森林土壤碳库和碳汇效应变化及其碳氮耦合机制.结果表明: 杨桦次生林比原始阔叶红松林在土壤表层和亚表层(0～20 cm)积累了更多的有机碳和氮,其土壤C/N值也显著低于阔叶红松林;相对于阔叶红松林,杨桦次生林土壤(0～20 cm)有机碳储量平均增加了14.7 t·hm^-2,相当于29.4 g·m^-2·a^-1的土壤碳汇增益.土壤有机碳和全氮在不同林型的不同土层中均表现为极显著正相关,二者具有明显的协同积累特征.与阔叶红松林生态系统相比,相对富氮的杨桦次生林生态系统的上部土层中氮对有机碳的决定系数明显高于阔叶红松林,说明杨桦次生林土壤有机碳的积累在更大程度上依赖含氮有机质积累.在有机质最丰富的表层(0～10 cm),两种林型间轻组有机碳、氮储量无显著差异,但杨桦次生林重组有机碳、氮的含量、储量及分配比例均显著高于阔叶红松林,其中,重组有机碳储量平均增加了8.5 t·hm^-2,表明次生演替过程中土壤有机碳、氮库的增加主要在于矿物质结合态稳定性土壤有机碳、氮库的增容.凋落物分解和稳定性土壤有机质形成中的碳氮耦合机制是次生演替过程中土壤有机碳、氮库变化的重要驱动机制.     

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.