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.     

Effects of imidazole-4-carboxamide and 2-azahypoxanthine on the growth and ectomycorrhizal colonization of Pinus densiflora seedlings inoculated with Tricholoma matsutake

Imidazole-4-carboxamide (ICA) and 2-azahypoxanthine (AHX) obtained from Lepista sordida inhibit and promote the growth of herbaceous plants, respectively. In this study, we examined the effects of these compounds on the growth and ectomycorrhizal (EM) colonization of Pinus densiflora seedlings inoculated with Tricholoma matsutake that forms EM associations with pines. The EM colonization by T. matsutake was observed on the root systems of P. densiflora seedlings treated with and without ICA and AHX. The growth of both non-EM and EM P. densiflora seedlings was inhibited by ICA, regardless of the EM colonization. In contrast, AHX promoted the growth of non-EM P. densiflora seedlings, but not of EM seedlings, suggesting that EM colonization interferes with the effect of AHX on P. densiflora growth.     

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.     

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.     

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.     

Recombination and genetic differentiation among natural populations of the ectomycorrhizal mushroom Tricholoma matsutake from southwestern China

Effective conservation and utilization strategies for natural biological resources require a clear understanding of the natural populations of the target organisms. Tricholoma matsutake is an ectomycorrhizal mushroom that forms symbiotic associations with plants and plays an important ecological role in natural forest ecosystems in many parts of the world. It is also an economically very important gourmet mushroom. Because no artificial cultivation is available, natural populations of this species are under increasing threats, primarily from habitat disturbance and destruction. Despite its economical and ecological importance, little is known about its genetics and population biology. Here, using 14 polymerase chain reaction–restriction fragment length polymorphism markers, we analysed 154 strains from 17 geographical locations in southwestern China, a region where over 25% of the global T. matsutake harvest comes from. Our results revealed abundant genetic variation within individual populations. The analyses of gene and genotype frequencies within populations indicated that most loci did not deviate from Hardy–Weinberg equilibrium in most populations and that alleles among loci were in linkage equilibrium in the majority of the local populations. These results are consistent with the hypothesis that sexual reproduction and recombination play an important role in natural populations of this species. Our analyses indicated low but significant genetic differentiation among the geographical populations, with a significant positive correlation between genetic distance and geographical distance. We discuss the implications of our results to the ecology and resource management of this species.     

Preparation and the antitumor activity in vitro of polysaccharides from Tricholoma matsutake

Tricholoma matsutake is the most valuable mushroom exhibiting a characteristic and delicate flavor as well as several biological activities among the various mushroom species. Extraction procedure and antitumor activity of polysaccharides from Tricholoma matsutake mycelium were studied in this work. Single factor tests and Response Surface Methodology (RSM) based on a three-level three-factor Box-Behnken design were applied to optimize the water extraction conditions. The polysaccharides yields increased when the ratio of water to sample increased from 10:1 to 30:1, extraction time from 1 to 4 h and temperature from 60 to 90°C as invested in single factor tests. To enhance further polysaccharides extraction, the extraction conditions were studied with RSM. The optimum extraction conditions were confirmed as follows:extraction time 4.3 h, the ratio of water to sample 29.2:1, extraction temperature 93.8°C. The extraction rate of polysaccharides under this condition could reach the highest value of 18.43% with one time extraction, and, 21.63% with two times extraction. Furthermore, the polysaccharides presented significant antitumor activity against B16 human melanoma cells in vitro in a dose-dependent manner. At the concentration of 10 mg/ml, the inhibition rate of polysaccharides got 67%.     

Intra- and inter-specific variations in the copy number of two types of retrotransposons from the ectomycorrhizal basidiomycete Tricholoma matsutake

To explore intra- and inter-specific variations of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces the fruit body matsutake, we carried out real-time PCR analysis based on two types of retrotransposons, one designated marY1, which resembles a retrovirus carrying the long terminal repeat (LTR) and the other marY2N, which resembles mRNA carrying the polyadenylated tail. Calculation based on the average genome size of homobasidiomycetes (34 Mbp) shows that ca. 5.5% of the total genome of T. matsutake isolated from Asia is made up of these retrotransposons, whereas they occupy ca. 1.4% in the isolates from Morocco, ca. 0.8% in isolates from Mexico, and ca. 0.5% in Tricholoma magnivelare, the species which produces American matsutake. Other Tricholoma spp. that produce fruit bodies similar to those of T. matsutake, such as T. bakamatsutake, T. fulvocastaneum, and T. robustum, carry them in the region less than 0.05% of their total genome. Copy number of LTR of marY1 is consistently and markedly higher than that of the coding regions of marY1 and marY2N. Data suggest that retrotransposons are deeply involved in evolution of the ectomycorrhizal symbiont.     

Spatially variable natural selection and the divergence between parapatric subspecies of lodgepole pine (Pinus contorta, Pinaceae)

? Premise of the study: Plant populations arrayed across sharp environmental gradients are ideal systems for identifying the genetic basis of ecologically relevant phenotypes. A series of five uplifted marine terraces along the northern coast of California represents one such system where morphologically distinct populations of lodgepole pine (Pinus contorta) are distributed across sharp soil gradients ranging from fertile soils near the coast to podzolic soils ca. 5 km inland. ? Methods: A total of 92 trees was sampled across four coastal marine terraces (N = 10-46 trees/terrace) located in Mendocino County, California and sequenced for a set of 24 candidate genes for growth and responses to various soil chemistry variables. Statistical analyses relying on patterns of nucleotide diversity were employed to identify genes whose diversity patterns were inconsistent with three null models. ? Key results: Most genes displayed patterns of nucleotide diversity that were consistent with null models (N = 19) or with the presence of paralogs (N = 3). Two genes, however, were exceptional: an aluminum responsive ABC-transporter with F(ST) = 0.664 and an inorganic phosphate transporter characterized by divergent haplotypes segregating at intermediate frequencies in most populations. ? Conclusions: Spatially variable natural selection along gradients of aluminum and phosphate ion concentrations likely accounted for both outliers. These results shed light on some of the genetic components comprising the extended phenotype of this ecosystem, as well as highlight ecotones as fruitful study systems for the detection of adaptive genetic variants.     

Seasonal changes in needle water content and needle ABA concentration of Japanese red pine, Pinus densiflora, in declining forests on Mt. Gokurakuji, Hiroshima prefecture, Japan

To evaluate the effects of air pollution on the decline of Pinus densiflora forests, various research has been conducted around Mt. Gokurakuji (34° 23N, 132° 19E, 693 m a.s.l.) north of the Seto Inland Sea, west Japan. To investigate the mechanisms responsible for decreases in photosynthesis (Pn) and stomatal conductance (gl), ¹³C of needles and seasonal changes in the water content (WC) and abscisic acid concentration (ABA) of needles were measured in various stands. The ¹³C values were less negative in declining stands and younger needles. ABA and WC were not correlated with each other. WC decreased consistently with needle age while the ABA showed a minimum in August and a smaller content in older needles. Monthly precipitation and the daily maximum vapor pressure were not correlated with ABA and WC. In declining stands, WC and ABA tended to be higher and lower, respectively, than in nondeclining stands. These results suggest that the trees in declining stands received less water stress than those in nondeclining stands and the differences in gl and ¹³C are not caused by the difference in water stress. The possibilities of the effects of air pollution and the infection of pine-wood nematode on the physiological decline on the pine needles are discussed.     

Lignosulfonate promotes the interaction between Scots pine and an ectomycorrhizal fungus Pisolithus tinctorius in vitro

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.     

Different soil moisture conditions change the outcome of the ectomycorrhizal symbiosis between Rhizopogon species and Pinus muricata

The outcome of species interactions often depends on the environmental conditions under which they occur. In this study, we tested how different soil moisture conditions affected the outcome of the ectomycorrhizal symbiosis between three Rhizopogon species and Pinus muricata in a factorial growth chamber experiment. We found that when grown in 7% soil moisture conditions, ectomycorrhizal plants had similar biomass, photosynthesis, conductance, and total leaf nitrogen as non-mycorrhizal plants. However, when grown at 13% soil moisture, ectomycorrhizal plants had significantly greater shoot biomass, higher photosynthetic and conductance rates, and higher total leaf nitrogen than non-mycorrhizal plants. The differences in plant response by mycorrhizal status in the two soil moisture treatments corresponded with evidence of water limitation experienced by the fungi, which had much lower colonization at 7% compared to 13% soil moisture. Our results suggest that the outcome of the ectomycorrhizal symbiosis can be context-dependent and that fluctuating environmental conditions may strongly affect the way plants and fungi interact. Peter G. Kennedy and Kabir G. Peay contributed equally to this work and order was determined by a coin toss.     

New evidence from mitochondrial DNA of a progenitor-derivative species relationship between black spruce and red spruce (Pinaceae)

Mitochondrial DNA (mtDNA) markers were used to assess the genetic diversity in allopatric populations of black spruce (Picea mariana [Mill.] BSP) and red spruce (P. rubens Sarg.). Patterns of mitochondrial haplotypes (mitotypes) were strikingly different between the two species. All mtDNA markers surveyed were polymorphic in black spruce, revealing four different mitotypes and high levels of mtDNA diversity (P(p) = 100%, A = 2.0, H = 0.496). In contrast, populations of red spruce had only two mitotypes and harbored low levels of ggenetic diversity (P(p) = 13.2%, A = 1.1, H = 0.120). When the southernmost allopatric populations of red spruce were considered, only one mitotype was detected. As previously reported for nuclear gene loci, the diversity observed for mtDNA in red spruce was a subset of that found in black spruce. Comparison of present and previously published data supports the hypothesis of a recent progenitor-derivative relationship between these species, red spruce presumably being derived by allopatric speciation of an isolated population of black spruce during the Pleistocene.     

In vitro pollen germination of species and two interspecific hybrids from the genus Brassica

In vitro pollen germination of five species and two interspecific hybrids from the genus Brassica was tested in four media. Genetically fixed differences in the demands for optimal pollen germination among species were found. The experiments were designed to define optimal content of mineral salts, sugar, and PEG for every investigated species or hybrids. The differences found among species are discussed in relation to the evolutionary trend.