Advances in the cultivation of the highly-prized ectomycorrhizal mushroom Tricholoma matsutake

Matsutake, the fruiting body of Tricholoma matsutake, is among the most economically important edible ectomycorrhizal (EM) mushrooms worldwide. This EM fungus develops “shiros”, which are mycelial aggregations that develop in association with the roots of EM coniferous trees and soil particles in well-drained and nutrient-poor forest soil. The fruiting bodies occur on the periphery of the outward-growing shiro. In spite of vast research, the cultivation of matsutake has been mostly unsuccessful. Commercial demand is therefore met by harvesting the fruiting bodies that naturally occur in forests of EM coniferous trees, mainly Pinus densiflora. Recent inoculation studies have produced mycorrhiza and shiro structures, and the host range of Tr. matsutake and associated species has been clarified. It has also become possible to identify strains of Tr. matsutake by DNA sequencing, which has also been useful to determine the origins of matsutake species in Asia and to elucidate the genetic structure of shiro. In this review, basic research and the outcomes of various trial of matsutake cultivation are discussed.     

Detection of the ectomycorrhizal fungusTricholoma matsutake and some related species with specific ITS primers

Oligonucleotide primers (Tm1 and Tm4) were designed to amplify a 447–448 base pair fragment, comprising sections of the rDNA internal transcribed spacers (ITS) and the entire 5.8S rDNA, ofTricholoma matsutake. PCR products of predicted size were produced for six of eight isolates ofT. matsutake from across its natural range in Asia, and for isolates of some closely related fungi includingT. bakamatsutake, T. magnivelare, andT. caligatum. The closely relatedT. robustum could be discriminated fromT. matsutake by PCR fragment size. No PCR products were produced where the primers were tested against 16 species of ectomycorrhizal fungi associated withPinus spp. in the Southern Hemisphere. The specific primers were also used successfully to produce PCR products from matsutake infected roots collected in natural forests in China and Japan, and from pure culture synthesisedPinus radiata-T. matsutake material. These primers will be useful in research directed at establishing matsutake in the Southern Hemisphere, and also have the potential to be applied to the study of matsutake within its natural range.     

Matsutake Trade in Yunnan Province,China: An Overview

Matsutake Trade in Yunnan Province, China: An Overview. Matsutake are economically important wild mushrooms that contribute greatly to rural livelihoods and local economies in many parts of the northern hemisphere. This paper provides an overview of the matsutake trade in Yunnan province, China, where increased attention is being given to the sustainable utilization of nontimber forest products. Topics covered include the distribution, production, and export of matsutake in Yunnan, and the market chain for matsutake in Yunnan, whereby matsutake are harvested from the wild and exported to Japan within 48 hours.     

Bidding Customs and Habitat Improvement for Matsutake (<Emphasis Type="Italic">Tricholoma matsutake</Emphasis>) in Japan

Bidding Customs and Habitat Improvement for Matsutake (Tricholoma matsutake) in Japan. A brief history of Japan’s matsutake production, use, and management is presented, with emphasis on habitat improvement efforts and the land use tradition of iriai. Bidding systems for allotment of matsutake gathering rights are discussed. Three villages with different bidding systems are compared to see what effects the bidding systems have on village finances, matsutake production, and matsutake habitat enhancement. Contrary to expectations, habitat for matsutake was not improved when land owners were guaranteed the gathering and selling rights to matsutake growing on their own lands. Instead, habitat improvement was most successful and matsutake production was highest on community-owned lands in Oka Village, where the iriai tradition is strongest.     

Mobile DNA distributions refine the phylogeny of “matsutake” mushrooms, Tricholoma sect. Caligata

“Matsutake” mushrooms are formed by several species of Tricholoma sect. Caligata distributed across the northern hemisphere. A phylogenetic analysis of matsutake based on virtually neutral mutations in DNA sequences resolved robust relationships among Tricholoma anatolicum, Tricholoma bakamatsutake, Tricholoma magnivelare, Tricholoma matsutake, and Tricholoma sp. from Mexico (=Tricholoma sp. Mex). However, relationships among these matsutake and other species, such as Tricholoma caligatum and Tricholoma fulvocastaneum, were ambiguous. We, therefore, analyzed genomic copy numbers of σ _marY1 , marY1, and marY2N retrotransposons by comparing them with the single-copy mobile DNA megB1 using real-time polymerase chain reaction (PCR) to clarify matsutake phylogeny. We also examined types of megB1-associated domains, composed of a number of poly (A) and poly (T) reminiscent of RNA-derived DNA elements among these species. Both datasets resolved two distinct groups, one composed of T. bakamatsutake, T. fulvocastaneum, and T. caligatum that could have diverged earlier and the other comprising T. magnivelare, Tricholoma sp. Mex, T. anatolicum, and T. matsutake that could have evolved later. In the first group, T. caligatum was the closest to the second group, followed by T. fulvocastaneum and T. bakamatsutake. Within the second group, T. magnivelare was clearly differentiated from the other species. The data suggest that matsutake underwent substantial evolution between the first group, mostly composed of Fagaceae symbionts, and the second group, comprised only of Pinaceae symbionts, but diverged little within each groups. Mobile DNA markers could be useful in resolving difficult phylogenies due to, for example, closely spaced speciation events.     

Vuilleminia erastii sp. nov. (Corticiales), an amphi-Beringian species and revision of the occurrence of Vuilleminia comedens in North America

Vuilleminia is a basidiomycete genus the species of which have resupinate, corticioid fruiting bodies. It is apparently a North Hemisphere genus, and the majority of its species are distributed in Europe and western Asia. In North America, there are two reports of Vuilleminia comedens. Detailed study of North American specimens and comparisons with additional collections led to the conclusion that they belong to a new lineage named Vuilleminia erastii sp. nov., whose distribution extends from western North America to East Asia, Siberia, and Finland. The species is recognized by the decorticating fruiting bodies with preference for species of Betulaceae in the boreal zone, relatively small allantoid basidiospores, and little-developed cystidia with apical appendix.     

Saprobic potential of <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>: growth over pine bark treated with surfactants

Saprotrophic growth of Tricholoma matsutake isolates was investigated over Pinus densiflora bark fragments either on soil or on agar media. Preferential colonization of pine bark fragments by hyphae, in glucose-deprived environments suggested that Matsutake was able to extract some nutrients to sustain its growth. This was confirmed in glucose-free liquid nutrient medium, where bark as sole carbon source significantly stimulated (up to two-fold) growth of T. matsutake isolates. The addition of surfactants (Tween 80 and Tween 40) in liquid medium further stimulated mycelium growth over pine bark by up to 55%. Such growth stimulation was associated with a sharp increase in protein and beta-glucosidase excretion by hyphae in culture filtrates. As T. matsutake has some saprotrophic ability, the initiation and extension of Matsutake Shiro in forest soil might require simultaneously nutrients derived from the host plant and from soil organic compounds. Data reported here may contribute to the formulation of new culture substrates adapted to the co-culture of T. matsutake and its host plant under controlled conditions.     

Tigridia amatlanensis (Tigridieae: Iridaceae), a new species from Oaxaca, Mexico

Tigridia amatlanensis is described and illustrated as a new species from Mexico. It is recognized by its nodding flowers and its inner and outer tepals cirrhous and markedly dimorphic in length. It is most closely related toT. hallbergii subsp.hallbergii andT. meleagris. Other morphologically similar species areT. catarinensis andT. molseediana.     

A qPCR assay that specifically quantifies <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> biomass in natural soil

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, “matsutake” mushrooms along densely developed mycelia, called “shiro,” in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A₀ horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.     

A new Lewisia (Portulacaceae) from the Sierra Nevada of California

Lewisia serrata, known from only three localities in the American River drainage of Eldorado and Placer counties, California, is described as a new species. Its closest relatives areL. cantelowii J. T. Howell andL. columbiana (T. Howell) Rob.     

KEYS TO STERILE,CRUSTOSE SAXICOLOUS AND TERRICOLOUS LICHENS OCCURRING IN THE BRITISH ISLES

A set of artificial keys is presented to all those crustose lichens (except most species ofLeprariaandLeproloma) which commonly occur without fruiting bodies on rock or soil in the British Isles. A number of predominantly corticolous species that occasionally also occur on rocks are included, as well as several undescribed but well-understood entities.     

Effect of Temperature,pH, and Media on the Mycelial Growth of Tuber koreanum

Members of the genus Tuber are ectomycorrhizal fungi; this genus includes more than 180 species worldwide. In the present study, the optimal pH, temperature, and medium suitable for the mycelial growth of the Korean truffle, Tuber koreanum, were determined. Mycelium of T. koreanum, isolated from fruiting bodies collected in Korea, was used to investigate the effects of these environmental factors. The results showed that malt extract agar and potato dextrose agar were the most suitable for the mycelial growth of T. koreanum when cultured at a pH of 6.0 at 25 °C for 30 days.     

Traceability of Marketable Japanese Shoro in New Zealand: Using Multiplex PCR To Exploit Phylogeographic Variation among Taxa in the Rhizopogon Subgenus Roseoli

Rhizopogon roseolus Corda (synonym Rhizopogon rubescens Tul.), an economically important edible mushroom associated with the Pinaceae (mostly Pinus sp.), has a global distribution resulting from the introduction of exotic trees into the Southern Hemisphere for plantation forestry. However, the marketability of R. roseolus varies with the place of origin. R. roseolus strains cultivated in New Zealand from local carpophores for the Japanese market are morphologically and biologically distinct from those produced in Japan and are consequently considered less valuable. In this study, the ITS1-5.8S-ITS2 rRNA (internal transcribed spacer [ITS]) region was used to examine the phylogenetic relationships of R. roseolus and other closely related fungi belonging to Rhizopogon subgenus Roseoli to determine the genetic basis for phenotypic differences among R. roseolus isolates from different geographic regions. Phylogenetic comparison revealed phylogeographic variation within Rhizopogon subgenus Roseoli. Collections from the United States and Europe grouped into four distinct clades. Rhizopogon roseolus isolates found in New Zealand were closely related to those from the United States, likely due to introduction of Pinus radiata from its native California in the United States. In contrast, Japanese R. roseolus isolates clustered closely with European collections. Phylogenetic differences between Japanese and New Zealand R. roseolus isolates may explain the morphological and biological properties attributed to these geographical variants. The ITS region was subsequently used to design a multiplex PCR for the simultaneous identification of Japanese and New Zealand R. roseolus isolates to track the establishment of ectomycorrhiza on P. radiata seedlings inoculated with commercially valuable R. roseolus. This diagnostic demonstrated the first fruiting of Japanese shoro cultivated on P. radiata in the Southern Hemisphere.Since the latter half of the 19th century, Northern Hemisphere species of exotic trees, in particular Pinus radiata, have been planted in the Southern Hemisphere (e.g., Chile, Argentina, South Africa, Australia, and New Zealand). Indeed, over 1,000,000 ha of exotic trees have been established in New Zealand alone (32, 9). Over 200 nonnative basidiomycete and ascomycete ectomycorrhizal (ECM) fungal species are associated with Pinus or Eucalyptus plantations in the Southern Hemisphere from the introduction of plants with intact root systems (46).The genus Rhizopogon Fries (Basidiomycota, Boletales) contains more than 100 species of hypogeous fungi (24), which form ECM associations mostly with members of the Pinaceae (44). The greatest diversity of Rhizopogon can be found in the coniferous forests of the Pacific northwestern United States (40, 30), although a number of species are known to occur in Europe (40) and Asia (3). At least four introduced species of Rhizopogon have also been reported in New Zealand, including R. clelandii Cunn., R. luteolus Fr., R. roseolus (7), and R. vinicolor A. H. Smith (5). At least two species, R. luteolus and R. roseolus, have been associated with P. radiata seedlings in forest nurseries in the North Island of New Zealand (6).In Japan, R. roseolus is known locally as shoro. Shoro is considered a delicacy (20), and its production is dependent on the collection of the carpophores in the field. In fact, only 200 years ago, shoro was the fourth most commonly consumed mushroom in Japan (34). However, the number of natural shoro has declined in the second half of the 20th century (15), resulting in its cultivation in forestry plantations since the late 1980s (50). To fulfill Japanese demand, plantations of P. radiata artificially inoculated with R. roseolus have been established in New Zealand since 1999 using spores from fruiting bodies collected locally (47). Three of the four plantations have subsequently produced fruiting bodies, but the crop has been deemed unsuitable due to consumer sensitivity in Japan to the origin of the products and doubts surrounding the authenticity of the fruiting bodies as Japanese shoro. The quality and market price of other edible fungi differ not only with species but also with their origin. In Japan, domestic Tricholoma matsutake is considered the premium source of matsutake, traded at $500/kg, while South Korean matsutake and those from China are considered less valuable ($250/kg and $100/kg, respectively) (31). Highly prized edible fungi have also been found to be contaminated with less valuable species. Tuber rufum Pico is a truffle species that is found alongside other valuable species in countries where truffles are commercially important. However, it is considered to be a poorly flavored species with no marketable value and is deemed a “contaminant” in truffières (4).Unfortunately, the taxonomy of the genus Rhizopogon is surrounded by many unresolved issues, which makes differentiation of shoro-like fungi found in different geographical locations difficult. In 1966, Smith and Zeller (40) completed the first taxonomic study of the genus Rhizopogon, dividing it into two subgenera, Rhizopogonella (subsequently transferred to Alpova [45]) and Rhizopogon. The subgenus Rhizopogon was further divided into four sections based on differences in the host plant, sporocarp morphology, and color of the peridium. All species associated with Pinus spp. were classified in Rhizopogon section Rhizopogon. Rhizopogon roseolus and R. rubescens were placed in subsection Angustispori, stirps Rubescens. A third species, Rhizopogon vulgaris Vittad., was placed in subsection Angustispori, stirps Vulgaris, due to differences in its spore morphology.Similarities between species in stirps Rubescens and stirps Vulgaris were recorded, with stirps Vulgaris considered a continuation of stirps Rubescens into the narrow-spore species. However, classification was based only on examinations of North American collections even though these species were originally described in Europe in the 19th century (40). Taxonomic reexamination of Rhizopogon using phylogenetic analyses of internal transcribed spacer (ITS) DNA sequences showed that Rhizopogon burlinghamii, R. roseolus, and R. vulgaris formed Rhizopogon section Rhizopogon clade C, separate from the other species sampled from section Rhizopogon (clades A and B). Their ITS sequences lacked insertions and deletions that are diagnostic of other section Rhizopogon clades. Rhizopogon roseolus and R. vulgaris were placed together under Rhizopogon subgenus Roseoli (13). Unfortunately, collections classified as R. rubescens were not included in the phylogenetic study. More recently, species concepts in the R. roseolus species group were examined by Martín and García (25). ITS sequence analyses separated the collections into five possible phylogenetic species.The continued taxonomic instability of Rhizopogon subgenus Roseoli has created ongoing confusion, and the correct species names are still not clear. In Japan, until now, shoro has been referred to as R. rubescens; however, R. rubescens is used widely as a synonym for R. roseolus. In this study, R. roseolus will be used to describe all collections unless specifically stated, as this taxonomic name appears to have precedence in previous phylogenetic studies of Rhizopogon (13, 19, 40) and since Mycobank (http://www.MycoBank.org/) considers R. rubescens to be a synonym of R. roseolus.Molecular diagnostic tools capable of distinguishing genetic differences in ECM fungi have been developed, allowing the differentiation of commercially important species from contaminants or similar species of less economic value. There are many studies where PCR primers designed for the amplification of the ITS region have been used to identify basidiomycetes (12, 18). Species-specific primers were created to identify and differentiate marketable boletes (28), to detect black truffle species (37), and to distinguish Asiatic black truffles from Tuber melanosporum in commercialized products (22). A multiplex PCR has also been developed to simultaneously detect different white truffle species and one of the most aggressive contaminant fungi for monitoring the persistence of a selected truffle in inoculated seedlings (1).Due to the sensitivity of consumers to the origins of shoro and the existing taxonomic complexity of the genus Rhizopogon, morphological and molecular methods were used to establish the diversity and genetic structure of Rhizopogon subgenus Roseoli. Phylogenetic relationships between shoro-like species (originally classified as R. vulgaris, R. rubescens, and R. roseolus) from different geographical locations were investigated to verify previously observed differences between shoro grown in Japan and New Zealand. A multiplex PCR was then developed for the rapid identification of ECMs and fruiting bodies grown from Japanese shoro in New Zealand to track the commercial production of this economically valuable edible fungus.     

A new aulopodid species,Hime microps,from the eastern South Pacific,with comments on geographic variations ofH. japonica

A new aulopodid fish,Hime microps, is described after two specimens from the Nazca submarine ridge (25°43′S, 85°29′W at 160–165 m; 25°42′S, 85°24′W at 162–168 m). This species is related toH. japonica andH. curtirostris but differs in having no pyloric caeca and smaller eyes with the horizontal diameter shorter than the snout length. Specimens ofH. japonica from off Japan, the Hawaiian submarine ridge and the Tasman Sea are compared and found to be morphologically identical.     

Studies in the cyperaceae of Thailand

Ten species ofCarex and one species ofKobresia from Thailand are taxonomically discussed, and some new range extensions into Thailand are noted. Described as new areCarex subinclinata andC. thailandica, both belonging to the sectionIndicae, andC. speciosa ssp.latifolia of the sectionRadicales. Two new combinations proposed areCarex helferi ssp.mapanifolia, andC. speciosa ssp.platyrhina.     

Successful large-scale production of fruiting bodies of Laetiporus sulphureus (Bull.: Fr.) Murrill on an artificial substrate

Laetiporus sulphureus is an edible wood-rotting basidiomycete fungus whose fruiting bodies contain substances with verified therapeutic evidences and large amounts of α-(1 → 3)-glucan which is used as an effective inducer of microbial α-(1 → 3)-glucanases. However, production of mature fruiting bodies of this species under artificially controlled conditions has not been reported until now. Here, we provide the first report of successful initiation and development of L. sulphureus fruiting bodies in large-scale experiments. Twelve Laetiporus strains were isolated from a natural habitat. A synthetic log production system with a substrate composed of a mixture of sawdust enriched with organic and inorganic additives was developed. It was found that shocking the fungus mycelium with cold water or low temperature was the only suitable method for forced fruiting of L. sulphureus strains. Primordia of two strains were initiated already after 5–6 days from induction, and after another 2 days, they began to develop into fruiting bodies. Carpophores appeared fastest on substrates with high organic supplementation (40–45 %) and a low moisture content (40 %). The resulting mature fruiting bodies reached a weight of 200–300 g. The method of cultivation presented in this paper opens the way to commercial production of this valuable basidiomycete.     

Modulation of androgens in southern hemisphere temperate breeding sparrows (Zonotrichia capensis): an altitudinal comparison

Most work investigating modulation of testosterone (T) levels in birds has focused on northern temperate and Arctic species, and to a lesser degree, tropical species. Studies exploring modulation of T in birds in temperate regions of the Southern Hemisphere are lacking. Here we explore patterns of T secretion across the breeding season in two populations of temperate Zonotrichia capensis in Chile, located only 130 km apart, but separated by 2000 m in elevation. We then compared these T profiles to those of conspecifcs in the tropics and congeners in northern zones. We measured baseline T levels during pre-breeding in lowland Z. c. chilensis, early breeding in highland Z. c. chilensis and mid-breeding in both populations. We also tested for social stimulation of T secretion during mid-breeding in both populations. Lastly, we challenged the hypothalamic-pituitary-gonadal (HPG) axis of the lowland population with gonadotropin-releasing hormone (GnRH) to determine maximum possible T production. We found that the highland population adjusted T secretion across the breeding season like northern species. Neither Z. c. chilensis population modulated T in response to social stimuli, nor the HPG axis of the lowland population was not maximally active during either pre- or mid-breeding. These results suggest that patterns of circulating T in the highland population of Z. c. chilensis in the Southern Hemisphere are similar to congeners in the temperate Northern Hemisphere, but those of the lowland population of Z. c. chilensis are not, and are more similar to conspecifics breeding in the tropics.     

The Houses That Matsutake Built<Superscript>1</Superscript>

The Houses That Matsutake Built. In the mountains of northwest Yunnan, China, a valuable mushroom, matsutake or song rong (Tricholoma matsutake) was commodified in the 1980s. Since that time, it has been exported in large quantities to Japan. The sale of matsutake now contributes more to the income of Shangri-la County (Diqing Autonomous Tibetan Prefecture) than any other crop, including timber and livestock. During the 1980s and 1990s, villagers in this remote region used their mushroom earnings to build spacious, beautiful new houses in the traditional local (Kham) style, and in some cases to buy motor vehicles or open businesses. In villages with access to productive matsutake habitat, virtually every household was able to build a new house; entire villages were transformed. During the 1990s, several villages developed locally based management regimes to enhance production and to address the problems and conflicts that arose from the harvest of such a valuable product. More recently, government agencies and NGOs have played a highly visible role in promoting “sustainable” harvest policies. The implications of their involvement are briefly examined and the future of matsutake harvest in Yunnan is discussed.     

Proetide trilobites from the Cystoid Limestone (Ashgill) of NW Spain,and the suprageneric classification of related forms

Few trilobites of the Order Proetida have been reported from the Ordovician»Selenopeltis Province«. Herein four species from the Ashgill Cystoid Limestone of the eastern Iberian Chains, NW Spain, are described and assigned to the generaRadnoria, Harpidella, Panarchaeogonus andRorringtonia. Two species,Radnoria carlsi andRorringtonia lenis, are new, and the others are described under open nomenclature. In the appendix, classification of Proetida belonging to the Aulacopleuridae, Brachymetopidae and the new family Rorringtoniidae is discussed, and all are included in the superfamily Aulacopleuracea.