Basidiome formation of an edible wild, putatively ectomycorrhizal fungus, Phlebopus portentosus without host plant.

Phlebopus portentosus is a popular wild edible ectomycorrhizal fungus in northern Thailand. In general ectomycorrhizal fungi produce basidiomes when associated with a host plant. In this paper mycelium growth and basidiome production of P. portentosus were examined in pure culture both in vitro and in pot-culture experiments. Five mycelial strains of P. portentosus were isolated from basidiomes and used in the experiments. The mycelia grew fastest on sorghum grains supplemented with fungal-host solution. The mycelia produced sclerotia-like structures after 3 wk incubation in darkness at 30 C. All strains of P. portentosus had the ability to form primordia. The primordia were formed under lowered temperature, high humidity and a 12 h photo-period. They developed to mature basidiomes after 8-12 d in in vitro. In the pot-culture primordia were found after 28-35 d incubation in the greenhouse and mature basidiomes released basidiospores within 6-8 d. Basidiospores were germinated on fungal-host medium and formed mycelial colonies. This fungus showed an ability to produce basidiomes even 2 y after the original isolation from tissues. This research provides valuable information concerning the techniques and protocols for the large scale commercial production of P. portentosus basidiomes in the absence of a host plant.

The cantharelloid clade: dealing with incongruent gene trees and phylogenetic reconstruction methods.

We reassessed the circumscription of the cantharelloid clade and identified monophyletic groups by using nLSU, nSSU, mtSSU and RPB2 sequence data. Results agreed with earlier studies that placed the genera Cantharellus, Craterellus, Hydnum, Clavulina, Membranomyces, Multiclavula, Sistotrema, Botryobasidium and the family Ceratobasidiaceae in that clade. Phylogenetic analyses support monophyly of all genera except Sistotrema, which was highly polyphyletic. Strongly supported monophyletic groups were: (i) Cantharellus-Craterellus, Hydnum, and the Sistotrema confluens group; (ii) Clavulina-Membranomyces and the S. brinkmannii-oblongisporum group, with Multiclavula being possibly sister of that clade; (iii) the Sistotrema eximum-octosporum group; (iv) Sistotrema adnatum and S. coronilla. Positions of Sistotrema raduloides and S. athelioides were unresolved, as were basal relationships. Botryobasidium was well supported as the sister taxon of all the above taxa, while Ceratobasidiaceae was the most basal lineage. The relationship between Tulasnella and members of the cantharelloid clade will require further scrutiny, although there is cumulative evidence that they are probably sister groups. The rates of molecular evolution of both the large and small nuclear ribosomal RNA genes (nuc-rDNA) are much higher in Cantharellus, Craterellus and Tulasnella than in the other cantharelloid taxa, and analyses of nuc-rDNA sequences strongly placed Tulasnella close to Cantharellus-Craterellus. In contrast analyses with RPB2 and mtSSU sequences placed Tulasnella at the base of the cantharelloid clade. Our attempt to reconstruct a "supertree" from tree topologies resulting from separate analyses that avoided phylogenetic reconstruction problems associated with missing data and/or unalignable sequences proved unsuccessful.

Species recognition in the truffle genus Tuber -- the synonyms Tuber aestivum and Tuber uncinatum.

The two morphologically similar truffles Tuber aestivum and T. uncinatum have caused confusion because T. uncinatum is regarded by different authors, as either a distinct species, variety, subspecies, or synonym of T. aestivum. A clarification of the relationship between the two truffles would help both conservation biology and cultivation. We aimed both to test the reliability of the only quantitative morphological character used to distinguish the two taxa, i.e. the height of the spore reticulum, and to compare sequences of the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region. Our study included 117 fruit bodies of T. aestivum and T. uncinatum, originating from eight European countries. The results showed that the spore reticulum height is not diagnostic. The phylogenetic analysis of ITS sequences from 81 fruit bodies and an additional 32 sequences from GenBank showed that T. aestivum and T. uncinatum were intermingled in one highly supported (100% bootstrap) monophyletic clade, separate from its sister species Tuber mesentericum. We conclude that T. aestivum and T. uncinatum are synonyms and the species should be named T. aestivum, as the oldest name has priority. For traders, T. aestivum syn. T. uncinatum should be used until conformity has been reached.

Tuber aestivum (syn. T. uncinatum) biotopes and their history on Gotland, Sweden.

This study aimed at testing the hypothesis that the genetically distinct Tuber aestivum population on the island of Gotland, Sweden, is adapted to habitats different from French T. aestivum populations. The soil structure, soil chemistry, bedrock, climate, vegetation and host tree continuity of 18 T. aestivum sites on Gotland were analysed and compared with data from France. We conclude that T. aestivum can grow in soils with a broad soil structure range and that no striking differences in soil chemistry were found. No T. aestivum indicator plants other than the host trees were found, but the host tree continuity on the T. aestivum sites on Gotland was more than 300 yr. If the T. aestivum population on Gotland constitutes an ecotype it is rather an adaptation to the colder and drier climate on Gotland. Selecting local T. aestivum inoculum for truffle orchards in northern Europe could be important for successful truffle production.

The population of the hypogeous fungus Tuber aestivum syn. T. uncinatum on the island of Gotland.

The aim of our study was to examine the genetic variation within Tuber aestivum on the Baltic island of Gotland, Sweden. Variation in such a limited geographical area should help illuminate the dispersal abilities of T. aestivum. Knowledge of the genetic variation in this northern outpost could also be useful in the selection of inoculum for the establishment of truffle orchards. Genetic structure and homogeneity of the population were studied using principal component and parsimony analyses of randomly amplified polymorphic DNA data. Our inventories showed that T. aestivum is abundantly distributed in suitable habitats on Gotland. The genetic variation observed suggests sexual reproduction and slow dispersal on the island. It is possible that the present population was established from one introduction, which may be due to ability to survive in this habitat rather than to rare colonising events. The T. aestivum population on Gotland may be an ecotype adapted to the climate and soil conditions on the island.

Cultivation of Oudemansiella tanzanica nom. prov. on agricultural solid wastes in Tanzania.

The edible mushroom Oudemansiella tanzanica nom. prov., which is new to science, has been studied as a potential crop to reduce agricultural solid wastes and increase domestic mushroom production. The substrates sawdust, sisal waste and paddy straw supplemented with chicken manure resulted in the highest biological efficiencies of any mushroom cultivated in Tanzania so far. In addition, the mushroom has one of the shortest cultivation cycles at 24 d. Despite the fact that the mushroom extracts substantial amounts of nutrients, the spent substrate can be used as fodder, as a soil conditioner and fertilizer and in bioremediation.

Albatrellus citrinus sp. nov., connected to Picea abies on lime rich soils.

Field observations indicated that a morphotype of Albatrellus subrubescens seemed connected to Picea abies and lime rich soils, while the original morphotype seemed connected with Pinus sylvestris and indifferent to lime. We conducted a molecular study (ITS sequencing of 22 Albatrellus specimens) to test the hypothesis that we in fact had discovered a new species. Our results confirmed the hypothesis, i.e. the Picea taxon (604 bp ITS) had little intraspecific variation in spite of 1600 km distance between samples, but compared with the Pinus taxon (598 bp ITS) the sequence difference was constantly 5.6% regardless of close distance (75 km). We describe the new species A. citrinus, which apart from ecology and ITS sequence, is different from A. subrubescens by a distinct yellowing with age, lack of dark spots of the cap, a mild taste and somewhat narrower spores. A. citrinus seems to be more related to A. ovinus than to A. subrubescens, and A. syringae may not even be a true Albatrellus.

Physiological and genetic characterization of fluorescent Pseudomonas associated with Cantharellus cibarius.

Fluorescent Pseudomonas spp. isolated from fruiting bodies (FB) of Cantharellus cibarius were characterized physiologically and genetically and were compared with fluorescent Pseudomonas from forest soil and with sequences from the GenBank database. Pseudomonas spp. from FB differed physiologically from isolates from soil lacking FB and had some similarities with the strains obtained from soil underneath the FB. Analyses of the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) patterns and partial sequencing analysis of the 16S-rDNA region indicated that the bacteria isolated from these environments were different. However, there was no specific Pseudomonas genotype restricted to the FB environment. Utilization of the reported fungal exudates trehalose and mannitol may explain how millions of bacteria survive in the C. cibarius FB without deteriorating the fungal mycelium. The importance of the metabolic characterization of bacteria and the possible mechanisms involved in the association with C cibarius are discussed. Our study showed that standard processes for bacterial identification, e.g., Biolog and 16S-rDNA are insufficient until databases for different ecosystems are created.

Use of different nitrogen sources by the edible ectomycorrhizal mushroom Cantharellus cibarius.

The growth of three strains of Cantharellus cibarius on liquid media containing ammonium, nitrate and bovine serum albumin (BSA) in different combinations was determined. The most readily utilisable source of N was ammonium. BSA utilisation was limited compared with media containing ammonium. Growth on nitrate was also poor, suggesting a limited capacity of C. cibarius to metabolise this nitrogen source. There was some indication of considerable intraspecific variation within C. cibarius in the utilisation of nitrogen sources. Possible links between atmospheric nitrogen deposition and the observed decrease of sporocarp formation by C. cibarius in Europe are discussed. We highlight the potential ecological significance of bacteria associated with C. cibarius which may circumvent the need for fungal extracellular enzymes to access complex nitrogen sources.

A 13C-NMR study of exudation and storage of carbohydrates and amino acids in the ectomycorrhizal edible mushroom Cantharellus cibarius.

(13)C-NMR analyses of Cantharellus cibarius growth media were performed. We found exudation of trehalose and mannitol, which may explain the phenomenon of reproducing Pseudomonas bacteria observed inside fruit bodies. Exudation varied with strain and environment. NMR analyses of stored (13)C was also performed. Trehalose, mannitol, and arginine were revealed. The mannitol pathway seems to play an important role for trehalose production in this species. This is the first study of the fate of the photosynthetically derived carbon in the highly appreciated edible ectomycorrhizal mushroom Cantharellus cibarius.