Zoltán Bratek

Mycorrhizae between black locust (Robinia pseudoacacia) and Terfezia terfezioides

Abstract In the present paper we report a mycorrhizal association between the hypogeous white truffle Terfezia terfezioides and the black locust (Robinia pseudoacacia) growing at various sites in Hungary. The mycorrhiza can be considered as being of the endo- or ectendo-type, since both mantle and Hartig net are absent. Morphological features of the septate hyphae colonizing cortical root cells were investigated by light microscopy on cryosections and on ultra-thin sections studied by transmission electron microscopy. Artificial infection of micropropagated black locust plantlets with the mycelium of the fungus was successful and had the same characteristics as naturally occurring associations.

Tests of species concepts of the small, white, European group of Tuber spp. based on morphology and rDNA ITS sequences with special reference to Tuber rapaeodorum

Several taxonomic problems arise in the group of small, white European truffles, probably due to the over-emphasized significance of certain morphological features of ascomata. The distinction between Tuber rapaeodorum Tul. & C. Tul. and Tuber borchii Vittad. and Tuber puberulum Berk. & Broome has not been accepted in several recent studies. Furthermore, the existence of T. rapaeodorum been questioned in some recent synopses of the genus. We conducted microscopic and ITS sequence investigations of 31 herbarium specimens. Using morphological features such as peridium structure, form and size of spores and dermatocystidia and spore numbers per ascus, we could distinguish T. borchii, T. foetidum Vittad., T. maculatum Vittad., Tuber puberulum, and T. rapaeodorum. Analysis of whole ITS sequences showed sharp differences among the morphologically separated groups. Neighbour-joining and parsimony methods produced highly supported branches and confirmed the identity of these species.

Changes in the Content of Modified Nucleotides in Wheat rRNA during Greening

The modified nucleotide content of the ribosomal RNAs in wheat is greatly influenced by light. The rRNAs of etiolated seedlings contain far fewer modified derivatives. The modified nucleotide composition characteristic of green plants develops gradually as a result of irradiation. In the course of the experiments changes in the state of modification of 5.8S and 18S rRNAs were examined during the greening of etiolated wheat seedlings. Three types of minor nucleotides, O2′-methyladenosine, O2′-methylguanosine and pseudouridine were found in the 5.8S rRNA of green wheat leaves, none of which was detected in etiolated wheat. The minor nucleotides appeared in the 5.8S rRNA only after 48 h irradiation. The sequences of 5.8S rDNA, TTS1, ITS2 and 18S rDNA were also determined and the presence of the hyper-modified nucleotide 1-methyl-3-(α-amino-α-carboxypropyl)-pseudouridine was detected in green wheat 18S rRNA. This minor component was not demonstrable in etiolated wheat 18S rRNA, but appeared after irradiation for 48 h.

Species Delimitation in Taxonomically Difficult Fungi: The Case of Hymenogaster

Background False truffles are ecologically important as mycorrhizal partners of trees and evolutionarily highly interesting as the result of a shift from epigeous mushroom-like to underground fruiting bodies. Since its first description by Vittadini in 1831, inappropriate species concepts in the highly diverse false truffle genus Hymenogaster has led to continued confusion, caused by a large variety of prevailing taxonomical opinions. Methodology In this study, we reconsidered the species delimitations in Hymenogaster based on a comprehensive collection of Central European taxa comprising more than 140 fruiting bodies from 20 years of field work. The ITS rDNA sequence dataset was subjected to phylogenetic analysis as well as clustering optimization using OPTSIL software. Conclusions Among distinct species concepts from the literature used to create reference partitions for clustering optimization, the broadest concept resulted in the highest agreement with the ITS data. Our results indicate a highly variable morphology of H. citrinus and H. griseus, most likely linked to environmental influences on the phenology (maturity, habitat, soil type and growing season). In particular, taxa described in the 19th century frequently appear as conspecific. Conversely, H. niveus appears as species complex comprising seven cryptic species with almost identical macro- and micromorphology. H. intermedius and H. huthii are described as novel species, each of which with a distinct morphology intermediate between two species complexes. A revised taxonomy for one of the most taxonomically difficult genera of Basidiomycetes is proposed, including an updated identification key. The (semi-)automated selection among species concepts used here is of importance for the revision of taxonomically problematic organism groups in general.

A novel mitovirus from the hypogeous ectomycorrhizal fungus Tuber excavatum

The evolution of mycorrhizal mutualism had a profound impact on establishment of terrestrial life [19]. Fossil records indicate that the earliest land plants, which had no proper roots, were already colonized by hyphal fungi, which formed vesicles and arbuscules strikingly similar to modern vesicular-arbuscular mycorrhizas. Today, over 90% of plants form mycorrhizal associations [19]. Although saprotrophic fungi play a central role in the forest ecosystem by cycling, translocating and sequestrating decomposed material, mycorrhizal fungi render inorganic decomposed litter accessible to their plant hosts. In return for inorganics, they receive organic compounds, in particular from coniferous and deciduous trees [14, 19]. In spite of their ecological importance, ectomycorrhizal fungi have largely been ignored by virologists interested in studying mycoviruses [10, 20–22]. Such viruses infecting a broad range of fungi may show a high degree of specialization to their hosts, since some genera exclusively infect fungi, whereas others associated with certain virus families infect multior unicellular organisms [10]. Mycoviruses have been reported from almost the entire range of fungal phyla [9, 15, 17] and possess single-stranded (ssRNA) genomes (genera Botrexvirus, Mycoflexivirus; families Narnaviridae, Endornaviridae, Hypoviridae) [9, 11, 12] or doublestranded (dsRNA) genomes (families Barnaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Metaviridae, Partitiviridae, Pseudoviridae, Reoviridae and Totiviridae). Members of the Birnaviridae and Cystoviridae are reported to infect fungal hosts [9, 15, 21], but they are not listed to infect fungi in the current ICTV master species list 2009 (version 9; http://talk.ictvonline.org/files/ictv_documents/m/msl/1231.aspx) [7, 10]. Until recently, the family Narnaviridae contained viruses exclusively found in fungi [1]. Since Narnaviridae contains records recently added to the INSDC database of sequences derived from plant material (e.g., ‘grapevine associated narnavirus-1’; GenBank accession GU108586), an alternative explanation for these findings would be that these sequences were actually derived from endophytic filamentous fungi and yeast associated with the plant tissues, which has also been suggested and partially proven by Al Rwahnih et al. [1]. Moreover, given the fact that true plant-pathogenic viruses of the genus Ourmiavirus have been proposed to belong to the family Narnaviridae challanges the current classification of virus genera and the host specificity of members of the family Narnaviridae [18]. The genera Mitovirus and Narnavirus include the simplest mycoviruses, which lack coat proteins and are characterized by a single open reading frame (ORF) coding for a viral RNA-directed RNA polymerase (RdRp) [2–6, 16]. Mitoviruses are strictly associated with mitochondria and possess characteristic 50and 30-terminal untranslated regions (UTRs) of variable length, which are most likely responsible for replicase recognition [3, 4]. While most mitoviruses are found in plant-pathogenic fungi, such as Ophiostoma, Chrysoporthe or Botrytis, members of a small J. B. Stielow CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

Phylogeny and phylogeography of the Tuber brumale aggr.

The Tuber brumale (winter truffle) is a black truffle reported from most European countries, belonging to the Melanosporum group. Its significance in the economy is ambivalent as the winter truffle has been shown to be a frequent contaminant species in the orchards of the Perigord truffle and occasionally in those of the summer truffle, yet owing to its delicate fragrance, its trade is worthy of note. The phylogeny and phylogeography of economically important truffles are relatively well-explored; however, no thorough research has been published on these aspects of the winter truffle. Therefore, here, we report the first phylogeographic analyses based on samples representing the entire distribution of the species. ITS sequences were used in this survey for haplotype and coalescent analyses, while phylogenetic analyses were based on the ITS, LSU and PKC loci. According to all loci, the samples clustered into two big clades imply the existence of two phylogenetic species. Based on our results, one of these appears to be endemic to the Carpathian Basin. In the other more widespread species, two main phylogeographic groups can be distinguished that show east-west separation with a zone of overlap in the Carpathian Basin, suggesting that they survived the latest glacial period in separate refugia.

Intraspecific invariability of the internal transcribed spacer region of rDNA of the truffle Terfezia terfezioides in Europe

ITS regions (internal transcribed spacers—ITS1 andITS2—with the 5.8S gene of the nuclear rDNA) of 25 fruit body samples ofTerfezia terfezioides, originating from Hungary and Italy, were compared. The amplification and sequencing of the ITS region was successful with both theITS1-ITS4 andITSIF-ITS4 primer pairs. No differences of the restriction fragment length polymorphism profiles were detected among 19 samples collected in one place at the same time. The sequences of the ITS region of 9 samples collected in different localities were highly invariable, differing in only two bases. Thus the intraspecific homogeneity of the ITS region seems to be an important species-specific characteristic ofT. terfezioides in contrast to otherTerfezia species. As the samples of the species were collected from different and distant localities of Europe, the ITS sequence ofT. terfezioides can be considered a very conservative, reliable molecular marker of the fungus. *** DIRECT SUPPORT *** A00EN076 00008

The most recent results on orchid mycorrhizal fungi in Hungary

Symbionts and endophytes of Hungarian orchids were studied at diverse habitats. Mycobionts of roots and in situ germinated protocorms of 15 orchid species were identified by molecular methods. Four fungal groups could be distinguished from orchids living at diversely wet treeless habitats: Ceratobasidiaceae, Epulorhiza 1, Epulorhiza 2 and Sebacinaceae. While the groups Ceratobasidiaceae and Sebacinaceae were detected only at habitats with medium water supply, members of clade Epulorhiza occurred at all of the treeless study sites. These observations suggest that fungi belonging to the genus Epulorhiza are more tolerant of water-stress than the other investigated genera. An ascomycetous fungus from the family Pezizaceae could be identified from the roots of Orchis coriophora. Further Ascomycetes were identified at forest habitats. Tuber maculatum was detected from the roots of Epipactis helleborine and Cephalanthera damasonium, and Tuber excavatum from Epipactis microphylla.

A new tool for rural development: Truffle cultivation

Our research has lead to the revelation that soil parameters of the Hungarian truffle orchards are different from those of natural habitats. In spite the result fructification in the plantation could not be excluded concerning the wide distribution of Tuber aestivum in the World (from Sweden to Morocco and from Ireland to Turkey). Therefore it is not sufficient the analysis of truffle habitats in Hungary in order to decide the suitability of a location for the establishment of a truffle orchard but habitats under extreme soil conditions should be examined to determine the ecological limits of this species.

Lecophagus vermicola sp. nov., a nematophagous hyphomycete with an unusual hunting strategy

Lecophagus vermicola sp. nov. is described and illustrated as a predacious (carnivorous) hyphomycete living in bark fissures of living trees of Platanus and other angiosperm and gymnosperm trees, recorded in Hungary, Luxembourg and France. The fungus captures nematodes unlike other Lecophagus species, which are predators of rotifers and tardigrades. The morphology of the sessile, adhesive knobs differ from all previously described species of the genus which form adhesive pegs. Molecular data confirms that the new species belongs to the Lecophagus clade but without matching existing sequences. The fungus captures victims with adhesive knobs and colonizes its prey with a mycelium of rather broad hyphae on which, again, adhesive knobs are formed which penetrate the cuticule of the victim. Clusters of colonized nematodes form a network utilized to capture more prey. The fungus lives in the xeric, ephemerally aquatic habitat of bark fissures of standing, living or dead, corticated trunks and branches. The genus Haptocara is compared, which has similar adhesive knobs capturing nematodes and similar broad hyphae, but for which no molecular data was available.