Morphological and molecular diversity and abundance of tomentelloid ectomycorrhizae in broad-leaved forests of the Hungarian Plain

The most common representatives of tomentelloid ectomycorrhizae (EM) collected in broad-leaved forests (Populus and Quercus) of the Hungarian Plain during a 4-year project are demonstrated. Eighteen specimens of nine tomentelloid EM morphotypes were investigated. Five of these, introduced here for the first time, were characterized by microscopical–morphological and anatomical methods. Molecular identification was carried out using sequence analysis of the nrDNA ITS region. Altogether, 54 ITS sequences (6 previously published and 12 new sequences from our mycorrhizae and 36 from GenBank derived from fruitbodies) were compared by phylogenetic analyses using neighbor-joining, maximum parsimony and maximum likelihood methods. Identification to species level was successful in the case of six EM morphotypes (Tomentella galzinii, T. subtestacea, T. sublilacina, T. pilosa, T. ferruginea and T. stuposa), and the possible taxonomic position of the remaining three was approached. These results are supported by morphology, as compared with literature data. Relative abundance of the EM morphotypes within the soil samples was estimated. Our results confirm that tomentelloid EM are constant, diverse and abundant members of the EM communities in temperate-continental broad-leaved forests.     

Morphological-anatomical characterization and molecular identification of Tomentella stuposa ectomycorrhizae and related anatomotypes

Species in the genus Tomentella (Thelephoraceae) belong to the most frequent and widespread ectomycorrhizal (EM) fungi found in temperate and boreal forests. Although several unidentified tomentelloid morphotypes have been presented as common members of EM communities in coniferous and broad-leaved forests, few tomentelloid EM have been identified and described in detail. In this study, ten tomentelloid EM isolates collected from Populus alba, Quercus cerris and Picea abies stands in Hungary and Germany are characterized and documented by morphological-anatomical methods using light microscopy. The investigated ectomycorrhizae belong to the same brown-black tomentelloid morphotype but form two different anatomotype groups (At I and At II). Molecular taxonomical identification was accomplished using phylogenetic analysis (neighbor joining method) of 49 Tomentella nrDNA-ITS nucleotide sequences including the 10 new and 39 GenBank sequences. The EM isolates clustered into two adjoining clades identical with the two anatomotypes. At II clustered with Tomentella stuposa while At I could not be identified to species. Based on the morphological similarity and the low genetic difference it must be a closely related taxon. A comparison of the recently known tomentelloid EM to T. stuposa is presented. Ecological questions involving abundance and host relationships are discussed.     

Current state and perspectives of truffle genetics and sustainable biotechnology

Mycorrhizal fungi belonging to the genus Tuber produce, after the establishment of a productive interaction with a plant host, hypogeous fruitbodies of great economic value known as ‘‘truffles’’. This review summarizes the state of art on life cycle, genetic, and biotechnological investigations of Tuber spp. The ascocarp formation in truffles is a consequence of the activation of the sexual phase of the biological cycle. The formation of a dikaryotic secondary mycelium and the karyogamy in the ascal cell (followed by meiosis with ascospores formation) have been hypothesized by several authors but some doubts yet arise from the Tuber cycle by considering that a series of abnormalities have been pointed out in respect to other Ascomycetes. It is unclear if binucleated hyphal cells are derived from the fusion of mononucleated cells belonging to mycelia from different mating types or from one only. According to the karyotypes of Tuber melanosporum, Tuber magnatum, and Tuber borchii, the numbers of hyphal chromosomes suggest a chromosome number of eight (2n); these values are in the range of those of several Ascomycetes and observed for Tuber aestivum (2n=10). The importance and growth in interest during the last years in the fungi protoplasts isolation and transformation techniques can be related to current developments in Tuber genetics and biotechnology. T. borchii could be transformed through liposome-mediated delivery of genetic material as mycelial protoplasts isolation and fusion with liposomes has already been established. On the other hand, Agrobacterium-mediated transformation has been successfully established for T. borchii.     

Analysis of gene expression in the vegetative and fructification phases of the white truffle, Tuber borchii Vittad., by mRNA differential display

The mRNA differential display technique was used to compare mRNA populations from fruit body and mycelium of a white truffle species in the attempt to identify and clone differentially expressed genes. The differential expression of five out of 30 amplicons was confirmed. One fragment (Tbm 56) corresponded to a part of the ribosomal genes. Three cDNA fragments (Tbf 12, Tbf 20, Tbf 21) were expressed only in the fructification phase, while the other cDNA (Tbf 55) was expressed strongly in fruit body and also detectable in the mycelium. These clones correspond to part of the single-copy genes in the Tuber borchii Vittad. genome.     

Morphological and genetic study of Prorocentrum donghaiense Lu from the East China Sea, and comparison with some related Prorocentrum species

In this paper, the detailed morphology of Prorocentrum donghaiense Lu from both field samples and cultures was examined, and a taxonomic comparison was made between P. donghaiense and some related Prorocentrum spp. using morphological and molecular data and other published information. There were distinct differences among these species in morphological characteristics that historically have been presented as conservative features. The discrepancies extended beyond that of individual variations within the same species due to environmental factors. Therefore, these morphological features may not be conservative but, rather, polymorphic depending on environmental conditions. Based on this analysis, we suggest that the high-biomass bloom-forming species in the East China Sea, previously reported as Prorocentrum dentatum Stein, is P. donghaiense Lu. The species reported from the East China Sea and Japanese and Korean waters appear to be the same species. Molecular data also suggest that P. dentatum (CCMP1517) and P. donghaiense are genetically identical. Therefore, the geographic distribution of P. donghaiense may be much wider than expected.     

Morphological and molecular characterization of Pisolithus occurring in Hokkaido Island, Northern Japan

Pisolithus basidiomes were found under different forest trees in Hokkaido Island, Japan. These basidiomes were characterized morphologically and molecularly. Although presenting different basidiome morphology and growing under different hosts, specimens presented similar spores ornamentation, and diameters. These spores had coarse, crowded, and blunted spines with three to eight basidiospores per basidium. Ribosomal DNA-based phylogenetic analysis indicated that variability of Pisolithus in this area is low. Phylogenetic analysis showed that Pisolithus analyzed in this study did not group with Pisolithus specimens from other geographical origins. These results suggest that Pisolithus from this area should be taxonomically distinguished from other Pisolithus.     

Morphological and molecular analyses support the existence of host-specific Peronospora species infecting Chenopodium

About 20 species of Peronospora have been reported to cause downy mildew on Chenopodium, but, particularly in plant pathology literature, only one species, P. farinosa, is considered to be involved. We performed sequence analysis of the ITS rDNA to reveal the phylogenetic relationships of Peronospora specimens from five species of Chenopodium, viz. C. album, C. ambrosioides, C. bonus-henricus, C. hybridum, and C. polyspermum. The five clades corresponded to particular Chenopodium species, and showed a high level of sequence divergence. Differences in the morphology of the conidia and ultimate branchlets also supported the separation of the five groups at the host species level. These results suggest that the names P. variabilis, P. boni-henrici, P. chenopodii, and P. chenopodii-polyspermi should be used for the four downy mildew pathogens specific to C. album, C. bonus-henricus, C. hybridum, and C. polyspermum, respectively. The Peronospora on C. ambrosioides was found to be an independent species.     

2种块菌的物种分子鉴定与系统地位分析

以采自内蒙古贺兰山地区的2种块菌为实验材料,分别运用形态学和分子生物学方法进行物种鉴定,并通过与GenBank数据库信息比对,利用Mega5.1软件构建系统进化树,分析其进化关系并确定2种块菌的进化地位。结果表明:两菌株子囊果都为地下生;其中,菌株order-a为灰黄色,近球形到不规则形,较硬,表面被覆微绒毛;菌株tuber-b呈黄褐色,质地均匀,表面有疣状物,顶端呈凹陷状,全株带有干果般特殊的香气。经分子生物学方法分析认为菌株order-a为块菌属的一种,tuber-b为冬块菌(Tuber brumale),并初步确定为内蒙古新记录种。     

Morphological,mycorrhizal and molecular characterization of Finnish truffles belonging to the Tuber anniae species-complex

The truffle species Tuber anniae was originally described from the U.S. Pacific Northwest and is purported to be uncommon. Here, we report for the first time on the fruiting of closely related taxa in Baltic Rim countries. These truffles were found in a forest dominated by Scots pine in eastern Finland. Mycorrhizal analyses confirmed its symbiosis with Pinus sylvestris. Morphological observations of ascomata and mycorrhizae, and phylogenetic analyses confirmed that these white truffles belong within the group of Tuber puberulum (i.e., Puberulum clade). Further, they group in Clades II and III of the T. anniae species-complex. With the inclusion of sequences from GenBank we are able to demonstrate that the previously unnamed environmental clade (Clade II) has been found as ectomycorrhiza in symbiosis with pine, birch, oak, aspen and even orchids in Europe. Thus, the T. anniae species-complex as a whole (and two of the three clades within) exhibit considerable geographic disjuncts: Northwestern North America and the Baltic Rim of Europe. Clade II, which was collected in agricultural soils in Finland and along roadsides in Alaska, may also be adapted for colonization into new habitats. This may help to explain its presence in New Zealand (where Tuber is not native), which most likely resulted from human-mediated dispersal of these fungi through forestry or the nursery trade. Based on our results, we hypothesize that management practices such as organic and lime amendments, along with aeration, are beneficial to the fruiting of T. anniae. Further research is needed to determine the edibility of these species and whether commercial markets can be developed.     

New data on ectomycorrhizae and soils of the Chinese truffles Tuber pseudoexcavatum and Tuber indicum, and their impact on truffle cultivation

Chinese truffles serve as a good complement to the market for Tuber melanosporum (Périgord black truffle). However, Chinese truffles could be introduced accidentally or fraudulently into the plantations of Mediterranean truffles, and they could have a negative effect on truffle production and natural ecosystems. The study of Tuber species from China which are commercialized in Europe began 14 years ago. Tuber pseudoexcavatum was proposed as a new species, and this has been validated by some authors based on molecular and phylogenetic studies. We synthesize their ectomycorrhizae using samples from the type collection, and we compare T. pseudoexcavatum and Tuber indicum ectomycorrhizae. The ectomycorrhizae of these species have a morphology which is related to the ectomycorrhizae of T. melanosporum. We provide useful information for the rapid screening of the above-mentioned Chinese truffles ectomycorrhizae, for the quality control of commercial plants mycorrhized with Tuber. Moreover, we analyze the soil tolerance and the host plant affinity of T. pseudoexcavatum and T. indicum, in order to assess the capacity of both Chinese truffles to penetrate T. melanosporum plantations and habitats.     

Morphological, physiological and molecular genetic characterization ofArabidopsis himalaica, with reference toA. thaliana

Arabidopsis himalaica (Edgeworth) O.E. Schulz, a poorly characterized species typical of HimalayanArabidopsis, was analyzed in terms of its morphology, physiology, chromosome number and molecular genetics, in comparison withA. thaliana which is the standard species in the genusArabidopsis. From view point of developmental genetics, several features which are specific toA. himalaica seem not to be derived by single-gene mutations inA. thaliana. Phylogenetic analyses based onrbcL sequences suggested that genusArabidopsis is not monophyletic. The detailed characterization ofA. himalaica should provide clues to understand the trait of evolution of particular features of Himalayan species ofArabidopsis and their genetic basis.     

Morphological evolution and ecological diversification of the forest-dwelling poppies (Papaveraceae: Chelidonioideae) as deduced from a molecular phylogeny of the ITS region

Sequences of the ITS region of nrDNA were analyzed for the seven genera of Papaveraceae subf. Chelidonioideae s.str. Three major clades can be recognized. These are 1.Chelidonium/Hylomecon/Stylophorum, 2.Eomecon/Sanguinaria, and 3.Bocconia/Macleaya. The monophyly of genera in the first of these three clades is doubtful, and clades two and three are sister to each other. Use of the ITS phylogeny of the subfamily to trace its morphological and ecological evolution shows that morphological change is concentrated in theBocconia/Macleaya clade, and probably related to the evolution of wind-pollination from insect-pollination in these two genera after habitat shift.     

Morphological differences and molecular phylogeny of freshwater blooming species, Peridiniopsis spp. (Dinophyceae) from China

In 2008–2010, several freshwater dinoflagellate blooms caused by Peridiniopsis spp. were observed in China. P. penardii and P. niei sampled from various geographical localities were examined by means of light and scanning electron microscopy. After comparing morphological and molecular differences, the new freshwater variety Peridiniopsis penardii var. robusta var. nov. (Peridiniales, Dinophyceae) found in Manwan Reservoir, Yunnan Province was described. The new variety differed from P. penardii since it possessed numerous robust antapical spines and a conspicuous apical spine. Molecular phylogenetic analyses based on SSU, LSU and ITS indicated P. niei, P. penardii and P. penardii var. robusta were closely related with P. kevei, and clustered into a monophyletic clade. The new variety possessed an endosymbiotic diatom which was similar to P. penardii and P. kevei, whereas the endosymbiont was not present in cells of P. niei. The endosymbiont SSU and ITS phylogenies showed that the endosymbionts of these three dinoflagellates were closely related to members of Thalassiosirales. Furthermore it was concluded that the endosymbionts might originate from Discostella-like species.     

植物菌根块菌分子鉴定及菌丝培养基优化的研究

块菌属(Tuber)是由真菌感染其适宜植物根系形成,部分块菌如法国黑孢块菌(T.melanosporum)和中国印度块菌(T.indicum)等为珍稀药食两用菌,具有很高的经济价值.对块菌的过度挖掘造成野生块菌资源匮乏,块菌人工栽培技术则是实现块菌产业可持续性发展的重要基础.目前国内外人工栽培主要采用块菌子实体匀浆制成...     

Natural hybridization origin of <Emphasis Type="Italic">Rhododendron agastum </Emphasis>(Ericaceae) in Yunnan,China: inferred from morphological and molecular evidence

The natural hybridization that occurs between two sympatric species of Rhododendron subgenus Hymenanthes in Yunnan, China, was investigated. The assumed parents, Rhododendron delavayi Franch. and R. decorum Franch., are morphologically distinct, and the putative hybrid species, R. agastum Balf. f. et W. W. Smith, has an intermediate morphology. We used the main morphological characters, sequences of the nuclear ribosomal DNA ITS region, and the chloroplast DNA trnL-F intronspacer to analyze the three species, and compared these morphological and molecular data with an artificial hybrid between R. decorum (♀) × R. delavayi (♂). From the results, we conclude that R. agastum is a natural hybrid between a female R. delavayi and a male R. decorum.     

Microsatellite primed-PCR to select molecular markers for Tuber species

The direct microsatellite-primed PCR and the RAMPO techniques were applied to detect inter-specific polymorphisms in Tuber species and to select species specific fragments. A T. borchii marker was identified and specific primers were selected.