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This contribution of Mycological Research News features: Can edible mushrooms promote sustainability in Beijing?; and Mycorrhizal fungi increase available calcium.
Molecular phylogenetic papers in this issue concern the phylogeny of Trichoderma based on a multigene approach, the molecular phylogeny of Nephroma, and monophyletic groups in Parmelia saxatilis. The synthesis and resynthesis of the lichen Baeomyces rufus has been examined molecularly and morphologically. Strains of Coniothyrium minitans are compared by morphological, physiological and molecular methods. Laccase genes from Gaeumannomyces graminis var. graminis, and polygalacturonases from Botrytis cinerea, have been characterized. Genetic transformation has been successfully achieved to insert markers into Clonostachys rosea, and ISSR-PCR fingerprinting used to examine the distribution of dark septate endophytes.
Three papers focus on hitherto undescribed fungi, a new genus of Harpellales on mayfly nymphs, and new species of Gaeumannomyces on Stenotaphrum and of Phytophthora on Ipomoea.
The following new scientific names are introduced: Tectimyces gen. nov.; and Gaeumannomyces wongoonoo, Phytophthora ipomoeae, T. leptophlebiidarum, and T. robustus spp. nov.
The phylogeny of Trichoderma and the phylogenetic relationships of its species was investigated by maximum parsimony analysis and distance analysis of DNA sequences from multiple genetic loci. 18S rDNA sequence analysis suggests that the genus Trichoderma evolved at the same time as Hypomyces and Fusarium and thus about 110 Myr ago. 28S rDNA sequence analysis shows that the genus Trichoderma is part of a monophyletic branch within the Hypocreaceae, which also includes Arachnocrea and Aphysiostroma in basal positions. Gene trees inferred from a combined analysis of the nuclear ribosomal internal transcribed spacer (ITS1 and 2), the D1 and D2 region of the 28S rDNA, the small subunit of the mitochondrial rDNA (mitSSU), the fifth and part of the sixth exon of translation elongation factor 2 (tef1), and a fragment of ech42 provide strong statistical support for a phylogeny consistent with the existence of four clades: clade A comprises species of Bissett's (1991) sect. Trichoderma but also T. hamatum, T. pubescens, T. asperellum, and T. strigosum; clade C comprises all the species contained in section Longibrachiatum as revised by Samuels et al. (1998), and clade D contains only T. aureoviride which is genetically most distant to all other species. Clade B, on the other hand, contains a large and taxonomically heterogeneous mixture of species, among which several subclades could be identified: subclade B1 containing H. lactea, H. citrina, H. citrina var. americana, H. lutea, and an unnamed T. sp. 1; subclade B2 containing T. stromaticum, and an unnamed T. sp. PPRI3559; subclade B3 containing T. fertile, T. oblongisporum, and H. hunua; subclade B5 containing T. polysporum, T. croceum, Hypocrea pilulifera, and T. minutisporum; and a larger subclade (B4), in which three strain clusters could be distinguished: one comprising T. harzianum, T. inhamatum, H. vinosa, and T. aggressivum, another one containing T. fasciculatum, T. longipile, and T. strictipile; and a third containing T. virens, T. flavofuscum, and T. crassum. The position of the remaining species of subclade B4 (T. spirale, T. cfr aureoviride, H. tawa, and T. tomentosum) was not resolved. A comparison of the topologies of the individual gene trees was concordant with the topology of the combined tree in most cases, but also revealed incongruent positions for a few species (T. oblongisporum, T. longipile, T. fasciculatum) which was most pronounced in the ITS1 and 2 tree. The results confirm the recent concept for sects Longibrachiatum and Trichoderma, indicate that the sects Hypocreanum and Pachybasium cannot be distinguished phylogenetically, and provide a first phylogenetic basis for dissection of the latter two sections.
The symbiotic phenotype of a lichen arises through the interaction and cooperation of two or more genetically unrelated partners. Ultrastructural and molecular methods were used to investigate the changes that take place during early stages of the lichenization process. The resynthesis of prethallus stages of Baeomyces rufus was studied by co-culturing under sterile conditions the isolated, axenically grown mycobiont and its green algal photobiont Elliptochloris bilobata. The lichenization process was monitored by SEM. One day after co-culture, symbionts were bound together by a newly secreted mucilage. By day 12, photobiont induced, morphological changes in the mycobiont were visible. Aerial hyphae grew around photobiont cells, showed a high frequency of branching and formed appressoria on the algal wall surface. By day 28, many photobiont cells were completely engulfed by hyphae and soredia-like clusters were observed. These morphological developments resemble lichenized structures formed in the natural lichen. cDNA-AFLP was used to investigate gene expression profiles on day 12 of co-culture. Differential gene expression patterns revealed that few genes were induced, and many fungal and algal genes seemed to be suppressed in the early stages of lichenization.
The phylogeny of Nephroma was studied by nucleotide sequences of the mitochondrial ribosomal small subunit (mtSSU rDNA) and the internal transcribed spacers of the nuclear ribosomal repeat (ITS), together with chemical characters. The biological material included both bipartite and tripartite species and all Nephroma species native to northern Europe. Phylogenetic analyses demonstrated that all Nephroma species form a monophyletic group and that Peltigera constitutes the sister group to Nephroma. The two gene regions revealed qualitatively similar relationships within Nephroma and chemical characters had a minor impact on tree topologies. The results demonstrated that tripartite Nephroma species do not form a monophyletic group within the genus, this being in agreement with previous findings from bi- and tripartite Peltigera species. The results also indicated that N. resupinatum does not form a monophyletic group with all other bipartite Nephroma species but form a sister group to the studied Nephroma taxa. Furthermore, N. helveticums. lat. is highly variable and seems to represent aggregates of closely related taxa. Also N. laevigatum and N. resupinatum are genetically variable. All European Nephroma species can be rapidly and accurately identified on the basis of their fungal ITS sequences. This will prove useful in ecological and environmental studies.
A considerable number of species of lichen-forming fungi have wide geographical distributions, but studies of their genetic variability are minimal. ITS rDNA sequences of 32 populations of Parmelia saxatilis from five continents revealed two monophyletic groups. β-tublin gene sequences from a subset of nine collections supported these conclusions. While the number of collections sequenced is limited, one monophyletic group (the Atlantic Population. AtP) was recognized as occurring in Arctic and Antarctic regions and also included collections from more atlantic sites. Samples from more mesic environments in the Mediterranean region belonged to a second monophyletic group (the Mediterranean Population, MeP). In addition, four subgroups were distinguishable within the Atlantic Population. Norstictic and protocetraric acids are reported from the species for the first time, the norstictic acid only being found in the Atlantic Population. Living thalli from the Atlantic Population were provenance-tested; specimens transported from the UK to central Spain where the Mediterranean Population occurs showed adverse symptoms after six months. These results demonstrate that there can be substantial large-scale genotypic variability within widespread lichen phenospecies, something which has implications for comparative ecological, physiological, and air pollution sensitivity studies as well as for lichen conservation.
Eight strains of the mycoparasite Coniothyrium minitans were analysed for intraspecific variation by three different approaches: analysis of morpho-physiological characteristics; random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis of the genomic DNA. A high variability in colony colour, gross morphology and conidial size was found depending on strain and culture conditions. All individual strains could be distinguished using the three type of analyses. However, a close correspondence between RAPD, AFLP patterns and morpho-physiological characteristics of C. minitans strains was not observed. Our results suggest AFLP analysis constituted a more efficient and reliable tool than RAPD analysis or morpho-physiological studies to detect intraspecific variability in C. minitans and to follow its fate after application in the field.
Recently we cloned and characterized three laccase genes from Gaeumannomyces graminis var. tritici, an important pathogen of wheat. Here we report cloning and characterization of two laccase genes from G. graminis var. graminis, a weak pathogen of rice and turf grasses. LAC1 and LAC2 genes were present in both varieties of the fungus. The genes were 94–95% identical, and intron positions were conserved between the two varieties. Our data demonstrated that laccases might be useful for phylogenetic studies to detect fine differences between G. graminis subspecies, varieties, or strains of the fungus that cannot be detected by traditional sequencing of 18S rRNA genes or ITS regions. We previously characterized two G. graminis var. graminis melanin mutants with altered lytic enzyme secretion patterns. Here we demonstrate altered transcription patterns of laccase genes between the two varieties and between the wild type and melanin mutants of G. graminis var. graminis. Transcription of LAC2 was downregulated in the over-melanized mutant as compared to wild-type G.graminis var. graminis and the unmelanized mutant, whereas transcription of LAC1 in planta was up-regulated in the over-melanized mutant, as compared to the wild type and the unmelanized mutant. In the unmelanized mutant transcription of both genes was similar to that observed in the wild type.
Marker genes were introduced in the biocontrol strain Clonostachys rosea IK726 (IBT 9371) as a tool for monitoring the strain in ecological studies. The β-glucuronidase (GUS) reporter gene and a gene encoding the green fluorescent protein (GFP) were, in separate experiments, integrated into the genome of IK726 using the Hygromycin B (HygB) resistance gene as selective marker. In order to select GUS and GFP transformants that resembled the wildtype strain, growth rate, production of enzymes and of metabolites of four GUS positive and six GFP positive transformants were tested in vitro. In addition, the biocontrol efficacy against disease caused by seed-borne Fusarium culmorum was evaluated on barley grown in sand. Compared to the wildtype, two selected GUS and GFP transformants, IK726c5 and IK726d11, did not vary in physiological properties. Both maintained the ability to colonize barley roots, and to reduce efficiently the severity of F. culmorum without affecting plant emergence. Quantification of GUS activity of IK726c5 in peat and vermiculite and on seeds was carried out. The GFP transformant, IK726d11, was visualized by epifluorescence and confocal scanning laser microscopy directly in soil, vermiculite, on carrot seed and roots, and on barley leaves. It was shown that C. rosea can thrive in very different niches. Conidia germination, colonization and conidiogenesis were demonstrated in vivo in all four environments. This is the first report on transformation of Clonostachys rosea with marker genes.
Polygalacturonase (PG) secretion was analysed from five independent isolates of Botrytis cinerea grown in minimal liquid media enriched with pectin as the only carbon source. To acquire information about the biochemical characteristics of the PGs secreted in vitro, we combined enzyme-activity assays with Western blot analysis and isoelectro-focusing (IEF). All isolates secreted at least four PGs isozymes with a similar electrophoretic patterns, easily distinguishable by western blots. IEF enabled the isoelectric point of the PGs for each isolate to be determined. Wide variability in isoelectric point was observed between the PGs of each isolate, ranging from above pH 9.0 to below pH 5.0. For one of the isolates, each PG secreted in vitro was further purified by chromatofocusing and analysed for endo/eso-polygalacturonase activity by thin layer chromatography. This combination of analytical techniques and enzyme assays provided a view of the pool of PGs produced by B. cinerea and defined the biochemical features of each isozyme.
In the present study we investigated the abundance and spatial distribution of dark septate root endophytes (DSE) in a 3×3 m plot in a spruce stand (Picea abies). A total of 144 DSE isolates were obtained by means of a hierarchical sampling design. Most roots were colonised, as DSE were isolated from 81.7% of root segments. ISSR–PCR fingerprinting was used to identify 21 unique ISSR types. Dominant types were isolated from adjacent points that covered an area of up to 6.8 m2 of the study plot, and ISSR types were intermingled extensively. Frequency of isolation of the different ISSR types was uneven with two dominant types that accounted for 38% and 28% of all DSE isolates, respectively. Seven DSE strains representing six different ISSR types were identified as Phialocephala fortinii based on the morphology of fertile conidiophores and/or ITS 1 and 2 sequence comparisons.
The genus Tectimyces gen. nov. (Harpellales: Legeriomycetaceae) is described with two species, T. leptophlebiidarum and T. robustus spp. nov., collected on the hindgut of the mayfly nymph Habroleptoides confusa (Ephemeroptera: Leptophlebiidae) in northern Spain. This is the second report of a trichomycete inhabiting a member of this family of ephemerids. Diagnostic for the new genus are type II zygospores and unappendaged trichospores, borne on long generative cells and carrying a very short collar after release. The position and morphological traits of the newly described taxa are discussed and compared with other genera and species, such as Bojamyces repens and Orphella spp.
A Phytophthora species was found on blighted foliage of Ipomoea longipedunculata, a morning glory native to the highlands of central Mexico. Based on host range, morphology, allozymes, mitochondrial DNA haplotype and rDNA sequences it is concluded that a new Phytophthora species, P. ipomoeae sp. nov., is the causal agent of leaf blight disease on I. longipedunculata.
The causal agent of a patch disease of buffalo grass (syn. St Augustine grass) is determined and described as a new species, Gaeumannomyces wongoonoo sp. nov. Glasshouse experiments have shown that it is pathogenic to buffalo grass (cultivar ST 1191) but not to maize or wheat. The pathogen is different to the varieties of G. graminis in having shorter ascospores (36–75 μm) and producing numerous perithecia readily on malt extract and potato dextrose agars. The disease is named ‘wongoonoo patch’ to distinguish it from take-all patch of turf grasses caused by G. graminis.