Identification and characterization of a new group of root-colonizing fungi within the Gaeumannomyces–Phialophora complex

A new group of darkly pigmented root-infecting fungi was isolated from cereal roots obtained from six different locations in northeastern Germany. Similar random amplified polymorphic DNA(RAPD) patterns and restriction profiles of amplified rDNA were used as a basis for classifying the isolates in a separate group. The isolates demonstrating mycelial and infection characteristics typical of Gaeumannomyces graminis could be differentiated from the varieties of G. graminis as well as from Gaeumannomyces cylindrosporus / Phialophora graminicola using RAPD Polymerase chain reaction (PCR) and rDNA Restriction-fragment length polymorphism (RFLP) analysis. Phylogenetic analysis of the Internal transcribed spacer (ITS) regions suggests that the isolates form a distinct group (named group 'E') situated within the Gaeumannomyces – Phialophora complex between the branch of the G. graminis varieties and Gaeumannomyces incrustans / Magnaporthe poae . Isolates of group E produced lobed hyphopodia and were shown in biotests to be non-pathogenic to wheat, oats, Italian Ryegrass and Chewings Fescue, suggesting it is a benign parasite which colonizes cereals or grasses without destroying vascular tissue. Furthermore, curved phialospores could be found. Summarizing the results presented, this new group could be classified as a new species of Phialophora . Although isolates of group E were found at only six of the 32 investigated locations, they composed up to 50% of total isolates of the Gaeumannomyces – Phialophora complex at these sites. Because of the non-pathogenic behaviour, the new group may be of value as biological control agents for pathogenic fungi.     

Comparison of fungi within the Gaeumannomyces-Phialophora complex by analysis of ribosomal DNA sequences.

Four ascomycete species of the genus Gaeumannomyces infect roots of monocotyledons. Gaeumannomyces graminis contains four varieties, var. tritici, var. avenae, var. graminis, and var. maydis. G. graminis varieties tritici, avenae, and graminis have Phialophora-like anamorphs and, together with the other Gaeumannomyces and Phialophora species found on cereal roots, constitute the Gaeumannomyces-Phialophora complex. Relatedness of a number of Gaeumannomyces and Phialophora isolates was assessed by comparison of DNA sequences of the 18S rRNA gene, the 5.8S rRNA gene, and the internal transcribed spacers (ITS). G. graminis var. tritici, G. graminis var. avenae, and G. graminis var. graminis isolates can be distinguished from each other by nucleotide sequence differences in the ITS regions. The G. graminis var. tritici isolates can be further subdivided into R and N isolates (correlating with ability [R] or inability [N] to infect rye). Phylogenetic analysis of the ITS regions of several oat-infecting G. graminis var. tritici isolates suggests that these isolates are actually more closely related to G. graminis var. avenae. The isolates of Magnaporthe grisea included in the analysis showed a surprising degree of relatedness to members of the Gaeumannomyces-Phialophora complex. G. graminis variety-specific oligonucleotide primers were used in PCRs to amplify DNA from cereal seedlings infected with G. graminis var. tritici or G. graminis var. avenae, and these should be valuable for sensitive detection of pathogenic isolates and for diagnosis of take-all.     

A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea

Magnaporthe oryzae is described as a new species distinct from M. grisea. Gene trees were inferred for Magnaporthe species using portions of three genes: actin, beta-tubulin, and calmodulin. These gene trees were found to be concordant and distinguished two distinct clades within M. grisea. One clade is associated with the grass genus Digitaria and is therefore nomenclaturally tied to M. grisea. The other clade is associated with Oryza sativa and other cultivated grasses and is described as a new species, M. oryzae. While no morphological characters as yet distinguish them, M. oryzae is distinguished from M. grisea by several base substitutions in each of three loci as well as results from laboratory matings; M.oryzae and M. grisea are not interfertile. Given that M. oryzae is the scientifically correct name for isolates associated with rice blast and grey leaf spot, continued use of M. grisea for such isolates would require formal nomenclatural conservation.     

Telomere-targeted retrotransposons in the rice blast fungus Magnaporthe oryzae: agents of telomere instability

The fungus Magnaporthe oryzae is a serious pathogen of rice and other grasses. Telomeric restriction fragments in Magnaporthe isolates that infect perennial ryegrass (prg) are hotspots for genomic rearrangement and undergo frequent, spontaneous alterations during fungal culture. The telomeres of rice-infecting isolates are very stable by comparison. Sequencing of chromosome ends from a number of prg-infecting isolates revealed two related non-LTR retrotransposons (M. oryzae Telomeric Retrotransposons or MoTeRs) inserted in the telomere repeats. This contrasts with rice pathogen telomeres that are uninterrupted by other sequences. Genetic evidence indicates that the MoTeR elements are responsible for the observed instability. MoTeRs represent a new family of telomere-targeted transposons whose members are found exclusively in fungi.     

禾顶囊壳4个变种对禾本科牧草的致病性研究

采用人工接种法测定了禾顶囊壳小麦变种、燕麦变种、玉米变种和禾谷变种对4种禾本科牧草鸭茅、披碱草、苇状羊茅和无芒雀麦的致病性。结果表明,除禾顶囊壳玉米变种Ggm01菌株对披碱草没有致病性外,4个变种对供试的其它牧草都有致病性,且能产生全蚀病的典型症状。其中小麦变种和燕麦变种对供试牧草的致病性强于禾谷变种,玉米变种致病性最弱。各变种的菌株存在着致病性分化。小麦变种Ggt9813菌株对苇状羊茅和披碱草的致病性强于燕麦变种,发病严重度均达到50％以上。目前在我国尚未发现燕麦变种,小麦变种主要出现在北方地区,在牧草上的发生亦不广泛,因此二者具有检疫重要性。     

全蚀病菌在玉米上的新变种

本文报道了玉米全蚀病菌禾顶囊壳菌(Gaeumannomyces graminis)的新变种——玉米变种[Gaeumannomyces graminis(Sacc.)Arx et Olivler var.maydis Yao Wang et Zhu var.nov.]。该变种在形态学、致病性、生物学及可溶性蛋白电泳谱带等方面,均不同于禾顶囊壳菌小麦变种[G.graminis var,tritici J.Walker)、水稻变种(G.graminis var.graminis Trans.)和燕麦变种[G.graminis var.avenae(Turner)Dennis]。模式标本保存在沈阳农业大学真菌标本室。     

Microorganisms in the rhizosphere of wheat colonized by the fungusGaeumannomyces graminis var.tritici

The population of microorganisms in wheat rhizosphere changed in the presence of the fungus Gaeumannomyces graminis var. tritici causing the take-all of wheat. In the majority of cases when the soil was artificially contaminated by the fungus, both the number of bacteria in the rhizosphere and the bacteria/fungi ratio temporarily increased. At the beginning bacteria growing in the presence of NH4+ predominated, later bacteria utilizing organic N-substances prevailed. Pseudomonas fluorescens and the related species colonized the rhizosphere and the soil to a greater extent in the presence of G. graminis. The wheat rhizosphere with G. graminis was found to contain a higher level of the slime-producing bacterium Agrobacterium spp.; this microorganism occurred on hyphal surfaces (in hyphosphere) of both G. graminis growing in soil and Mucor spp. Changes in microbial populations in the wheat rhizosphere during the first stage of colonization by G. graminis can be partly explained by a simultaneous rhizosphere colonization by microorganisms which accompany this fungus in soil. In the period of increase in the number of bacteria in rhizosphere a temporary stimulation of wheat growth was observed.     

Molecular phylogeny of the Blastocladiomycota (Fungi) based on nuclear ribosomal DNA

The Blastocladiomycota is a recently described phylum of ecologically diverse zoosporic fungi whose species have not been thoroughly sampled and placed within a molecular phylogeny. In this study, we investigated the phylogeny of the Blastocladiomycota based on ribosomal DNA sequences from strains identified by traditional morphological and ultrastructural characters. Our results support the monophyly of the Coelomomycetaceae and Physodermataceae but the Blastocladiaceae and Catenariaceae are paraphyletic or polyphyletic. The data support two clades within Allomyces with strains identified as Allomyces arbusculus in both clades, suggesting that species concepts in Allomyces are in need of revision. A clade of Catenaria species isolated from midge larvae group separately from other Catenaria species, suggesting that this genus may need revision. In the Physodermataceae, Urophlyctis species cluster with a clade of Physoderma species. The algal parasite Paraphysoderma sedebokerensis nom. prov. clusters sister to other taxa in the Physodermataceae. Catenomyces persicinus, which has been classified in the Catenariaceae, groups with the Chytridiomycota rather than Blastocladiomycota. The rDNA operon seems to be suitable for classification within the Blastocladiomycota and distinguishes among genera; however, this region alone is not suitable to determine the position of the Blastocladiomycota among other basal fungal phyla with statistical support. A focused effort to find and isolate, or directly amplify DNA from additional taxa will be necessary to evaluate diversity in this phylum. We provide this rDNA phylogeny as a preliminary framework to guide further taxon and gene sampling and to facilitate future ecological, morphological, and systematic studies.     

Genome organization and evolution of the AVR-Pita avirulence gene family in the Magnaporthe grisea species complex

The avirulence (AVR) gene AVR-Pita in Magnaporthe oryzae prevents the fungus from infecting rice cultivars containing the resistance gene Pi-ta. A survey of isolates of the M. grisea species complex from diverse hosts showed that AVR-Pita is a member of a gene family, which led us to rename it to AVR-Pita1. Avirulence function, distribution, and genomic context of two other members, named AVR-Pita2 and AVR-Pita3, were characterized. AVR-Pita2, but not AVR-Pita3, was functional as an AVR gene corresponding to Pi-ta. The AVR-Pita1 and AVR-Pita2 genes were present in isolates of both M. oryzae and M. grisea, whereas the AVR-Pita3 gene was present only in isolates of M. oryzae. Orthologues of members of the AVR-Pita family could not be found in any fungal species sequenced to date, suggesting that the gene family may be unique to the M. grisea species complex. The genomic context of its members was analyzed in eight strains. The AVR-Pita1 and AVR-Pita2 genes in some isolates appeared to be located near telomeres and flanked by diverse repetitive DNA elements, suggesting that frequent deletion or amplification of these genes within the M. grisea species complex might have resulted from recombination mediated by repetitive DNA elements.     

Widespread occurrence and phylogenetic placement of a soil clone group adds a prominent new branch to the fungal tree of life

Fungi are one of the most diverse groups of Eukarya and play essential roles in terrestrial ecosystems as decomposers, pathogens and mutualists. This study unifies disparate reports of unclassified fungal sequences from soils of diverse origins and anchors many of them in a well-supported clade of the Ascomycota equivalent to a subphylum. We refer to this clade as Soil Clone Group I (SCGI). We expand the breadth of environments surveyed and develop a taxon-specific primer to amplify 2.4kbp rDNA fragments directly from soil. Our results also expand the known range of this group from North America to Europe and Australia. The ancient origin of SCGI implies that it may represent an important transitional form among the basal Ascomycota groups. SCGI is unusual because it currently represents the only major fungal lineage known only from sequence data. This is an important contribution towards building a more complete fungal phylogeny and highlights the need for further work to determine the function and biology of SCGI taxa.     

Inflorescence diversification in the "finger millet clade" (Chloridoideae, Poaceae): a comparison of molecular phylogeny and developmental morphology

Within the Poaceae, inflorescence diversification and its bearing on phylogeny and evolution are exceedingly complex. We used phylogenetic information of the "finger millet clade," a group of grasses with digitate inflorescences, to study the inflorescence diversification. This clade appears monophyletic in the morphological and molecular phylogenetic analyses. Three well-supported clades are shown in our cpDNA-derived phylogeny, with clades I and III consisting of species of Chloris and Microchloa, respectively, and clade II including species of Cynodon, Dactyloctenium, and Eleusine. Variation appears at different times throughout development. Changes involving primordium number and arrangement occur very early, changes involving duration of primordium activity occur much later. Characters derived from the comparison of developmental sequences were optimized onto the most parsimonious tree. The developmental characters were congruent with the molecular phylogeny. Two developmental characters may not be homologous in the Chloris subclade and the Cynodon subclade.     

Effector-mediated suppression of chitin-triggered immunity by magnaporthe oryzae is necessary for rice blast disease

Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Development and application of a TaqMan real-time PCR assay for rapid detection of Magnaporthe poae

In North America, one of the most important root diseases of Poa and Festuca turf is summer patch, caused by Magnaporthe poae. Detection and identification of M. poae in infected roots by conventional culture-based methods is difficult and time consuming, typically taking 3 wk or longer to accomplish. In this study, a culture-independent, TaqMan real-time PCR assay was developed for the detection of M. poae from the roots of fungicide treated and non-treated Kentucky bluegrass (Poa pratensis) turf. The assay was validated with the target pathogen, closely related fungal species and a number of other microorganisms that inhabit the same host and soil environment. This assay was more sensitive (could detect as little as 3.88 pg genomic DNA of M. poae), rapid and accurate compared to direct microscopic observation and isolation on a selective medium. The real-time PCR detection results corresponded closely to visual assessments of disease severity in the field. Utilization of this assay in diagnostic laboratories will enable turfgrass managers to more quickly and effectively detect and potentially reduce fungicide usage through early and accurate identification of the pathogen.     

Phylogeny of the sea spiders (Arthropoda, Pycnogonida) based on direct optimization of six loci and morphology

Higher‐level phylogenetics of Pycnogonida has been discussed for many decades but scarcely studied from a cladistic perspective. Traditional taxonomic classifications are yet to be tested and affinities among families and genera are not well understood. Pycnogonida includes more than 1300 species described, but no systematic revisions at any level are available. Previous attempts to propose a phylogeny of the sea spiders were limited in characters and taxon sampling, therefore not allowing a robust test of relationships among lineages. Herein, we present the first comprehensive phylogenetic analysis of the Pycnogonida based on a total evidence approach and Direct Optimization. Sixty‐three pycnogonid species representing all families including fossil taxa were included. For most of the extant taxa more than 6 kb of nuclear and mitochondrial DNA and 78 morphological characters were scored. The most parsimonious hypotheses obtained in equally weighted total evidence analyses show the two most diverse families Ammotheidae and Callipallenidae to be non‐monophyletic. Austrodecidae + Colossendeidae + Pycnogonidae are in the basal most clade, these are morphologically diverse groups of species mostly found in cold waters. The raising of the family Pallenopsidae is supported, while Eurycyde and Ascorhynchus are definitely separated from Ammotheidae. The four fossil taxa are grouped within living Pycnogonida, instead of being an early derived clade. This phylogeny represents a solid framework to work towards the understanding of pycnogonid systematics, providing a data set and a testable hypothesis that indicate those clades that need severe testing, especially some of the deep nodes of the pycnogonid tree and the relationships of ammotheid and callipallenid forms. The inclusion of more rare taxa and additional sources of evidence are necessary for a phylogenetic classification of the Pycnogonida. © The Willi Hennig Society 2006.