Epichloë endophytes: fungal symbionts of grasses

Recent developments have increased our understanding of the evolution of mutualistic associations between Epichlo? endophytes and their grass hosts. Most of the asexual species appear to be interspecific hybrids. Although endophytes form compatible associations with their natural hosts, transfers to other hosts elicit a range of incompatible reactions. Recently, the genes involved in the synthesis of the secondary metabolites ergot alkaloids and indole-diterpenes, which confer protective benefits on the association, have been cloned.     

Sympatric Epichloë species and chemotypic profiles in natural populations of Lolium perenne

The distribution of different Epichloë species within eight natural populations of Lolium perenne was studied. In total, 40.2% of the asymptomatic plants were infected by Epichloë festucae var. lolii or by Epichloë typhina. Both species occurred in sympatry in seven grass populations, and some plants had dual infections by both taxa. No hybrid taxa such as Epichloë hybrida were detected. Epichloë festucae strains were classified into two morphotypes, M1 and M3, according to culture characters, both morphotypes occurred in sympatry in seven populations. Plants bearing stromata produced by Epichloë typhina were observed, but asymptomatic plants infected by this species also occurred in seven populations. The alkaloid profile of Lolium perenne plants was related to the morphotype of their infecting strains: most plants infected by M3-strains were characterized by lolitrem, and those with M1-strains contained either ergovaline or lolitrem. Plants infected by Epichloë typhina were characterized by high peramine content.     

A new species of Isoëtes (Isoëtaceae,Pteridophyta) for the Mediterranean

Abstract

Isoëtes iapygia from the Apulia region (southern Italy) is described as a new species and illustrated. It differs in several characters from the other Mediterranean Isoëtes species, but has some intermediate characteristics similar to terrestrial and amphibious Isoëtes. I. iapygia is similar to the amphibious species for the lack of phyllopodia, even if it has scales; it is similar to terrestrial species for the complete velum and tuberculate macrospores. A completely unique character is represented by the two air chambers. Its habitat is on limestone rocks, in temporary pools.     

A new species of Megischus Brullé (Hymenoptera, Stephanidae) from China, with a key to the Chinese species

A new species of Megischus Brullé from China, Megischus aplicatussp. n.,is described and illustrated. A key to the Chinese species of Megischus is added. The holotype is deposited in the Parasitic Hymenoptera Collection of Zhejiang University, Hangzhou.     

Chinese tomato yelIow Ieaf curl virus— a new species of geminivirus

Chine tomato yellow leaf curl virus (TYLCV-CHI) and other geminiviruses were analysed with 20 monoclonaI antibodies. It was shown that TYLCV-CHI is serclogicaIly close to Chinese tabacco Ieaf curl virus (TbLCV-CHI). The fragment of TYLCV-CHI DNA including the common region (CR), N-terminal of coat protein gene and AV1 gene was amplified by PCR and cloned, and its DNA sequence was determined. These raults showed that TYLCV-CHI is different from other known geminiviruses in the world, and is a new whitefly-transmitted gerninivirus.     

Identification of Epichloë endophytes in planta by a microsatellite-based PCR fingerprinting assay with automated analysis

Epichlo? endophytes are a group of filamentous fungi that include both sexual (Epichlo?) and asexual (Neotyphodium) species. As a group they are genetically diverse and form both antagonistic and mutualistic associations with temperate grasses. We report here on the development of a microsatellite-based PCR system for fingerprinting this group of fungi with template isolated from either culture or infected plant material. M13mp19 partial genomic libraries were constructed for size-fractionated genomic DNA from two endophyte strains. These libraries were screened with a mixture of DIG-labeled dinucleotide and trinucleotide repeat probes. Positive clones were sequenced, and nine unique microsatellite loci were identified. An additional microsatellite was serendipitously identified in the 3' untranscribed region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from N. lolii Lp19. Primers were designed for each locus and a panel of endophytes, from different taxonomic groupings, was screened to determine the degree of polymorphism. On the basis of these results a multiplex assay was developed for strain identification with fluorescently labeled primers for five of these loci. Using this system the size of the products amplified can be precisely determined by automated analysis, and an allele profile for each strain can be readily generated. The assay was shown to resolve endophyte groupings to the level of known isozyme phenotype groupings. In a blind test the assay was used successfully to identify a set of endophytes in planta. A reference database of allele sizes has been established for the panel of endophytes examined, and this will be expanded as new strains are analyzed.     

Contig assembly and microsynteny analysis using a bacterial artificial chromosome library for Epichloë festucae, a mutualistic fungal endophyte of grasses

We constructed and characterized a bacterial artificial chromosome (BAC) library for Epichlo? festucae, a genetically tractable fungal plant mutualist. The 6144 clone library with an average insert size of 87kb represents at least 18-fold coverage of the 29 Mb genome. We used the library to assemble a 110kb contig spanning the putative ornithine decarboxylase (odc) ortholog and subsequently expanded it to 228kb with a single walking step in each direction. Furthermore, we evaluated conservation of microsynteny between E. festucae and some model filamentous fungi by comparing sequence available from a 43kb region at the end of one BAC to publicly available fungal genome sequences. Orthologs to the 13 contiguous open reading frames (ORFs) identified in E. festucae are syntenic in Neurospora crassa and Magnaporthe grisea occurring in small sets of two, three or four colinear ORFs. This library is a valuable resource for research into traits important for the development and maintenance of a plant-fungus mutualistic symbiosis.     

Campanula xylocarpa — A new species of the seriesSaxicolae Witasek

In this paper, a newCampanula species is described from Eastern Slovakia. Its morphological, distributional and cytological relationships and ecological preferences are briefly discussed and affinity to the otherSaxicolae examined. The taxon is characterized by the following features: specific combination of constant morphological characters; diploid chromosome number (2n = 34); ecological specialization to the fissures of limestone and dolomitic rocks; relict occurrence in a small, sharply limited area; very late flowering. Geographically, it appears to be the northernmost representative of theSaxicolae.     

Formation of arthroconidia during regeneration and selection of transformed Epichloë festucae protoplasts

Transformation is an essential tool for modern fungal research and has played a fundamental role in gaining insight into gene function. Polyethylene glycol (PEG)-mediated transformation of protoplasts is the most commonly used method for genetic transformation of filamentous fungi. Selectable marker genes, that confer resistance to antibiotics, are generally incorporated with the DNA of interest, allowing transformed cells to grow through the antibiotic overlay. Colonies arising from transformed fungal cells are sub-cultured and further analysed. However, the morphological state of the fungal cells during the transformation procedure has been largely overlooked. We investigated the morphological appearance of transformed fungal cells prior to their emergence through the antibiotic overlay. Hyphae appeared to segment and bulge, reminiscent of arthroconidia, an asexual spore typically produced by segmentation of pre-existing hyphae. Selective expression of eGFP under the control of a spore specific promoter, PcatA, in these cells confirmed their spore-like nature. Reducing the oxygen availability to surface-grown cultures partially recapitulated this morphological form. A GFP fusion to the cell wall integrity MAP kinase MpkA localised to the arthroconidia nuclei suggesting the cell wall integrity signalling pathway modulates cell wall stress responses in arthroconidia. This dramatic morphological change was also observed in transformed Magnaporthe oryzae cells suggesting it may be a more general phenomenon in filamentous fungi. Given the changes in cellular structure and spore-like appearance, these observations may have technical implications for deleting genes involved in these processes in Epichloë festucae and, more broadly, a range of fungal species.     

Isoëtes sabatina (IsoëtaceaeLycopodiophyta), a new aquatic species from central Italy

The new species Isoëtes sabatina is described and illustrated. Morphological, anatomical, and ecological characters are given. It differs from the similar I. echinospora by several characters, such as megaspore ornamentation and size, microspore size, velum extension and form, and also for its ecology. A table showing the main differential characters of the new species compared with those of the other European aquatic species is presented. So far, the species is known from a single locality (Bracciano Lake, central Italy), having a peculiar ecology and within the Mediterranean bioclimate.