New neotyphodium endophyte species from the grass tribes stipeae and meliceae.

Several species of Achnatherum (grass tribe Stipeae) and Melica (tribe Meliceae) typically are infected by nonpathogenic, seed-transmissible fungi with characteristics of Neotyphodium species (anamorphic Clavicipitaceae). Molecular phylogenetic studies clearly have distinguished the endophytes from Achnatherum inebrians (from Xinjiang Province, China), A. robustum and A. eminens (both from North America) and indicate that the A. inebrians endophyte comprises a unique nonhybrid lineage within the Epichloe and Neotyphodium phylogeny, whereas the endophytes of A. robustum, and A. eminens are hybrids with multiple EpichlooY species (holomorphic Clavicipitaceae) as ancestors. Likewise distinct hybrid origins are indicated for Neotyphodium species from the European Melica species, M. ciliata and M. transsilvanica, the South African species M. decumbens and M. racemosa, and the South American species M. stuckertii. Neotyphodium species have been described from A. inebrians from Gansu Province, China, (N. gansuense), A. eminens (N. chisosum), M. stuckertii (N. tembladerae) and the South African Melica species (N. melicicola). However the endophytes from A. robustum and the European Melica species have not been described and the phylogenetic relationships of N. gansuense have not been investigated. Here we report a comprehensive study of morphological features and phylogenetic analyses of beta -tubulin and actin gene sequences on an expanded collection of endophytes from the Stipeae and Meliceae. These data provide a firm foundation for the description of two new Neotyphodium species, N. guerinii from M. ciliata and M. transsilvanica, and N. funkii from A. robustum. We also propose the new variety, N. gansuense var. inebrians for endophytes of A. inebrians from Xinjiang Province, which are morphologically and phylogenetically distinct from, yet clearly related to, N. gansuense from Gansu Province.

The relationship between mycotoxin synthesis and isolate morphology in fungal endophytes of Lolium perenne.

• Variability in the fungal endophytes of 83 natural populations of Lolium perenne (perennial ryegrass) from Europe was assessed. • One plant per population was used for endophyte isolation and mycotoxin analysis. Variability in three isozymes, colony morphology and growth rate on potato dextrose agar (PDA), and synthesis of ergovaline, lolitrem B and peramine was recorded. • Three species were found among 94 strains isolated: Neotyphodium lolii, Neotyphodium sp. (LpTG-2) and Gliocladium-like. The most frequent species was N. lolii, which showed high variability. In 12 populations, a single plant harboured two different endophytes. One-third of the isolates of N. lolii did not produce ergovaline whereas a few isolates did not produce lolitrem B. Ergovaline and lolitrem B-deficient strains, but not the few peramine-deficient isolates, had characteristic morphologies on PDA. No isolate was deficient for both ergovaline and lolitrem B synthesis. • Selection of ergovaline and lolitrem-deficient strains based only on the morphology of the isolates in culture may be possible.

Species delimitation in the African Armillaria complex by analysis of the ribosomal DNA spacers.

Thirteen Armillaria isolates, collected from various geographical areas in tropical Africa and previously characterized by cultural morphology, pairing tests and isozyme analysis, were evaluated using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). DNA regions corresponding to the intergenic spacer (IGS) and internal transcribed spacer (ITS) were amplified and analyzed by restriction enzyme digestion. The IGS amplification products were about 875 bp long and uniform in length among the isolates. The amplified-ITS region showed two different lengths corresponding to two groups. The first group included the isolates believed to belong to A. mellea ssp. africana and two Kenyan isolates (K11 and K12) belonging to a yet unnamed biological species. The second group included isolates identified as A. heimii and a Tanzanian isolate (T7). Each length variant of the ITS showed distinct RFLP banding patterns. Digestion with EcoRI confirmed the two polymorphic groups while the endonucleases AluI and NdeII discriminated the A. mellea isolates from the Kenyan isolates K11 and K12. In addition, the latter enzyme showed a slight dissimilarity between the A. heimii isolates from Western and Eastern Africa (C1 and Z1). Digestion with HinfI cleaved the isolates of A. heimii into two sub-groups corresponding to the heterothallic and homothallic forms. This endonuclease also indicated that the isolate T7, originating from Tanzania, was clearly similar to the heterothallic species A. heimii. Data presented support the maintenance of three distinct species of Armillaria in tropical Africa with A. heimii as a variable species, the isolates of which were separated in accordance with their sexual system. The results indicate that PCR-RFLP can be used as a simple and speedy taxonomical tool for the ecological studies of Armillaria species.

A comparison of different methods for the identification of genets of Armillaria spp.

In a previous study, somatic incompatibility (SI) was used to map the genotypes (or genets) of three Armillaria species (A. ostoyae, A. gallica, A. cepistipes) at four forest sites. A total of 109 genets was identified among 764 isolates of the three species. The object of the present study was to compare SI with three other methods of distinguishing genets: Random Amplified Polymorphic DNAs (RAPD), mating-type alleles, and isoenzyme analysis. RAPDs and mating-type alleles were used with 73 isolates belonging to 36 genets identified on the basis of SI reactions. A few isolates were assayed for isoenzyme polymorphism. RAPDs, mating-type alleles, and SI were equal in their ability to distinguish genets in the populations tested. In three cases, isolates distinguished by SI reactions included two subgroups with different RAPD phenotypes. In two cases groups distinguished by different SI reactions shared the same set of mating-type alleles. Isoenzyme analysis showed interspecific differences and a few differences between the genets of the same species. These results validate the use of SI as a routine method for epidemiological studies of Armillaria spp. The EcoR I fragment profiles of mitochondrial DNA of the same group of isolates were also assayed. The analysis regrouped the genets into 'mitochondrial types' sharing a common cytoplasm and possibly closely related to one another through sexual reproduction. Each mitochondrial type consisted of one to five different genets.

Occurrence of genets of Armillaria spp. in four mountain forests in Central France: the colonization strategy of Armillaria ostoyae.

Four forests each of 4-8 ha were investigated in Central France: (1) a pure beech stand harbouring only the saprophytic species Armillaria gallica and A. cepistipes; (2) a pure beech stand with the same species plus A. ostoyae; (3) an ancient beech stand to which conifers had been introduced 60 yr ago, and in which A. ostoyae was becoming the dominant species with A. gallica and A. cepistipes still present; (4) a young pine stand on an old conifer site, where A. ostoyae was exclusive and pathogenic. A total of 764 isolates was obtained from rhizomorphs, fruiting bodies and mycelia from stumps. Mapping of genets of the different Armillaria species was carried out on the four sites by testing for somatic incompatibility. This allowed comparison of the number, area and distribution of the genets of the different species. There was complete overlap of the genets of A. ostoyae with those of A. cepistipes or A. gallica, suggesting different colonization strategies. By contrast, the genets of A. gallica and A. cepistipes overlapped only rarely, suggesting similar colonization strategies. Two different genets of the same species intermingled only in a limited area along their border. In the conifer plantation, A. ostoyae tended to colonize the stumps whereas the other two species prevailed as rhizomorphs in the litter, but the competition was evolving in favour of A. ostoyae. At the fourth site, where the stumps had been uprooted and the pine seedlings had been left to grow, a large number of small genets of A. ostoyae was observed, suggesting recent infection by basidiospores.