Sylvie Pažoutová

Geosmithia Fungi are Highly Diverse and Consistent Bark Beetle Associates : Evidence from their Community Structure in Temperate Europe

Geosmithia spp. (Ascomycota: Hypocreales) are little-studied, dry-spored fungi that occur in galleries built by many phloeophagous bark beetles. This study mapped the distribution and environmental preferences of Geosmithia species occurring in galleries of temperate European bark beetles. One hundred seven host tree samples of 16 tree species infested with 23 subcortical insect species were collected from across Europe during the years 1997–2005. Over 600 Geosmithia isolates from the beetles were sorted into 17 operational taxonomic units (OTUs) based on their phenotype similarity and phylogeny of internal transcribed spacer (ITS) region of rDNA (ITS1-5.8S-ITS2). The OTUs represent six known species and eight undescribed taxa. Ninety-two samples infested with subcortical insects were characterized by the presence/absence of OTUs and the similarity among the samples was evaluated. Geographically distant populations of the same beetle species host relatively uniform Geosmithia communities across large geographic areas (ranging from southern Bulgaria to the Czech Republic). This suggests effective dispersal of Geosmithia spp. by bark beetles. Clustering of similar samples in ordination analysis is correlated predominantly with the isolation source (bark beetles and their respective feeding plant), but not with their geographical origin. The composition of the Geosmithia OTU community of each bark beetle species depends on the degree of isolation of the species’ niches. Thus, Geosmithia communities associated with regularly co-occurring bark beetle species are highly similar. The similarity decreases with decreasing frequency of beetle species’ co-occurrence, a pattern resembling that of entomochoric ophiostomatoid fungi. These findings suggest that: 1) communities of Geosmithia spp. are vector-specific; 2) at least in some cases, the association between Geosmithia OTUs and bark beetles may have been very stable and symbioses are likely to be a fundamental factor in the speciation of Geosmithia fungi; and 3) that even nonsticky spores of Geosmithia are suitable for maintaining an insect–fungus association, contrary to previous hypotheses.

Morphological and molecular characterisation of Geosmithia putterillii, G. pallida comb. nov. and G. flava sp. nov., associated with subcorticolous insects.

Geosmithia putterillii is an anamorphic fungus with connections to bark beetles. Genetic variability of 89 isolates traditionally grouped in G. putterillii and G. lavendula isolated from different geographical regions from subcorticolous insects and from other unspecific substrata was assessed using RAPD, sequencing of the ITS region (ITS1-5.8SrDNA-ITS2) and morphological characters. RAPD analysis revealed eight distinct groups. One group was represented by G. lavendula type strain and showed no relations to any other isolate. Five RAPD-types with similar ITS sequences and phenotype were related to the ex-type strain of Penicillium pallidum (generally given as a synonym of G. putterillii). Because of unique phylogenetic position and a phenotype markedly different from G. putterillii, the new combination G. pallida is made here. For another group of isolates formerly identified as G. putterillii the new species G. fiava is described based on a characteristic RAPD-type, a unique ITS sequences and a different phenotype. These newly recognized species are stable in culture and with worldwide distribution.

The phylogeny and evolution of the genus Claviceps

Phylogenetic trees of 16 Claviceps species were constructed based on alignments of 5.8S rDNA and the adjacent ITS1 and ITS2 spacers. Two highly supported clades were found: (1) C. paspali, C. zizaniae, C. grohii, C. sulcata, C. fusiformis , and C. purpurea ; and (2) C. citrina, C. phalaridis , two unidentified Claviceps spp. (isolates PM and SG), C. sorghicola, C. gigantea, C. sorghi, C. africana, C. viridis , and C. pusilla. No relationship was found between the species placement and its morphological markers. The probe from C. purpurea gene cpd1 for dimethylallyl tryptophan synthase, the first enzyme of alkaloid biosynthesis, was hybridized to Pst 1 digested genomes of the above species under non-stringent conditions. Hybridizing DNA was present in all species of clade 1, although the signal of the C. paspali gene was weaker. In clade 2, only C. africana, C. gigantea , and C. pusilla gave weak positive signals. Colorimetric detection found small amounts of alkaloids in cultures of Claviceps sp. SG and PM but despite that, no cpd1 hybridizing bands were found. The occurrence of two major clades of Claviceps and their biogeography suggests, that the genus originates from South America and that the evolution of its species was influenced by comigration with their hosts and with the global climatic changes that influenced spreading of grass subfamilies.

Phaeoacremonium rubrigenum: Hyphomycete associated with bark beetles found in Czechia

Phaeoacremonium is a recently described genus (Crouset al. 1996) associated with decline diseases of woody hosts and with human infections. During a study of surface mycobiota of the oak bark beetles, the hyphomycetous microfungusPhaeoacremonium rubrigenum was isolated from larvae ofScolytus intricatus (Coleoptera: Scolytidae) and their galleries onQuercus robur and also from an adult ofLeperisinus fraxini found under the bark ofFraxinus excelsior. The species determination was confirmed by rDNA (ITS1-5.8S-ITS2) comparison.P. rubrigenum was recorded for the first time in Czechia. The records on oak are considered to be the first ones in the world. A description of morphological features and figures of the fungus are given. Representative strains are maintained in theCulture Collection of Fungi (CCF), Faculty of Science, Charles University (Prague, Czechia).

Assessing the Pathogenic Effect of Fusarium, Geosmithia and Ophiostoma Fungi from Broad-Leaved Trees

Phytopathogenic effect ofGeosmithia pallida, G. langdonii, Ophiostoma grandicarpum, O. querci, two isolates ofO. piceae, and two isolates ofFusarium solani was compared using plant growth test (stem and root length of garden cress plants seeded on mycelium-covered potato carrot agar);Ophiostoma spp. andF. solani were isolated from oak,Geosmithia spp. from galleries ofScolytus intricatus on beech. All fungi inhibited more the root elongation than that of stems.F. solani led to plant collapse after briefly stimulating the growth of stem and in one case also root.G. langdonii inhibited stem and root growth to 20 % and led to plant collapse.G. pallida inhibited root growth to 25 % whereas stem growth was almost unimpaired.Ophiostoma spp. reduced stem growth to ∼60–80 % and root growth to 25–60 %O. piceae andO. querci caused plant collapse after 15–20 d.

Delimitation of cryptic species inside Claviceps purpurea

Claviceps purpurea is an ovarian parasite infecting grasses (Poaceae) including cereals and forage plants. This fungus produces toxic alkaloids and consumption of contaminated grains can cause ergotism in humans and other mammals. Recent molecular genetics studies have indicated that it included three cryptic species (G1, G2, G3). In this study, reproductive isolation amongst these groups and among material from Phragmites and Molinia was tested using gene flow statistics for five polymorphic loci, and to support these data, phylogenetic affiliations based on gene trees and a multigene phylogeny were used. The four recognized species are characterized based on morphology and host spectrum and formal taxonomic names are proposed. Claviceps purpurea sensu stricto (G1 group) represents a typical rye ergot, but infects various other grasses. Typical hosts of Claviceps humidiphila (new name for G2 species), like Phalaris arundinacea, belong to grasses preferring humid locations. Claviceps spartinae (G3) is specific to chloridoid grasses from salt barches. The material from Phragmites and Molinia can be authenticated with the species Claviceps microcephala for which the new name Claviceps arundinis is proposed here. The divergence time between species was estimated and the tools for species identification are discussed.

Specialised population of Claviceps purpurea from salt marsh Spartina species

The Claviceps purpurea population colonising British Spartina salt marsh stands is characterised by unusually long cylindrical conidia (average 9.5–12 μm) and sclerotia floating on the water surface. RAPD, AFLP and rDNA comparison defined these isolates as the third genetically distinct homogenous population (G3) of C. purpurea. The same morphological and genetical markers were found also in S. alterniflora isolates from Spartina from the USA. All G3 isolates belonged to a chemotype producing ergocristine and ergocryptine. In phylogenetic trees based on rDNA and AFLP, a G1 population from fields and meadows appeared as the sister clade to the one formed by G3 ( Spartina ) and G2 (wet and shady habitats), both with floating sclerotia and elongated conidia. British stands of S. anglica were probably colonised by isolates introduced from America, rather than by isolates from species of neighbouring biotopes.

Claviceps citrina sp. nov., a parasite of the halophytic grass Distichlis spicata from Mexico.

The occurrence of an undescribed ergot species, Claviceps citrina, on the halophytic chloridoid grass Distichlis spicata, in the Texcoco region of central Mexico, is reported. RAPD and ITS1 sequences of this fungus were compared with other Claviceps species and the morphological uniqueness of the fungus was reinforced. Its sclerotia do not contain any alkaloids of the ergoline type. Germination of sclerotia occurred immediately after placing them on humid sand. After removal of the capitula, the remaining stipe was able to regenerate the capitulum.

Hydroxylated Anthraquinones Produced by Geosmithia species

Geosmithia fungi are little known symbionts of bark beetles. Secondary metabolites of lilac colored species G. lavendula and other nine Geosmithia species were investigated in order to elucidate their possible role in the interactions of the fungi with environment. Hydroxylated anthraquinones (yellow, orange, and red pigments), were found to be the most abundant compounds produced into the medium during the submerged cultivation. Three main compounds were identified as 1,3,6,8-tetrahydroxyanthraquinone (1), rhodolamprometrin (1-acetyl-2,4,5,7-tetrahydroxyanthraquinone; 2), and 1-acetyl-2,4,5,7,8-pentahydroxyanthraquinone (3). Compounds 2 and 3 (representing the majority of produced metabolites) inhibited the growth of G+-bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentration of 64–512 μg/mL. Anti-inflammatory activity detected as inhibition of cyclooxygenase-2 was found only for compound 3 at 1 and 10 μg/mL. Compound 2 interfered with the morphology, compound 3 with cell-cycle dynamics of adherent mammalian cell lines.

Pleomorphic conidiation in Claviceps

Types of asexual sporulation in 17 Claviceps species and the closely related Corallocytostroma ornicopreoides were revised in relation to the phylogeny of clavicipitaceous fungi. We observed: (1) enteroblastic conidiation from branched phialidic conidiophores typical of the genus (anamorph Sphacelia) in all species including Corallocytostroma; (2) widespread and often sequential formation of terminal holoblastic secondary conidia on tapering hyphae arising from sphacelial macroconidia: and (3) in addition to sphacelial conidiation, sympodial holoblastic conidiation of the Ephelis-type in cultures of C. zizaniae and in both the culture and sphacelial tissue of C. citrina. Secondary conidiation was not found in C. purpurea, C. citrina and C. sorghicola. During sphacelial fructification, most species produced macroconidia and microconidia. Only macroconidia formed in planta underwent secondary conidiation whereas microconidia did not germinate at all. In C. phalaridis, the formation of holoblastic 2-3 celled appendaged conidia was observed, similar to that of Aciculosporium and Neoclaviceps. In dendrograms based on ITS1-5.8S rDNA-ITS2 sequences, genera and species with appendaged conidia grouped on a highly supported clade with ancestral Corallocytostroma. The clade was placed inside a group of tropical species of Claviceps, without any relationship to Balansiae.