Eeva J. Vainio

Genetic variation in Phlebiopsis gigantea as detected with random amplified microsatellite (RAMS) markers

Random Amplified Microsatellite (RAMS) markers were used to detect variation among Phlebiopsis gigantea isolates from seven European countries. The scoring of 26 bands revealed five markers common to all isolates and 21 variable markers, of which six occurred in only one strain. A considerable amount of variation was observed among the progeny isolates of single strains. The analysis of variable markers resulted in 75 different banding patterns among the 86 isolates studied. The high degree of polymorphism and heterozygosity observed in RAMS markers suggests that the amount of genetic variation within this fungus is considerable. The equal distribution of markers in strains from different locations and the lack of country-specific markers suggests that genetic differentiation among the populations is low. The lack of distinct groups of similar banding patterns supports the idea of P. gigantea being a true biological species consisting of a single intersterility group.

Characterization of Cyanobacteria by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene

Abstract Planktonic, filamentous cyanobacterial strains from different genera, both toxic and nontoxic strains, were characterized by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene. Total protein pattern analysis revealed the mutual relationships at the genus level. Restriction fragment length polymorphism (RFLP) of the 16S rRNA gene with reference strains proved to be a good method for the cyanobacterial taxonomy. The nonheterocystous strains outgrouped from the nitrogen-fixing ones. With both methods, Aphanizomenon clustered with Anabaena, and Nodularia with Nostoc. In the RFLP study of Anabaena, the neurotoxic strains were identical, but the hepatotoxic ones formed a heterogeneous group. Genetic distances found in the RFLP study were short, confirming that close genotypic relationships underlie considerable diversity among cyanobacterial genera.

Genetic differentiation between European and North American populations of Phlebiopsis gigantea

We investigated the degree of genetic dif- ferentiation between geographically isolated popula- tions of the saprophytic fungus Phlebiopsis gigantea from Europe and North America using two different molecular approaches: (i) denaturing gradient gel electrophoresis (DGGE) and sequence analyses of the ITS region, and (ii) genetic fingerprinting using random amplified microsatellite (RAMS) markers. The analysis included 84 homokaryotic single spore isolates of 28 European strains, 13 heterokaryotic strains from North America, and the Rotstop strain used as a biocontrol agent against Heterobasidion an- nosum. Three different ITS-alleles (designated as ITS/A, ITS/B and ITS/C) were separable by the

Viruses accumulate in aging infection centers of a fungal forest pathogen

Fungal viruses (mycoviruses) with RNA genomes are believed to lack extracellular infective particles. These viruses are transmitted laterally among fungal strains through mycelial anastomoses or vertically via their infected spores, but little is known regarding their prevalence and patterns of dispersal under natural conditions. Here, we examined, in detail, the spatial and temporal changes in a mycovirus community and its host fungus Heterobasidion parviporum, the most devastating fungal pathogen of conifers in the Boreal forest region. During the 7-year sampling period, viruses accumulated in clonal host individuals as a result of indigenous viruses spreading within and between clones as well as novel strains arriving via airborne spores. Viral community changes produced pockets of heterogeneity within large H. parviporum clones. The appearance of novel viral infections in aging clones indicated that transient cell-to-cell contacts between Heterobasidion strains are likely to occur more frequently than what was inferred from genotypic analyses. Intraspecific variation was low among the three partitivirus species at the study site, whereas the unassigned viral species HetRV6 was highly polymorphic. The accumulation of point mutations during persistent infections resulted in viral diversification, that is, the presence of nearly identical viral sequence variants within single clones. Our results also suggest that co-infections by distantly related viral species are more stable than those between conspecific strains, and mutual exclusion may play a role in determining mycoviral communities.

Taxonomy, biogeography and importance of Heterobasidion viruses.

The genus Heterobasidion consists of several species of necrotrophic and saprotrophic fungi, and includes some of the most detrimental organisms in boreal conifer forests. These fungi host a widespread and diverse mycovirus community composed of more than 16 species of Partitiviridae, a species of Narnaviridae and one taxonomically unassigned virus related to the Curvularia thermal tolerance virus. These viruses are able to cross species borders, co-infect single host strains and cause phenotypic changes in their hosts. The abundance of viruses increases over time in Heterobasidion infection centers, and they are targeted by fungal RNA interference. Long-term field studies are essential for obtaining a comprehensive view of virus effects in the nature.

Description and prevalence of a putative novel mycovirus within the conifer pathogen Gremmeniella abietina

The European race of Gremmeniella abietina (Lagerberg) Morelet is the causal agent of stem canker and shoot blight on numerous conifers in Europe and North America. It comprises different species and biotypes in which the presence of mycoviruses has been determined. In this report, we describe the full-length sequence of the RNA-dependent RNA polymerase (RdRp) of a putative novel virus, Gremmeniella abietina RNA virus 6 (GaRV6), with 2165xa0nt and a GC content of 54.7xa0%. A BLASTp search using the deduced RdRp amino acid sequence confirmed GaRV6 to be related to members of a still unassigned virus taxon, which includes, e.g., Fusarium graminearum dsRNA mycovirus 4 (FgV-4) and the mutualistic Curvularia thermal tolerance virus (CThTV). The prevalence and genetic diversity of GaRV6 was also studied within the European race of G. abietina. We examined 162 isolates originating from Canada, the Czech Republic, Finland, Italy, Montenegro, Serbia, Spain, Switzerland, Turkey and the United States. According to direct specific reverse transcription (RT) PCR screening based on the RdRp sequence, the virus appears to be present only in Spain, where it is relatively abundant but genetically highly uniform.

Fusarium circinatum isolates from northern Spain are commonly infected by three distinct mitoviruses

Pitch canker is a serious disease of pines caused by the ascomycete fungus Gibberella circinata (anamorphxa0=xa0Fusarium circinatum). Three distinct mitovirus strains have been described in this fungus: Fusarium circinatum mitovirus 1 (FcMV1), FcMV2-1 and FcMV2-2. Here, we investigated the frequency and population variation of these viruses and closely related sequence variants in northern Spain using RT-PCR and sequencing. Each virus strain and similar sequence variants shared >95xa0% sequence identity and were collectively designated as virus types. All virus types were relatively common in Spain, with estimated prevalence of 18.5xa0%, 8.9xa0% and 16.3xa0% for FcMV1, FcMV2-1 and FcMV2-2, respectively.

Occurrence of similar mycoviruses in pathogenic, saprotrophic and mycorrhizal fungi inhabiting the same forest stand

Fungal viruses (mycoviruses) are considered to be highly host specific, but phylogenetic analysis supports the occasional occurrence of horizontal transmission between species. We used an extensive sampling strategy to investigate whether similar viruses occur in more than one fungal species of the same forest habitat. Mycelial samples were collected from in-growth mesh bags (N = 259), fruiting bodies (N = 173) and cultured isolates (N = 68) at a forest site where the spatial distribution of viral infections in clonal individuals of the wood decay fungus Heterobasidion parviporum was mapped in detail earlier. The investigation revealed previously known Heterobasidion viruses in ∼2% of the single or pooled mycorrhizal samples from mesh bags, ∼3% of the fruiting body samples and none of the fungal cultures analyzed. Novel virus strains distinct from known Heterobasidion viruses were detected in cultures of ectomycorrhizal fungi (Lactarius tabidus, L. rufus) and saprotrophic fungi (Megacollybia platyphylla, Mucoraceae spp.). Overall, our results support the view that mycoviruses do not readily cross species borders. Regarding potential virocontrol applications, the introduction of Heterobasidion viruses into natural habitats is not expected to cause a major infection pressure towards the indigenous fungal community. However, the ecological consequences of the putative interspecies virus transmission events detected require further investigation.

Erratum to: Distribution of Viruses Inhabiting Heterobasidion annosum in a Pine-Dominated Forest Plot in Southern Finland

We investigated the diversity and spatial distribution of viruses infecting strains of the root rot fungus Heterobasidion annosum collected from pine stumps at a heavily infected forest site. Four different partitiviruses were detected in 14 H. annosum isolates at the study site, constituting approximately 29% of all Heterobasidion isolates investigated (Nxa0=xa048). Two of the viruses detected were new partitiviruses designated here as Heterobasidion partitivirus 16 (HetPV16) and HetPV20, and two were previously known partitiviruses: HetPV7 and HetPV13. The two new partitiviruses found, HetPV16-an1 and HetPV20-an1, shared ~70% RdRp nucleotide sequence identity with the alphapartitivirus Rosellinia necatrix partitivirus 2, and less than 40% identity with known viruses of Heterobasidion spp. HetPV7-an1 was closely similar to HetPV7-pa1 isolated earlier from Heterobasidion parviporum, supporting the view of conspecific virus pools in different Heterobasidion species. Three fungal isolates were found to be co-infected with two different partitivirus strains (HetPV7-an1 and HetPV13-an2 or HetPV16-an1 and HetPV20-an1). Different isolates representing each host clone had variable virus compositions, and virus strains occurring in more than one host clone showed minor sequence variations between clones.

The complete genome sequence of HetPV20-an1, an alphapartitivirus infecting the conifer-pathogenic fungus Heterobasidion annosum

Root rot fungi of the genus Heterobasidion are highly destructive conifer pathogens in the northern Boreal forest region. This report describes the complete genome sequence of Heterobasidion partitivirus 20 infecting a Finnish strain of Heterobasidion annosum. The bisegmented dsRNA genome of HetPV20-an1 encodes a predicted RNA-dependent RNA polymerase of 605 amino acids (aa) and a capsid protein of 536 aa. Based on sequence similarity and phylogenetic analysis, this virus is a new member of the genus Alphapartitivirus. HetPV20-an1 shares ~65% RdRP aa sequence identity with the most similar virus strain, Rosellinia necatrix partitivirus 2, whereas the CP of HetPV20-an1 is most similar to that of rose partitivirus with ~27% overall aa sequence identity. HetPV20-an1 is only distantly related to previously known partitiviruses of Heterobasidion species and shares ~29% RdRP aa sequence identity and ~16% CP aa sequence identity with Heterobasidion partitivirus 1 from H. abietinum.