Characterization of small RNAs originating from mitoviruses infecting the conifer pathogen Fusarium circinatum.

Deep sequencing of small RNAs has proved effective in the diagnosis of mycovirus infections. In this study, the presence of mycoviruses in ten isolates of the phytopathogenic fungus Fusarium circinatum was investigated by high-throughput sequencing (HTS) of small RNAs. The contigs resulting from de novo assembly of the reads were aligned to viral genome sequences. The presence of each mycovirus detected in the isolates was confirmed by RT-PCR analysis with four previously described primer pairs and seven new pairs designed on the basis of sequencing data. The findings demonstrate the potential use of HTS for reconstructing previously identified mitoviruses infecting F. circinatum.

Heterobasidion wood decay fungi host diverse and globally distributed viruses related to Helicobasidium mompa partitivirus V70.

Viruses of the Partitiviridae family occur at a relatively low frequency in the fungal genus Heterobasidion, but show high genetic diversity. Here, we describe four novel partitivirus species that infect three Heterobasidion species that are pathogens of conifers: H. annosum, H. parviporum and H. irregulare. We show that these viruses, designated Heterobasidion partitivirus 12 (HetPV12), HetPV13, HetPV14 and HetPV15, form a phylogenetically distinct clade together with the previously described Heterobasidion partitivirus 3 (HetPV3) found in the H. insulare species complex and Helicobasidium mompa partitivirus V70, both members of the genus Alphapartitivirus. Closely related strains of HetPV13 (over 97% polymerase identity at the nucleotide level) occur in H. annosum and H. parviporum, suggesting recent transmission of this virus species between the two fungal host species. Moreover, the occurrence of nearly identical HetPV13 strains in Finland and Poland (ca 1400km apart) indicates that the dispersal capacity of Heterobasidion partitiviruses is high. Viruses related to HetPV3 have a global distribution but only ca 2.7% overall prevalence among isolates of Heterobasidion. In three cases, these HetPV3-related viruses co-infected their hosts with distantly related partitiviruses or Heterobasidion RNA virus 6.

Biogeographical patterns and determinants of invasion by forest pathogens in Europe.

A large database of invasive forest pathogens (IFPs) was developed to investigate the patterns and determinants of invasion in Europe. Detailed taxonomic and biological information on the invasive species was combined with country-specific data on land use, climate, and the time since invasion to identify the determinants of invasiveness, and to differentiate the class of environments which share territorial and climate features associated with a susceptibility to invasion. IFPs increased exponentially in the last four decades. Until 1919, IFPs already present moved across Europe. Then, new IFPs were introduced mainly from North America, and recently from Asia. Hybrid pathogens also appeared. Countries with a wider range of environments, higher human impact or international trade hosted more IFPs. Rainfall influenced the diffusion rates. Environmental conditions of the new and original ranges and systematic and ecological attributes affected invasiveness. Further spread of established IFPs is expected in countries that have experienced commercial isolation in the recent past. Densely populated countries with high environmental diversity may be the weakest links in attempts to prevent new arrivals. Tight coordination of actions against new arrivals is needed. Eradication seems impossible, and prevention seems the only reliable measure, although this will be difficult in the face of global mobility.

Morphological measurements and ITS sequences show that the new alder rust in Europe is conspecific with Melampsoridium hiratsukanum in eastern Asia.

Three species of Melampsoridium have been reported to infect hosts in genus Alnus. An epidemic of foliar rust affecting A. glutinosa and A. incana began in Europe in the mid-1990s, and the associated pathogen was identified as Melampsoridium hiratsukanum based on morphology. In this investigation we analyzed the morphology and genetic variation of alder rusts from Europe and Japan and the host specificity of the European epidemic rust. Our results showed that two rusts occur on the leaves of alders native to northern Europe; in Scotland an endemic rust indistinguishable from M. betulinum occurs, whereas alders in areas of Europe affected by the current epidemic were infected by M. hiratsukanum. M. hiratsukanum from naturally infected alder in Finland produced aecia on all Larix species tested but did not infect Betula leaves.

First Observations of Mycosphaerella pini on Scots Pine in Finland.

Red band needle blight of pines caused by Mycosphaerella pini (anamorph Dothistroma septosporum) has recently been recorded on Scots pine (Pinus sylvestris) at 14 rural districts in southern and central Finland. Scots pine is the most common and commercially most important tree species in Finland. Red bands with aggregations of conidial stromata on otherwise brown attached needles were frequently encountered on saplings and young trees in dense stands and sporadically on lower twigs of mature trees within 2 m of the ground. These symptoms and signs, typical for M. pini (1), were also observed on needles of P. contorta and P. cembra, which occur in Finland in low frequency. Symptoms of red band needle blight and abundant conidial stromata were found in March and April of 2008 after a mild and rainy winter. After this time, the frequency at which fresh acervuli were observed decreased. Conidia were isolated after squeezing conidial stromata into a drop of sterile water and rinsing out the drop onto water agar from where single conidia were picked up from under the microscope with a modified Pasteur pipette. The conidia were hyaline, smooth, thin walled, filiform, 2.0 to 2.7 (2.4) µm wide, and 15 to 37 (29.4) µm long. Germination of the conidia on water agar was 100%. The cultures grew slowly and reached a diameter of 4 to 10 mm within 3 weeks on modified orange serum agar (2) at 20°C and abundantly produced conidia. Complete sequences of the internal transcribed spacer (ITS) region including the 5.8S rRNA gene were obtained for three isolates from different rural districts: Hartola (61°34'N, 26°01'E), Kangasniemi (61°59'N, 26°39'E), and Suonenjoki (62°37'N, 27°07'E). These sequences are deposited in GenBank with Accession Nos. EU834294, EU834295, and EU834296 and are identical to each other and to more than 50 M. pini sequences in GenBank, including those of several Estonian and Austrian isolates. These isolates are deposited in the culture collection of the Finnish Forest Research Institute with identifiers Dot1, Dot2, and Dot8. Symptoms were reproduced after inoculation of 1-year-old Scots pine seedlings growing in seedling trays with 115 cm3 cavities. A conidial suspension (5 to 6·× 103 conidia ml-1) was prepared from two single-conidial cultures (Dot10 and Dot15), combined, and sprayed during July 2008 onto 192 seedlings until needles looked completely wet. Sixty-four seedlings were sprayed with distilled water as a control treatment. Seedlings were incubated outdoors in Suonenjoki and covered with a transparent plastic hood for the first 5 days after inoculation. The first symptoms (brown segments and red bands on needles) appeared on inoculated seedlings 1 month later, and conidial stromata appeared after another 2 to 4 weeks. M. pini was reisolated from the acervuli of 10 sample needles. Three months after inoculation, all inoculated seedlings showed symptoms while all noninoculated seedlings were healthy. It is possible that M. pini has spread recently from the south since it was identified for the first time in Estonia 2 years earlier (3). Although the Gulf of Finland separates Finnish pine forests from Estonian pine forests by approximately 50 to 100 km, spores may have been aerially disseminated over this distance. Alternatively, introduction of M. pini to Finland may have occurred on imported seedlings. References: (1) Anonymous. OEPP/EPPO Bull. 35, 303, 2005. (2) M. Hanso and R. Drenkhan. Plant Pathol. 57:170, 2008. (3) M. M. Müller et al. Mycol. Res. 98:593, 1994.

First Report of Phytophthora ramorum and P. inflata in Ornamental Rhododendrons in Finland.

Phytophthora ramorum was found for the first time in Finland during the spring of 2004 on marketed plants of Rhododendron spp. originating in other EU member states. During August of 2004, the pathogen was also found in one Finnish nursery on German Rhododendron catawbiense plants and several Finnish Rhododendron spp. cultivars. P. ramorum was detected by species-specific PCR and isolated (1). It was first characterized by an abundant production of chlamydospores on PARP and V8 agar, absence of oogonia and antheridia, and elongate, ellipsoid, deciduous, semipapillate sporangia produced in soil extract water (3). A partial sequence of the ß-tubulin gene was identical to that of P. ramorum deposited in GenBank. Despite strict regulations governing the movement of plants, the pathogen has been found every year since 2004 on materials transported to Finland from other EU countries. A total of 586 samples were taken from symptomatic plants of several susceptible species from 2004 to 2006. P. ramorum was detected in 51 rhododendron samples and the number of the outbreak sites was 28. In domestic plant production, P. ramorum was found in only one nursery. The infected plants in this nursery were destroyed in 2005 according to the EU regulation 2004/426/EG. During the 2006 growing season, 84 samples from trace-forward inspections and reinspections of the nursery were tested and P. ramorum was not detected in any of the samples. In 2005, surveys for P. ramorum on Finnish Rhododendron spp. cultivars with necrotic lesions on leaves and blackened tips yielded, in addition to P. ramorum, another Phytophthora sp. On V8 agar, this homothallic species showed a stellate growth pattern with sparse aerial mycelium. Oogonia had both paragynous and amphigynous antheridia, and sporangia produced in soil extract water were ellipsoid in shape and semipapillate. A 763-bp segment of the ß-tubulin gene was sequenced and was identical to the ß-tubulin sequence of P. inflata strain IMI342898 (GenBank), which was isolated in 1990 from Syringa sp. in the UK. It is likely that this P. inflata strain has been spreading in Europe with the ornamental plant trade. To fulfill Koch's postulates, rhododenrdon plants were inoculated (2) with P. inflata or P. ramorum, typical symptoms observed, and the pathogens were reisolated from inoculated plants. Both Phytophthora species also caused necrotic lesions on Alnus glutinosa, A. incana, and Betula pendula. Pinus sylvestris was resistant to both Phytophthora spp., whereas Picea abies was susceptible to P. inflata but not P. ramorum. References: (1) EPPO Bull. 36:145, 2006. (2) E. Hansen et al. Plant Dis. 89:63, 2005. (3) S. Werres et al. Mycol. Res. 105:1155, 2001.

Two unrelated double-stranded RNA molecule patterns in Gremmeniella abietina type A code for putative viruses of the families Totiviridae and Partitiviridae.

Two double stranded (ds) RNA molecule patterns, probably of viral origin, were sequenced from Gremmeniella abietina var. abietina type A. The genome of Gremmeniella abietina RNA virus L1 (GaRV-L1) from isolate HR2 was 5133 bp and contained two open reading frames (ORFs). The 5'-proximal ORF coded for a putative coat protein (CP) and the 3'-proximal ORF encoded putative RNA-dependent RNA polymerase (RdRp). GaRV-L1 had sequence similarities with a previously described totivirus (Helminthosporium victoriae 190S virus) and two unclassified members of family Totiviridae ( Sphaeropsis sapinea RNA virus 1 and Sphaeropsis sapinea RNA virus 2). The genome of Gremmeniella abietina RNA virus MS1 (GaRV-MS1) from isolate C5 was composed of three dsRNA molecules coding for a putative RdRp (dsRNA1), a putative CP (dsRNA2) and protein of unknown function (dsRNA3). The lengths of these dsRNA molecules were 1782, 1586 and 1181 bp, respectively. Sequence comparisons indicated that the GaRV-MS1 dsRNA pattern comprises a putative virus that is highly similar to Discula destructiva virus 1, Discula destructiva virus 2 and Fusarium solani virus 1 of the family Partitiviridae.

Gremmeniella abietina mitochondrial RNA virus S1 is phylogenetically related to the members of the genus Mitovirus.

A double stranded (ds) RNA genome of Gremmeniella abietina mitochondrial RNA virus S1 (GaMRV-S1) was sequenced. The length of the genome was 2572 base pairs, it had a very low GC content (30.6%), and sequence and length variations occurred in both ends of it. The genome coded for a putative 741 amino acid long RNA-dependent RNA polymerase (RdRp) using a mitochondrial translation code. Comparison of the putative amino acid sequences suggested that GaMRV-S1 is a putative member of the genus Mitovirus.

Phylogeography of the circumpolar Paranoplocephala arctica species complex (Cestoda: Anoplocephalidae) parasitizing collared lemmings (Dicrostonyx spp.).

The Paranoplocephla arctica complex (Cyclophyllidea, Anoplocephalidae), host-specific cestodes of collared lemmings Dicrostonyx, include two morphospecies P. arctica and P. alternata, whose taxonomical status now must be considered ambiguous. The genetic population structure and phylogeography of the P. arctica complex was studied from 83 individuals sampled throughout the Holarctic distribution range using 600 bp of the mitochondrial cytochrome c oxidase subunit I gene (COI). The mitochondrial DNA (mtDNA) phylogeny divides the species complex into one main Nearctic and one main Palaearctic phylogroup, corresponding to the main phylogenetic division of the hosts. In the Palearctic phylogroup, the parasite clades correspond to the host clades although the parasites from Wrangel Island form an exception as the host on this island, D. groenlandicus, belongs to the Nearctic phylogroup. In the Nearctic, northern refugia beyond the ice limit of the Pleistocene glaciations are proposed for the hosts. All reconstructions of parasite phylogeny show a genetically differentiated population structure that in the Canadian Arctic lacks strict congruence between phylogeny and geography. The parasite phylogeny does not show complete congruence with host relationships, suggesting a history of colonization and secondary patterns of dispersal from Beringia into the Canadian Arctic, an event not proposed by the host phylogenies alone.

Diversity of endophytic fungi of single Norway spruce needles and their role as pioneer decomposers.

The diversity of endophytic fungi within single symptomless Norway spruce needles is described and their possible role as pioneer decomposers after needle detachment is investigated. The majority (90%) of all 182 isolates from green intact needles were identified as Lophodermium piceae. Up to 34 isolates were obtained from single needles. Generally, all isolates within single needles had distinct randomly amplified microsatellite (RAMS) patterns. Single trees may thus contain a higher number of L. piceae individuals than the number of their needles. To investigate the ability of needle endophytes to act as pioneer decomposers, surface-sterilized needles were incubated on sterile sand inoculated with autoclaved or live spruce forest humus layer. The dry weight loss of 13-17% found in needles after a 20-week incubation did not significantly differ between the sterilized and live treatments. Hence, fungi surviving the surface sterilization of needles can act as pioneer decomposers. A considerable portion of the needles remained green during the incubation. Brown and black needles, in which the weight loss had presumably taken place, were invaded throughout by single haplotypes different from L. piceae. Instead, Tiarasporella parca, a less common needle endophyte, occurred among these invaders of brown needles. Needle endophytes of Norway spruce seem thus to have different abilities to decompose host tissues after needle cast. L. piceae is obviously not an important pioneer decomposer of Norway spruce needles. The diversity of fungal individuals drops sharply when needles start to decompose. Thus, in single needles the decomposing mycota is considerably less diverse than the endophytic mycota.

Genetic evidence for somatic haploidization in developing fruit bodies of Armillaria tabescens.

Armillaria spp. have vegetative hyphae with diploid uninucleate cells, but the fruit bodies of many species contain clamped dikaryotic hyphae. Earlier observations suggest that somatic haploidization takes place in developing fruit bodies. To verify this, a uninucleate diploid cell was isolated from each of the 49 mating combinations between single-spore isolates of Armillaria tabescens and they were fruited. Twenty-four isolates produced fruit bodies with at least a partially dikaryotic subhymenium. Dikaryotic hyphae were isolated from fruit-body primordia and homokaryons were obtained by micromanipulation or by protoplasting. Approximately half of the isolates proved to represent recombinant mating types in respect to parent homokaryons, and most of them contained recombinant haploid DNA, based on random-amplified microsatellite markers. The results show that the nuclei in dikaryotic hyphae found in fruit bodies result from somatic haploidization. The mechanism of haploidization remains unclear.

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

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.

Comparison of partial 16S rRNA gene sequences obtained from activated sludge bacteria.

The cultivated and uncultivated bacterial communities of an activated sludge plant were studied. Two samples were taken and a total of 516 bacterial isolates were classified into groups using their whole-cell protein patterns. The distribution of bacteria into protein-pattern groups differed significantly between the two samples, suggesting variation in culturable bacterial flora. Partial 16S rRNA gene sequences were determined for representatives of the commonest protein-pattern groups. Most of the sequences obtained were previously unknown, but relatively closely related to known sequences of organisms belonging to the alpha, beta or gamma subclasses of the proteobacteria, the first two subclasses being predominant. This classification of bacteria isolated on a diluted nutrient-rich medium differed from recent culture-dependent studies using nutrient-rich media. The uncultivated bacterial community was studied by analyzing ten partial 16S rRNA gene sequences cloned directly from activated sludge. None of the cloned sequences was identical to those determined for culturable organisms; or to those in the GenBank database. They were, however, related to the alpha or beta subclasses of the proteobacteria, or to the gram-positive bacteria with a high G + C DNA content.

Diversity within bacterial isolates hybridizing with Comamonas probe ppT.

Diversity within bacteria isolated from activated sludge and the Baltic sea and recognized by the rRNA targeted oligonucleotide probe ppT (designed by BRAUN-HOWLAND et al., 1993 to recognize C. testosteroni) was studied. The partial nucleotide sequences of 16S genes of the isolates revealed that the activated sludge and Baltic sea isolates each formed a separate group distinct from C. testosteroni. The analysis of phage and bacteriocin sensitivity as well as whole cell protein patterns revealed the same grouping, but in these characters also intragroup variation was observed. As a conclusion, neither of the studied groups were C. testosteroni, but they probably belong to two previously unknown species common in activated sludge or the Baltic sea.

Description of chlorophenol-degrading Pseudomonas sp. strains KF1T, KF3, and NKF1 as a new species of the genus Sphingomonas, Sphingomonas subarctica sp. nov.

Gram-negative polychlorophenol-degrading bacterial strains KF1T (T = type strain), KF3, and NKF1, which were described previously as Pseudomonas saccharophila strains, were studied by chemotaxonomic, genetic, and physiological methods and by electron microscopy and compared with selected xenobiotic compound-degrading bacteria. These strains contained sphingolipids with d-18:0, d-20:1, and d-21:1 as the main dihydrosphingosines, ubiquinone 10 as the main respiratory quinone, and spermidine as the major polyamine, and the DNA G + C content was 66 mol%. The cellular fatty acids included about 60% octadecenoic acid, 9% 2-hydroxymyristic acid, 14% cis-9-hexadecenoic acid, and 10% hexadecanoic acid. These strains exhibited less than 97% 16S ribosomal DNA sequence similarity to all of the other taxa studied. In the DNA-DNA reassociation studies the highest levels of reassociation between these strains and previously described species were less than 40%. Thin sections of cells of strains KF1T, KF3, and NKF1 were examined by electron microscopy, and the results showed that the cells had peculiar concentrically arranged layered membranous blebs that extruded from the outer membrane, especially at the cell division points. On the basis of the results of this study, polychlorophenol-degrading strains KF1T, KF3, and NKF1 are considered members of a new species of the genus Sphingomonas, Sphingomonas subarctica. The polycyclic aromatic hydrocarbon-degrading organism Sphingomonas paucimobilis EPA 505 was closely related to Sphingomonas chlorophenolica as determined by chemotaxonomic, phylogenetic, and physiological criteria. The xenobiotic compound degraders Alcaligenes sp. strain A175 and Pseudomonas sp. strain BN6 were identified as members of species of the genus Sphingomonas.

Bacterial diversity at surface water in three locations within the Baltic sea as revealed by culture-dependent molecular techniques.

Diversity of culturable bacteria inhabiting the Baltic sea surface waters was studied in three separate locations. Based on electrophoretically separated whole cell proteins the number of operational taxonomic units (OTU) within each sampling location was high. Most of the OTUs were unique to single locations. Within each sampling location 8-22% of isolates belonged to a single OTU. Rarefaction analysis revealed that the bacterial community was more divergent at a polluted location than at clean areas. Also the most common OTUs were different in clean locations compared to the polluted site suggesting that both diversity and species composition of the bacterial community is greatly affected by pollution. The partial 16S rRNA gene sequences of the isolates of the most common OTUs are unique. Intragroup variation and an OTU-specific bacteriocin system was observed among the isolates of the second common OTU. The bacteriocin activity was linked to restriction fragment length polymorphism grouping, although additional variation correlating to geographic origin of isolates was observed.

Presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The use of fecal streptococci as fecal indicators requires better knowledge of the ecology of these bacteria. We isolated 371 presumptive fecal streptococci from environmental samples--domestic wastewater, forest industry wastewater, contaminated surface and seawater, well water, cow dung, bird droppings, and pristine waters--and clustered them according to their protein profiles in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Some clusters could be tentatively identified with the help of reference strains. Samples from each environment had a typical composition of streptococcus types. Enterococcus faecalis was present, but not as a dominating enterococcal species, in samples in which fecal contamination was probable. Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii had protein profiles that were difficult to distinguish from each other. These bacteria were found in a variety of samples. Enterococcus casseliflavus and Enterococcus gallinarum had identical protein profiles. On the basis of the maximum temperatures for growth and pigment production, isolates of this protein profile group common in forest industry wastewaters were identified as E. casseliflavus. Lactococcus lactis subsp. lactis was also found in this environment. Nearly all strains from pristine waters belonged to protein profile groups which could not be identified with the aid of known Aerococcus, Enterococcus, Lactococcus, or Streptococcus strains. The maximum temperatures for growth and the results of fatty acid analysis were in general agreement within each protein profile group.

Bacteriophage PRD1 proteins: cross-linking and scanning transmission electron microscopy analysis.

Bacteriophage PRD1, a double-stranded DNA virus infecting Escherichia coli, has a membrane inside the protein capsid. Chemical cross-linking and scanning transmission electron microscopy showed that the multimeric major coat protein (P3) exists in a trimeric form. Cross-linking revealed, in addition, that protein P11, located between the protein coat and the membrane, exists also as a homotrimer. Minor protein P7 was associated with the major coat protein P3. Under nonreducing conditions the infectivity proteins P16 and P18 formed homomultimeric complexes which were dissociated upon addition of 2-mercaptoethanol.

Bacteriophage phi 6 envelope elucidated by chemical cross-linking, immunodetection, and cryoelectron microscopy.

Bacteriophage phi 6 is an enveloped dsRNA virus which infects the plant pathogenic Pseudomonas syringae bacterium. Using low dose cryoelectron microscopy we show that the nucleocapsid, spikeless virion, and intact virion have radii of 29, 35, and 43 nm, respectively. Thus, the membrane is 6 nm thick and the surface spikes of the receptor binding protein P3 extend 8 nm from the membrane surface. Cross-linking, immunological, and complementation evidence suggest that the spikes are formed of multimeric P3 molecules and that P3 is associated with membrane-bound protein P6. We observe that the envelope can accommodate up to 400 molecules of P3 but that the average virion contains less than one-fourth of this amount. Assembly of a very small number of P3 or truncated P3 molecules onto inactive virions restores infectivity, showing that only a few spikes are necessary for receptor binding and membrane fusion.

Ecology of bacteriophages infecting activated sludge bacteria.

Little is known about the endemic bacteriophages of activated sludge. In this investigation 49 virus-host systems were studied by isolating co-occurring bacteria and bacteriophages from the aeration basin of a sewage treatment plant during 5 successive weeks. The phage titers were high and fluctuated during the time period. The occurrence of phage-sensitive and -resistant hosts did not depend on the presence or absence of phages. Several phage-host systems expressed variable plating efficiencies. In addition, phages with broad host ranges were observed. These results show that phages are an active part of this ecosystem and that they may exert selection pressure for phage resistance on their bacterial host populations.