GENETIC DIFFERENTIATION IN SYMPATRIC BUT TEMPORALLY ISOLATED PINE BARK BUGS, ARADUS-CINNAMOMEUS (HETEROPTERA)

Peer reviewe

Tree invasions and biosecurity: eco-evolutionary dynamics of hitchhiking fungi

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RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine

RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine

Sydowia polyspora dominates fungal communities carried by two Tomicus species in pine plantations threatened by Fusarium circinatum

Producción CientíficaBark beetles (Coleoptera, Scolytinae) carry a diverse filamentous fungal community sometimes acting as vectors or carriers of phytopathogens. In this study, mycobiota carried by two Tomicus species (Tomicus piniperda and Tomicus destruens) were investigated through (i) morphological and molecular identification of taxa; (ii) taxonomic richness, diversity, evenness, dominance and phoresy indices; (iii) ecological network analysis and (iv) statistical co-occurrence analysis. The studied mycobiota were formed by eleven taxa and showed a moderate fungal diversity with low evenness. The fungus Sydowia polyspora was significantly abundant and dominated the community. All the fungal taxa were randomly associated. Both insect species (T. piniperda and T. destruens) were collected from plantations of Pinus radiata infected by Fusarium circinatum. The ecological factors that could drive community ecology and phoretic links between fungi and bark beetles are discussed.Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (project AGL2015-69370-R)Ministerio de Economía, Industria y Competitividad (project AGL2012-39912)Junta de Castilla y León - Fondo Europeo de Desarrollo Regional (grant ORDEN EDU/1083/2013

Preventing mycelial spread of Heterobasidion annosum in young Scots pine stands using fungal and viral biocontrol agents

Heterobasidion annosum is one of the most important causal agents of root rot of pines in Europe. The timing of cuttings to wintertime or stump treatment by control agents in summertime are used to prevent the spread of the fungus to new forest sites via aerial spores. However, there are no efficient control treatments for an already established infection except for changing the tree species to a resistant one, which often is not possible. In this study, we tested whether treating stumps around Heterobasidion disease centres by the biocontrol fungus Phlebiopsis gigantea could reduce the spread of the pathogen. In addition, we tested whether the infection of H. annosum by a debilitating mycovirus, HetPV13-an1, would affect the control efficacy. The results showed that the enlargement of disease centres was reduced by P. gigantea, and that this biocontrol effect was enhanced by the virus application. Furthermore, the results showed that the growth rate of P. gigantea varies not only between root systems, but also among different roots of a single stump

Identification of Spanish isolates of Rhizoctonia solani from potato by anastomosis grouping, ITS-RFLP and RAMS-fingerprinting

Anastomosis grouping, restriction fragment length polymorphism (RFLP) of the ITS regions including the 5.85 rDNA, and random amplified microsatellites (RAMS) were used to characterize isolates of Rhizoctonia solani collected from Spain and Finland. There was a high similarity between the results obtained with the three techniques. RAMS markers revealed more genetic variation among isolates of R. solani than RFLP. The anastomosis group (AG)–3 isolates were clearly separated from isolates belonging to other AGs by RAMS, RFLPs and anastomosis grouping. Almost all the isolates sampled from potato belonged to AG–3. No differences were observed between Spanish and Finnish AG–3 isolates

Phytophthora cactorum and Colletotrichum acutatum: Survival and Detection

Phytophthora cactorum and Colletotrichum acutatum are pathogens which are transported with plant material as latent infections and can also survive in soil and plant debris. Since the beginning of 1990’s P. cactorum caused losses in strawberries in Finland and increased culling of silver birch seedlings in forest nurseries because of stem lesions. In this study primers specific for the pathogen were designed, and in a simple PCR they gave an amplification product from pure cultures only when P. cactorum was used as a template. No cross reactions were found with other Phytophthoras in group I or other microbes. Inoculated strawberry plants gave also a clear band in PCR-analyses when the template concentration was diluted. However, amplification was not always reproducible with birch seedlings. With soil samples the best result was gained by a combination of baiting and isolation. C. acutatum is a quarantine pathogen on strawberry in the European Union and thus the infected plants are destroyed in Finland to avoid further spread of the pathogen. The pathogen has earlier been found to survive over one winter in infected plant debris and soil. In the survival test (2003-2005) done in this study, specific amplification products were obtained from test plants inoculated with artificially infected plant residues after 20 months of storage outdoors on soil surface. More positive results were achieved from bait plants grown in soil collected from the field where infected plants had been destroyed two years before, than from samples collected a year after the plant destruction