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

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

Defoliation of Tilia cordata trees associated with Apiognomonia errabunda infection in Finland

We investigated the causative agent of a disease outbreak affecting small-leaved limes (Tilia cordata Mill.) and resulting in darkening of the leaf petioles and excessive defoliation during summer 2016 in southern Finland. The fungal species composition of the symptomatic petioles was examined by culture isolation and molecular identification using ITS rDNA sequences, which revealed the most prevalent fungal species present in the petioles as Apiognomonia errabunda (Roberge) Hhn. Based on reviewing curated herbarium specimens deposited at the Universities of Helsinki and Turku, A. errabunda is native and widely distributed in small-leaved limes in Finland, and occasionally infects also other broadleaved trees, including Quercus robur L. and ornamental species of Tilia L. and Fagus L. The ITS sequence analysis conducted during this study revealed minor within-species polymorphisms similar to those observed earlier in the Central European and Russian populations of A. errabunda, and reports the first nucleotide sequences of this species from the Nordic countries.Peer reviewe

Intraspecific comparative genomics of isolates of the Norway spruce pathogen (Heterobasidion parviporum) and identification of its potential virulence factors

Background: Heterobasidion parviporum is an economically most important fungal forest pathogen in northern Europe, causing root and butt rot disease of Norway spruce (Picea abies (L.) Karst.). The mechanisms underlying the pathogenesis and virulence of this species remain elusive. No reference genome to facilitate functional analysis is available for this species. Results: To better understand the virulence factor at both phenotypic and genomic level, we characterized 15 H. parviporum isolates originating from different locations across Finland for virulence, vegetative growth, sporulation and saprotrophic wood decay. Wood decay capability and latitude of fungal origins exerted interactive effects on their virulence and appeared important for H. parviporum virulence. We sequenced the most virulent isolate, the first full genome sequences of H. parviporum as a reference genome, and re-sequenced the remaining 14 H. parviporum isolates. Genome-wide alignments and intrinsic polymorphism analysis showed that these isolates exhibited overall high genomic similarity with an average of at least 96% nucleotide identity when compared to the reference, yet had remarkable intra-specific level of polymorphism with a bias for CpG to TpG mutations. Reads mapping coverage analysis enabled the classification of all predicted genes into five groups and uncovered two genomic regions exclusively present in the reference with putative contribution to its higher virulence. Genes enriched for copy number variations (deletions and duplications) and nucleotide polymorphism were involved in oxidation-reduction processes and encoding domains relevant to transcription factors. Some secreted protein coding genes based on the genome-wide selection pressure, or the presence of variants were proposed as potential virulence candidates. Conclusion: Our study reported on the first reference genome sequence for this Norway spruce pathogen (H. parviporum). Comparative genomics analysis gave insight into the overall genomic variation among this fungal species and also facilitated the identification of several secreted protein coding genes as putative virulence factors for the further functional analysis. We also analyzed and identified phenotypic traits potentially linked to its virulence.Peer reviewe

ICTV Virus Taxonomy Profile: \u3cem\u3ePartitiviridae\u3c/em\u3e

The Partitiviridae is a family of small, isometric, non-enveloped viruses with bisegmented double-stranded (ds) RNA genomes of 3–4.8 kbp. The two genome segments are individually encapsidated. The family has five genera, with characteristic hosts for members of each genus: either plants or fungi for genera Alphapartitivirus and Betapartitivirus, fungi for genus Gammapartitivirus, plants for genus Deltapartitivirus and protozoa for genus Cryspovirus. Partitiviruses are transmitted intracellularly via seeds (plants), oocysts (protozoa) or hyphal anastomosis, cell division and sporogenesis (fungi); there are no known natural vectors. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Partitiviridae, which is available at www.ictv.global/report/partitiviridae

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

Bunyaviruses Affect Growth, Sporulation, and Elicitin Production in Phytophthora cactorum

Phytophthora cactorum is an important oomycetous plant pathogen with numerous host plant species, including garden strawberry (Fragaria × ananassa) and silver birch (Betula pendula). P. cactorum also hosts mycoviruses, but their phenotypic effects on the host oomycete have not been studied earlier. In the present study, we tested polyethylene glycol (PEG)-induced water stress for virus curing and created an isogenic virus-free isolate for testing viral effects in pair with the original isolate. Phytophthora cactorum bunya-like viruses 1 and 2 (PcBV1 & 2) significantly reduced hyphal growth of the P. cactorum host isolate, as well as sporangia production and size. Transcriptomic and proteomic analyses revealed an increase in the production of elicitins due to bunyavirus infection. However, the presence of bunyaviruses did not seem to alter the pathogenicity of P. cactorum. Virus transmission through anastomosis was unsuccessful in vitro

A Framework for the Evaluation of Biosecurity, Commercial, Regulatory, and Scientific Impacts of Plant Viruses and Viroids Identified by NGS Technologies

Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios.Peer reviewe