Ecological impacts of Phlebiopsis gigantea biocontrol treatment against Heterobasidion spp. as revealed by fungal community profiling and population analyses

Wood decay fungi belonging to the species complex Heterobasidion annosum sensu lato are among the most common and economically important species causing root rot and stem decay in conifers of the northern temperate regions. New infections by these pathogens can be suppressed by tree stump treatments using chemical or biological control agents. In Finland, the corticiaceous fungus Phlebiopsis gigantea has been formulated into a commercial biocontrol agent called Rotstop (Verdera Ltd.). This thesis addresses the ecological impacts of Rotstop biocontrol treatment on the mycoflora of conifer stumps. Locally, fungal communities within Rotstop-treated and untreated stumps were analyzed using a novel method based on DGGE profiling of small subunit ribosomal DNA fragments amplified directly from wood samples. Population analyses for P. gigantea and H. annosum s.l. were conducted to evaluate possible risks associated with local and/or global distribution of the Rotstop strain. Based on molecular community profiling by DGGE, we detected a few individual wood-inhabiting fungal species (OTUs) that seemed to have suffered or benefited from the Rotstop biocontrol treatment. The DGGE analyses also revealed fungal diversity not retrieved by cultivation and some fungal sequence types untypical for decomposing conifer wood. However, statistical analysis of DGGE community profiles obtained from Rotstop-treated and untreated conifer stumps revealed that the Rotstop treatment had not caused a statistically significant reduction in the species diversity of wood-inhabiting fungi within our experimental forest plots. Locally, ISSR genotyping of cultured P. gigantea strains showed that the Rotstop biocontrol strain was capable of surviving up to six years within treated Norway spruce stumps, while in Scots pine stumps it was sooner replaced by successor fungal species. In addition, the spread of resident P. gigantea strains into Rotstop-treated forest stands seemed effective in preventing the formation of genetically monomorphic populations in the short run. On a global scale, we detected a considerable level of genetic differentiation between the interfertile European and North American populations of P. gigantea. These results strongly suggest that local biocontrol strains should be used in order to prevent global spread of P. gigantea and hybrid formation between geographically isolated populations. The population analysis for H. annosum s.l. revealed a collection of Chinese fungal strains that showed a high degree of laboratory fertility with three different allopatric H. annosum s.l. taxa. However, based on the molecular markers, the Chinese strains could be clearly affiliated with the H. parviporum taxonomical cluster, which thus appears to have a continuous distribution range from Europe through southern Siberia to northern China. Keywords: Rotstop, wood decay, DGGE, ISSR fingerprinting, ribosomal DNAJuurikäävän biologisen torjunnan vaikutukset lahopuun sienilajiston monimuotoisuuteen Juurikääpien sukuun kuuluvat lahottajasienet (Heterobasidion spp.) ovat taloudellisesti merkittävin havupuiden juuri- ja tyvilahon aiheuttaja Pohjois-Euroopassa. Juurikääpätartuntoja voidaan kuitenkin estää tehokkasti hyödyntämällä juurikäävälle antagonistista harmaaorvakkasientä (Phlebiopsis gigantea), josta on Suomen oloihin kehitetty biologinen kantokäsittelyaine, Rotstop (Verdera Oy). Tämä tutkimuskokonaisuus käsittelee harmaaorvakkakäsittelyn vaikutuksia havupuun kannoissa esiintyvän sienilajiston monimuotoisuuteen. Työn puitteissa kehitettiin menetelmä, jolla ympäristönäytteiden sienilajistoa voidaan tarkastella suoraan lahosta puuaineksesta kullekin sienilajille ominaisten ribosomaalisten merkkimolekyylien (SSU rDNA) monistuksen ja DGGE-analyysin perusteella. Tutkimuksessa osoitettiin lahopuusta suoraan eristettyjen DNA-näytteiden paljastavan lajistoa, jota ei tavoitettu vertailevassa viljelyanalyysissä. Rotstopilla käsiteltyjä ja käsittelemättömiä kantoja vertailtaessa osa lajistosta näytti hyötyvän tai kärsivän käsittelystä, mutta sienidiversiteetin yleistasossa ei kuitenkaan havaittu tilastollisesti merkitsevää laskua käsitellyillä koealoilla. Harmaaorvakan osalta kantokäsittelyn vaikutuksia paikallisten populaatioiden monimuotoisuuteen tarkasteltiin havupuiden kannoista eristetyistä puhdasviljelmistä vertaillen Rotstopilla käsiteltyjä sekä käsittelemättömiä koealoja. ISSR-merkkimolekyylien perusteella voitiin todentaa Rotstop-genotyypin säilyminen kuusen kannoissa kuusi vuotta käsittelyn jälkeen sekä leviäminen myös saman koealan käsittelemättömiin kantoihin. Toisaalta Rotstop-kannan ei havaittu levinneen samoilta koealoilta viiden vuoden kuluttua käsittelystä kaadettujen puiden kantoihin. Lisäksi harmaaorvakan luontainen levintä käsitellyille koealoille näytti estävän tehokkaasti perinnöllisesti monomorfisen populaation syntymisen lyhyellä aikavälillä. Harmaaorvakkasienen lajinsisäisen muuntelun tasoa selvitettäessä havaittiin selvä maantietellinen erilaistuminen sienen eurooppalaisten ja pohjois-amerikkalaisten populaatioiden välillä. Tulosten perusteella biologiseen torjuntaan tulisi käyttää ainoastaan paikallisia harmaaorvakkakantoja, jotta voidaan välttyä geneettisen aineksen siirtymiseltä ja mahdollisten hybridikantojen muodostumiselta. Juurikäävän populaatioanalyysin perusteella voitiin osoittaa Kiinasta peräisin olevien juurikääpäkantojen kuuluvan samaan taksonomiseen ryhmään kuin eurooppalainen kuusenjuurikääpä (H. parviporum). Siten tällä patogeenilajilla näyttäisi olevan katkeamaton levinneisyys ulottuen Euroopasta Etelä-Siperian kautta aina pohjoiseen Kiinaan asti. Asiasanat: Harmaaorvakka, Rotstop, kantokäsittely, juuri- ja tyvilaho, DGGE, diversiteett

Sienivirusten potentiaali juurikäävän torjunnassa

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Towards the Forest Virome: High-Throughput Sequencing Drastically Expands Our Understanding on Virosphere in Temperate Forest Ecosystems

Thanks to the development of HTS technologies, a vast amount of genetic information on the virosphere of temperate forests has been gained in the last seven years. To estimate the qualitative/quantitative impact of HTS on forest virology, we have summarized viruses affecting major tree/shrub species and their fungal associates, including fungal plant pathogens, mutualists and saprotrophs. The contribution of HTS methods is extremely significant for forest virology. Reviewed data on viral presence in holobionts allowed us a first attempt to address the role of virome in holobionts. Forest health is dependent on the variability of microorganisms interacting with the host tree/holobiont; symbiotic microbiota and pathogens engage in a permanent interplay, which influences the host. Through virus–virus interplays synergistic or antagonistic relations may evolve, which may drastically affect the health of the holobiont. Novel insights of these interplays may allow practical applications for forest plant protection based on endophytes and mycovirus biocontrol agents. The current analysis is conceived in light of the prospect that novel viruses may initiate an emergent infectious disease and that measures for the avoidance of future outbreaks in forests should be considered.Peer Reviewe

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

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

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

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