RESUMO
Fungi harbor a vast diversity of mobile genetic elements (MGEs). Recently, novel fungal MGEs, tentatively referred to as 'ambiviruses,' were described. 'Ambiviruses' have single-stranded RNA genomes of about 4-5 kb in length that contain at least two open reading frames (ORFs) in non-overlapping ambisense orientation. Both ORFs are conserved among all currently known 'ambiviruses,' and one of them encodes a distinct viral RNA-directed RNA polymerase (RdRP), the hallmark gene of ribovirian kingdom Orthornavirae. However, 'ambivirus' genomes are circular and predicted to replicate via a rolling-circle mechanism. Their genomes are also predicted to form rod-like structures and contain ribozymes in various combinations in both sense and antisense orientations-features reminiscent of viroids, virusoids, ribozyvirian kolmiovirids, and yet-unclassified MGEs (such as 'epsilonviruses,' 'zetaviruses,' and some 'obelisks'). As a first step toward the formal classification of 'ambiviruses,' the International Committee on Taxonomy of Viruses (ICTV) recently approved the establishment of a novel ribovirian phylum, Ambiviricota, to accommodate an initial set of 20 members with well-annotated genome sequences.
Assuntos
Genoma Viral , Fases de Leitura Aberta , Viroides , Viroides/genética , Viroides/classificação , Filogenia , RNA Viral/genética , Vírus de RNA/genética , Vírus de RNA/classificação , Fungos/genética , Fungos/virologia , RNA Polimerase Dependente de RNA/genética , Micovírus/genética , Micovírus/classificação , Micovírus/isolamento & purificaçãoRESUMO
Here, we report the discovery and complete genome sequence of a novel virus, designated as "Phytophthora heveae alphaendornavirus 1" (PhAEV1), from a single isolate of the plant pathogenic oomycete Phytophthora heveae (kingdom Stramenipila) isolated from a tropical evergreen lowland rainforest in northern Vietnam. PhAEV1 was detected by both cellulose affinity chromatography of dsRNA and high-throughput sequencing of total RNA, and its presence and sequence were confirmed by RT-PCR and Sanger sequencing. The PhAEV1 genome, 12,820 nucleotides (nt) in length, was predicted to encode a single large polyprotein with the catalytic core domain of viral (superfamily 1) RNA helicase (HEL, amino acid [aa] positions 1,287-1,531), glycosyltransferase (GT, aa positions ca. 2,800-3,125), and RNA-directed RNA polymerase (RdRp, aa positions 3,875-4,112). PhAEV1 is the most similar to Phytophthora cactorum alphaendornavirus 3, sharing 39.4% and 39.1% nt and aa sequence identity, respectively. In addition to the first 5'-terminal AUG codon, three additional in-frame methionine codons were found in close proximity (nt 14-16, 96-98, and 176-178), suggesting potential additional translation initiation sites. Conserved RdRp motifs (A-E) similar to those detected in related endornaviruses were identified in PhAEV1, as well as in several previously described alphaendornaviruses from other Phytophthora species in which these motifs had not been identified previously. Phylogenetic analysis showed that PhAEV1 clusters with members of the genus Alphaendornavirus in the family Endornaviridae and is basal to two other alphaendornaviruses described from another oomycete, Phytophthora cactorum. PhAEV1 is the first virus reported in P. heveae.
Assuntos
Phytophthora , Vírus de RNA , Phytophthora/genética , Filogenia , Vírus de RNA/genética , Sequência de Aminoácidos , RNA Polimerase Dependente de RNA/genética , Genoma Viral , Fases de Leitura Aberta , RNA Viral/genéticaRESUMO
The genome sequence of a mitovirus found in an isolate of Diaporthe rudis, one of the causal agents of Phomopsis dieback on grapevines, was determined by two high-throughput sequencing approaches, small RNA and total RNA sequencing. The genome of this mitovirus is 2,455 nt in length and includes a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). A BLASTx comparison of the full-length genome sequence showed the highest similarity (54.15%) with that of Colletotrichum falcatum mitovirus 1 (CfMV1). Our results reveal a new member of the genus Mitovirus first detected in D. rudis (Fr.) Nitschke, with the proposed name "Diaporthe rudis mitovirus 1" (DrMV1).
Assuntos
Micovírus/genética , Genoma Viral , Filogenia , RNA Polimerase Dependente de RNA/genética , Saccharomycetales/virologia , Proteínas Virais/genética , Micovírus/classificação , Micovírus/isolamento & purificação , Expressão Gênica , Tamanho do Genoma , Fases de Leitura Aberta , Doenças das Plantas/microbiologia , Vitis/microbiologia , Sequenciamento Completo do GenomaRESUMO
Hymenoscyphus fraxineus mitovirus 1 (HfMV1) occurs in the fungus Hymenoscyphus fraxineus, an introduced plant pathogen responsible for the devastating ash dieback epidemic in Europe. Here, we explored the prevalence and genetic structure of HfMV1 to elucidate the invasion history of both the virus and the fungal host. A total of 1298 H. fraxineus isolates (181 from Japan and 1117 from Europe) were screened for the presence of this RNA virus and 301 virus-positive isolates subjected to partial sequence analysis of the viral RNA polymerase gene. Our results indicate a high mean prevalence (78.7%) of HfMV1 across European H. fraxineus isolates, which is supported by the observed high transmission rate (average 83.8%) of the mitovirus into sexual spores of its host. In accordance with an expected founder effect in the introduced population in Europe, only 1.1% of the Japanese isolates were tested virus positive. In Europe, HfMV1 shows low nucleotide diversity but a high number of haplotypes, which seem to be subject to strong purifying selection. Phylogenetic and clustering analysis detected two genetically distinct HfMV1 groups, both present throughout Europe. This pattern supports the hypothesis that only two (mitovirus-carrying) H. fraxineus individuals were introduced into Europe as previously suggested from the bi-allelic nature of the fungus. Moreover, our data points to reciprocal mating events between the two introduced individuals, which presumably initiated the ash dieback epidemic in Europe.
Assuntos
Ascomicetos/virologia , Micovírus/genética , Genética Populacional , Europa (Continente) , Fraxinus/microbiologia , Variação Genética , Japão , Filogenia , Doenças das Plantas/microbiologiaRESUMO
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 2165 nt and a GC content of 54.7 %. 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.
Assuntos
Ascomicetos/virologia , Doenças das Plantas/microbiologia , Vírus de RNA/isolamento & purificação , Traqueófitas/microbiologia , Ascomicetos/fisiologia , Variação Genética , Dados de Sequência Molecular , Filogenia , Prevalência , Vírus de RNA/classificação , Vírus de RNA/genética , Proteínas Virais/genéticaRESUMO
Fusarium circinatum Nirenberg & O'Donnell (teleomorph = Gibberella circinata) is the causal agent of pitch canker disease of pines. Since 2004 it has been present in Europe, particularly in northern Spain, affecting P. radiata and P. pinaster in plantations and nurseries. The disease has now also spread to other European countries, including France, Italy and Portugal. In this report, we describe three novel members of the genus Mitovirus from a Spanish isolate of F. circinatum: Fusarium circinatum mitovirus 1 (FcMV1), FcMV2-1 and FcMV2-2. Using a mitochondrial translation table, the complete 2419-bp genome of FcMV1 encodes an RNA-dependent RNA polymerase of 731 amino acids (GC-content ca 30 %). The partial genomes of FcMV2-1 and FcMV2-2 (2193 and 1973 bp, respectively) share ca 48 % RdRp sequence similarity at the aa level and might be regarded as conspecific, while FcMV1 is clearly distinct, showing 32-35 % polymerase similarity to the other strains. However, FcMV1 shared 46 % protein-level similarity with Thielaviopsis basicola mitovirus. This is the first study to report viruses in F. circinatum, as well as the first time that mitovirus genome sequences are described from Fusarium spp.
Assuntos
Fusarium/virologia , Prunus/microbiologia , Vírus de RNA/classificação , Vírus de RNA/isolamento & purificação , Genoma Viral , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/microbiologia , Doenças das Plantas/terapia , Vírus de RNA/genética , Vírus de RNA/fisiologiaRESUMO
Members of the genus Armillaria are widespread forest pathogens against which effective protection has not yet been developed. Due to their longevity and the creation of large-scale cloning of Armillaria individuals, the use of mycoviruses as biocontrol agents (BCAs) against these pathogens could be an effective alternative. This work describes the detection and characterization of viruses in Armillaria spp. collected in the Czech Republic through the application of stranded total RNA sequencing. A total of five single-stranded RNA viruses were detected in Armillaria ostoyae and A. cepistipes, including viruses of the family Tymoviridae and four viruses belonging to the recently described "ambivirus" group with a circular ambisense genome arrangement. Both hammerhead (HHRz) and hairpin (HpRz) ribozymes were detected in all the ambiviricot sequences. Armillaria viruses were compared through phylogenetic analysis and confirmed their specific host by direct RT-PCR. One virus appears to infect both Armillaria species, suggesting the occurrence of interspecies transmission in nature.
Assuntos
Armillaria , Micovírus , Genoma Viral , Filogenia , RNA Viral , República Tcheca , Armillaria/genética , Armillaria/virologia , Micovírus/classificação , Micovírus/genética , Micovírus/isolamento & purificação , RNA Viral/genética , Vírus de RNA/genética , Vírus de RNA/classificação , Vírus de RNA/isolamento & purificação , Doenças das Plantas/virologia , Doenças das Plantas/microbiologia , Análise de Sequência de RNARESUMO
We explored the virome of the "Phytophthora palustris complex", a group of aquatic specialists geographically limited to Southeast and East Asia, the native origin of many destructive invasive forest Phytophthora spp. Based on high-throughput sequencing (RNAseq) of 112 isolates of "P. palustris" collected from rivers, mangroves, and ponds, and natural forests in subtropical and tropical areas in Indonesia, Taiwan, and Japan, 52 putative viruses were identified, which, to varying degrees, were phylogenetically related to the families Botybirnaviridae, Narnaviridae, Tombusviridae, and Totiviridae, and the order Bunyavirales. The prevalence of all viruses in their hosts was investigated and confirmed by RT-PCR. The rich virus composition, high abundance, and distribution discovered in our study indicate that viruses are naturally infecting taxa from the "P. palustris complex" in their natural niche, and that they are predominant members of the host cellular environment. Certain Indonesian localities are the viruses' hotspots and particular "P. palustris" isolates show complex multiviral infections. This study defines the first bi-segmented bunya-like virus together with the first tombus-like and botybirna-like viruses in the genus Phytophthora and provides insights into the spread and evolution of RNA viruses in the natural populations of an oomycete species.
RESUMO
High-throughput virome analyses with various fungi, from cultured or uncultured sources, have led to the discovery of diverse viruses with unique genome structures and even neo-lifestyles. Examples in the former category include splipalmiviruses and ambiviruses. Splipalmiviruses, related to yeast narnaviruses, have multiple positive-sense (+) single-stranded (ss) RNA genomic segments that separately encode the RNA-dependent RNA polymerase motifs, the hallmark of RNA viruses (members of the kingdom Orthornavirae). Ambiviruses appear to have an undivided ssRNA genome of 3â¼5 kb with two large open reading frames (ORFs) separated by intergenic regions. Another narna-like virus group has two fully overlapping ORFs on both strands of a genomic segment that span more than 90% of the genome size. New virus lifestyles exhibited by mycoviruses include the yado-kari/yado-nushi nature characterized by the partnership between the (+)ssRNA yadokarivirus and an unrelated dsRNA virus (donor of the capsid for the former) and the hadaka nature of capsidless 10-11 segmented (+)ssRNA accessible by RNase in infected mycelial homogenates. Furthermore, dsRNA polymycoviruses with phylogenetic affinity to (+)ssRNA animal caliciviruses have been shown to be infectious as dsRNA-protein complexes or deproteinized naked dsRNA. Many previous phylogenetic gaps have been filled by recently discovered fungal and other viruses, which haveprovided interesting evolutionary insights. Phylogenetic analyses and the discovery of natural and experimental cross-kingdom infections suggest that horizontal virus transfer may have occurred and continue to occur between fungi and other kingdoms.
Assuntos
Micovírus , Vírus de RNA , Animais , Genoma Viral , Filogenia , RNA de Cadeia Dupla , RNA Viral , RNA Polimerase Dependente de RNARESUMO
Phytophthora castaneae, an oomycete pathogen causing root and trunk rot of different tree species in Asia, was shown to harbor a rich diversity of novel viruses from different families. Four P. castaneae isolates collected from Chamaecyparis hodginsii in a semi-natural montane forest site in Vietnam were investigated for viral presence by traditional and next-generation sequencing (NGS) techniques, i.e., double-stranded RNA (dsRNA) extraction and high-throughput sequencing (HTS) of small RNAs (sRNAs) and total RNA. Genome organization, sequence similarity, and phylogenetic analyses indicated that the viruses were related to members of the order Bunyavirales and families Endornaviridae, Megabirnaviridae, Narnaviridae, Totiviridae, and the proposed family "Fusagraviridae." The study describes six novel viruses: Phytophthora castaneae RNA virus 1-5 (PcaRV1-5) and Phytophthora castaneae negative-stranded RNA virus 1 (PcaNSRV1). All six viruses were detected by sRNA sequencing, which demonstrates an active RNA interference (RNAi) system targeting viruses in P. castaneae. To our knowledge, this is the first report of viruses in P. castaneae and the whole Phytophthora major Clade 5, as well as of the activity of an RNAi mechanism targeting viral genomes among Clade 5 species. PcaRV1 is the first megabirnavirus described in oomycetes and the genus Phytophthora.
RESUMO
Marine oomycetes have recently been shown to be concurrently infected by (-)ssRNA viruses of the order Bunyavirales. In this work, even higher virus variability was found in a single isolate of Phytophthora condilina, a recently described member of Phytophthora phylogenetic Clade 6a, which was isolated from brackish estuarine waters in southern Portugal. Using total and small RNA-seq the full RdRp of 13 different potential novel bunya-like viruses and two complete toti-like viruses were detected. All these viruses were successfully confirmed by reverse transcription polymerase chain reaction (RT-PCR) using total RNA as template, but complementarily one of the toti-like and five of the bunya-like viruses were confirmed when dsRNA was purified for RT-PCR. In our study, total RNA-seq was by far more efficient for de novo assembling of the virus sequencing but small RNA-seq showed higher read numbers for most viruses. Two main populations of small RNAs (21 nts and 25 nts-long) were identified, which were in accordance with other Phytophthora species. To the best of our knowledge, this is the first study using small RNA sequencing to identify viruses in Phytophthora spp.
Assuntos
Phytophthora/virologia , Vírus de RNA/classificação , Vírus de RNA/genética , Pequeno RNA não Traduzido/genética , RNA Viral/genética , Análise de Sequência de RNA , Viroses/virologia , Biologia Computacional , Genoma Viral , Fases de Leitura Aberta , Filogenia , Portugal , Vírus de RNA/isolamento & purificação , RNA-Seq , Análise de Sequência de DNARESUMO
We investigated the incidence of RNA viruses in a collection of Halophytophthora spp. from estuarine ecosystems in southern Portugal. The first approach to detect the presence of viruses was based on the occurrence of dsRNA, typically considered as a viral molecule in plants and fungi. Two dsRNA-banding patterns (â¼7 and 9 kb) were observed in seven of 73 Halophytophthora isolates tested (9.6%). Consequently, two dsRNA-hosting isolates were chosen to perform stranded RNA sequencing for de novo virus sequence assembly. A total of eight putative novel virus species with genomic affinities to members of the order Bunyavirales were detected and their full-length RdRp gene characterized by RACE. Based on the direct partial amplification of their RdRp gene by RT-PCR multiple viral infections occur in both isolates selected. Likewise, the screening of those viruses in the whole collection of Halophytophthora isolates showed that their occurrence is limited to one single Halophytophthora species. To our knowledge, this is the first report demonstrating the presence of negative (-) ssRNA viruses in marine oomycetes.
RESUMO
The European race of the ascomycetous species Gremmeniella abietina (Lagerberg) Morelet includes causal agents of shoot blight and stem canker of several conifers in Europe and North America, which are known to host a diverse virome. GaRV6 is the latest and sixth mycovirus species reported within G. abietina. Before its description, one victorivirus and one gammapartitivirus species were described in biotype A, two mitoviruses in both biotypes A and B and a betaendornavirus in biotype B. Possible phenotypic changes produced by mycoviruses on G. abietina mycelial growth have been reported in Spanish mitovirus-free and GaRV6-hosting G. abietina isolates, which had higher growth rates at the optimal temperature of 15 °C, but no other major differences have been observed between partitivirus-like dsRNA and dsRNA-free isolates. In this review, we reappraise the diversity of viruses found in G. abietina so far, and their relevance in clarifying the taxonomy of G. abietina. We also provide evidence for the presence of two new viruses belonging to the families Fusariviridae and Endornaviridae in Spanish isolates.
Assuntos
Ascomicetos/virologia , Biodiversidade , Micovírus/classificação , Micovírus/isolamento & purificação , Europa (Continente) , América do Norte , Doenças das Plantas/microbiologia , Traqueófitas/microbiologiaRESUMO
The population genetics of the family Partitiviridae was studied within the European race of the conifer pathogen Gremmeniella abietina. One hundred sixty-two isolates were collected from different countries, including Canada, the Czech Republic, Finland, Italy, Montenegro, Serbia, Spain, Switzerland, Turkey and the United States. A unique species of G. abietina RNA virus-MS1 (GaRV-MS1) appears to occur indistinctly in G. abietina biotypes A and B, without a particular geographical distribution pattern. Forty-six isolates were shown to host GaRV-MS1 according to direct specific RT-PCR screening, and the virus was more common in biotype A than B. Phylogenetic analysis based on 46 partial coat protein (CP) cDNA sequences divided the GaRV-MS1 population into two closely related clades, while RNA-dependent RNA polymerase (RdRp) sequences revealed only one clade. The evolution of the virus appears to mainly occur through purifying selection but also through recombination. Recombination events were detected within alignments of the three complete CP and RdRp sequences of GaRV-MS1. This is the first time that recombination events have been directly identified in fungal partitiviruses and in G. abietina in particular. The results suggest that the population dynamics of GaRV-MS1 do not have a direct impact on the genetic structure of its host, G. abietina, though they might have had an innocuous ancestral relationship.
Assuntos
Ascomicetos/virologia , Vírus de RNA/classificação , Vírus de RNA/isolamento & purificação , Ascomicetos/isolamento & purificação , Canadá , Análise por Conglomerados , Europa (Continente) , Evolução Molecular , Genótipo , Dados de Sequência Molecular , Filogenia , Vírus de RNA/genética , RNA Viral/genética , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência , Estados UnidosRESUMO
The genetic structure of the genus Mitovirus community hosted by the European pathogenic conifer fungus Gremmeniella abietina var. abietina was investigated. Gremmeniella abietina is a species complex with a divergent mycovirus community, composed mainly of Totivirus, Partitivirus, and Mitovirus species. In this work, the total doubled-stranded (ds)RNA from 353 isolates from Canada, Finland, Spain, Switzerland, Turkey, and USA was extracted to look for the presence of a ca. 2.5 kb band typical of mitoviruses' genomes. Based on the banding data, 60 partial RNA-dependent RNA polymerase (RdRp) DNA sequences (ca. 500 bp) were amplified with reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. Two distantly related mitovirus groups (species) were observed in the clustering analysis, one of them related to GMV1-1 and the other one related to a new putative species described in this study, GMV2-1. Viruses in these two clusters seemed to be subjected to purifying selection. The cluster with GMV1-1 included viruses observed in the Finnish biotype A and Spanish strains, whereas the cluster including GMV2-1 was composed of viruses of the Finnish biotype B and one from the Spanish population. Thereby, the Spanish population of G. abietina harboured mitovirus strains occurring in both biotype A and B strains, and it is the first one hosting distantly related mycoviruses of a single genus in one population of G. abietina. This may suggest that horizontal transmission of viruses could have occurred between biotype B and the Spanish population.
Assuntos
Ascomicetos/virologia , Vírus de RNA/isolamento & purificação , Ascomicetos/classificação , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Europa (Continente) , Especificidade de Hospedeiro , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/microbiologia , Vírus de RNA/classificação , Vírus de RNA/genética , Vírus de RNA/fisiologia , Árvores/microbiologiaRESUMO
Alternative and ecological strategies are necessary and demanded for disease management in order to reduce the use of pesticides in agriculture. Thus, the use of biological control agents such as plant growth-promoting rhizobacteria (PGPR) or several strains of the beneficial fungus Trichoderma spp. to combat plant diseases is the basis of biocontrol of plant pathogens and is a good approach to reach this healthy and environmentally adequate objective.
Assuntos
Agentes de Controle Biológico , Biotecnologia , Técnicas de Transferência de Genes , Plantas Geneticamente Modificadas/genética , Trichoderma/genética , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas/microbiologiaRESUMO
Using the TrichoEST database, generated in a previous functional genomics project from the beneficial filamentous fungus Trichoderma harzianum, a gene named Thkel1, which codes for a putative kelch-repeat protein, was isolated and characterized. Silencing of this gene in T. harzianum leads to a reduction of glucosidase activity and mycelial growth under abiotic stress conditions. Expression of this gene in Arabidopsis enhances plant tolerance to salt and osmotic stresses, accompanied by an increase in glucosidase activity and a reduction of abscisic acid levels compared to those observed in wild-type plants. Data presented throughout this article suggest the high value of T. harzianum as a source of genes able to facilitate the achievement of producing plants resistant to abiotic stresses without alteration of their phenotype.
Assuntos
Adaptação Fisiológica , Arabidopsis/fisiologia , Glucosidases/metabolismo , Trichoderma/genética , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes Fúngicos , Vetores Genéticos , Glucosidases/genética , Pressão Osmótica , Filogenia , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/metabolismo , Plantas Tolerantes a Sal/fisiologia , Cloreto de Sódio/metabolismoRESUMO
Genetic structure of the European Gremmeniella abietina var. abietina was analyzed in this study. Ninety-two Spanish isolates, six Swiss isolates of Alpine biotype, 76 Finnish isolates of biotype A and 54 Finnish and seven Russian isolates of biotype B were collected. Genetic variation of different populations was analyzed using sequence analysis of specifically amplified markers GAAA1000, GAAA800 and ACA900. Variation in the GAAA1000 marker was significant, and composed of 33 alleles divided into the following four studied populations: five alleles in the Alpine type, 12 in biotype B, 16 in biotype A and two in the Spanish population. Based on variation in GAAA1000 marker, a subset of isolates were further analyzed using GAAA800 and ACA900 sequences, which showed lower overall genetic variability, and no variation among the Spanish population. Genetic differentiation analysis revealed a high genetic differentiation among populations. Finally, clustering analysis of GAAA1000 sequences showed that the Spanish isolates clearly separated from the rest of the biotypes, whereas the Alpine type was closely related to the B type. However, one of the A-type isolates had an identical GAAA1000 allele with the prevailing allele among Spanish isolates. Altogether, our data suggest that the Spanish population is genetically highly differentiated from any other G. abietina population in Europe with a probable A-type origin.