The ADnet Bayesian belief network for alder decline: Integrating empirical data and expert knowledge.

The globalization in plant material trading has caused the emergence of invasive pests in many ecosystems, such as the alder pathogen Phytophthora ×alni in European riparian forests. Due to the ecological importance of alder to the functioning of rivers and the increasing incidence of P. ×alni-induced alder decline, effective and accessible decision tools are required to help managers and stakeholders control the disease. This study proposes a Bayesian belief network methodology to integrate diverse information on the factors affecting the survival and infection ability of P. ×alni in riparian habitats to help predict and manage disease incidence. The resulting Alder Decline Network (ADnet) management tool integrates information about alder decline from scientific literature, expert knowledge and empirical data. Expert knowledge was gathered through elicitation techniques that included 19 experts from 12 institutions and 8 countries. An original dataset was created covering 1189 European locations, from which P. ×alni occurrence was modeled based on bioclimatic variables. ADnet uncertainty was evaluated through its sensitivity to changes in states and three scenario analyses. The ADnet tool indicated that mild temperatures and high precipitation are key factors favoring pathogen survival. Flood timing, water velocity, and soil type have the strongest influence on disease incidence. ADnet can support ecosystem management decisions and knowledge transfer to address P. ×alni-induced alder decline at local or regional levels across Europe. Management actions such as avoiding the planting of potentially infected trees or removing man-made structures that increase the flooding period in disease-affected sites could decrease the incidence of alder disease in riparian forests and limit its spread. The coverage of the ADnet tool can be expanded by updating data on the pathogen's occurrence, particularly from its distributional limits. Research on the role of genetic variability in alder susceptibility and pathogen virulence may also help improve future ADnet versions.

Susceptibility of germinating seedlings of European and Eurasian populations of Pinus sylvestris to damping-off caused by Fusarium circinatum.

The effect of inoculation with Fusarium circinatum on survival of seed and seedlings of 19 populations of Pinus sylvestris was examined under environmentally controlled conditions, with four treatments (0, 50, 103, 106 spores ml-1). A single seed source of P. radiata was included as a positive control. Germination (emergence of the plumule above the compost) and health of seedlings was assessed daily, for 85 days. Spore density had a significant effect on germination: at 50 spores ml-1, only germination of a Northeast Scotland population was reduced. Treatment with 1000 spores ml-1, however, reduced germination of six populations of P. sylvestris and of P. radiata. Survival of emerged seedlings also varied with inoculum dose. Approximately 75% of seedlings survived 85 days after germination after inoculation with 50 spores ml-1. Seedlings of all populations were killed within 12-16 days of germination by the 103 and 106 spores ml-1 treatments. Emerged seedlings of the Austrian populations showed the highest susceptibility to F. circinatum following treatment with 50 spores ml-1, although 15% of seedlings of one Austrian population (AU3) survived to the end of the experiment (85 days after germination). There was no clear pattern in survival rates of the P. sylvestris seedlings from other populations treated with 1000 or 1 million spores ml-1 due to death of all emerged seedlings within a short period. Variations in susceptibility of different populations of P. sylvestris to F. circinatum may be used in future selection and breeding programmes to reduce the impact of the pathogen as it spreads over wider areas in Europe and Eurasia.

Co-Infections by Fusarium circinatum and Phytophthora spp. on Pinus radiata: Complex Phenotypic and Molecular Interactions.

This study investigated the complex phenotypic and genetic response of Monterey pine (Pinus radiata) seedlings to co-infections by F. circinatum, the causal agent of pine pitch canker disease, and the oomycetes Phytophthora xcambivora and P. parvispora. Monterey pine seedlings were wound-inoculated with each single pathogen and with the combinations F. circinatum/P. xcambivora and F. circinatum/P. parvispora. Initially, seedlings inoculated only with F. circinatum showed less severe symptoms than seedlings co-inoculated or inoculated only with P. xcambivora or P. parvispora. However, 30 days post-inoculation (dpi), all inoculated seedlings, including those inoculated only with F. circinatum, showed severe symptoms with no significant differences among treatments. The transcriptomic profiles of three genes encoding pathogenesis-related proteins, i.e., chitinase (PR3), thaumatin-like protein (PR5), phenylalanine ammonia-lyase (PAL), and the pyruvate decarboxylase (PDC)-encoding gene were analyzed at various time intervals after inoculation. In seedlings inoculated with single pathogens, F. circinatum stimulated the up-regulation of all genes, while between the two oomycetes, only P. xcambivora induced significant up-regulations. In seedlings co-inoculated with F. circinatum and P.xcambivora or P. parvispora none of the genes showed a significant over-expression 4 dpi. In contrast, at 11 dpi, significant up-regulation was observed for PR5 in the combination F. circinatum/P.xcambivora and PDC in the combination F. circinatum/P. parvispora, thus suggesting a possible synergism of multiple infections in triggering this plant defense mechanism.

Dual RNA-Sequencing Analysis of Resistant (Pinus pinea) and Susceptible (Pinus radiata) Hosts during Fusarium circinatum Challenge.

Fusarium circinatum causes one of the most important diseases of conifers worldwide, the pine pitch canker (PPC). However, no effective field intervention measures aiming to control or eradicate PPC are available. Due to the variation in host genetic resistance, the development of resistant varieties is postulated as a viable and promising strategy. By using an integrated approach, this study aimed to identify differences in the molecular responses and physiological traits of the highly susceptible Pinus radiata and the highly resistant Pinus pinea to F. circinatum at an early stage of infection. Dual RNA-Seq analysis also allowed to evaluate pathogen behavior when infecting each pine species. No significant changes in the physiological analysis were found upon pathogen infection, although transcriptional reprogramming was observed mainly in the resistant species. The transcriptome profiling of P. pinea revealed an early perception of the pathogen infection together with a strong and coordinated defense activation through the reinforcement and lignification of the cell wall, the antioxidant activity, the induction of PR genes, and the biosynthesis of defense hormones. On the contrary, P. radiata had a weaker response, possibly due to impaired perception of the fungal infection that led to a reduced downstream defense signaling. Fusarium circinatum showed a different transcriptomic profile depending on the pine species being infected. While in P. pinea, the pathogen focused on the degradation of plant cell walls, active uptake of the plant nutrients was showed in P. radiata. These findings present useful knowledge for the development of breeding programs to manage PPC.

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

Fungal endophytic communities on twigs of fast and slow growing Scots pine (Pinus sylvestris L.) in northern Spain.

Most plant species harbour a diverse community of endophytic, but their role is still unknown in most cases, including ecologically and economically important tree species. This study describes the culturable fungal endophytic community of Pinus sylvestris L. twigs in northern Spain and its relationship with diametric growth of the host. In all, 360 twig samples were collected from 30 Scots pines in fifteen stands. Isolates were obtained from all twig samples and 43 fungal taxa were identified by morphogrouping and subsequent ITS rDNA sequencing. All isolates were Ascomycetes, being Dothideomycetes and Sordariomycetes the most abundant classes. Half of the species were host generalists while the others were conifer or pine specialists. We found three new endophytic species for the Pinaceae: Biscogniauxia mediterranea, Phaeomoniella effusa and Plectania milleri, and additional six new species for P. sylvestris: Daldinia fissa, Hypocrea viridescens, Nigrospora oryzae, Ophiostoma nigrocarpum, Penicillium melinii and Penicillium polonicum. The endophytic community of fast and slow growing trees showed differences in species composition, abundance and evenness, but not in diversity. Phoma herbarum was associated to fast growing trees and Hypocrea lixii to those growing slow. Our results support the hypothesis that some endophytic species may affect growth of P. sylvestris.

Description and prevalence of a putative novel mycovirus within the conifer pathogen Gremmeniella abietina.

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.

Fusarium circinatum isolates from northern Spain are commonly infected by three distinct mitoviruses.

Pitch canker is a serious disease of pines caused by the ascomycete fungus Gibberella circinata (anamorph = Fusarium circinatum). Three distinct mitovirus strains have been described in this fungus: Fusarium circinatum mitovirus 1 (FcMV1), FcMV2-1 and FcMV2-2. Here, we investigated the frequency and population variation of these viruses and closely related sequence variants in northern Spain using RT-PCR and sequencing. Each virus strain and similar sequence variants shared >95 % sequence identity and were collectively designated as virus types. All virus types were relatively common in Spain, with estimated prevalence of 18.5 %, 8.9 % and 16.3 % for FcMV1, FcMV2-1 and FcMV2-2, respectively.

The European race of Gremmeniella abietina hosts a single species of Gammapartitivirus showing a global distribution and possible recombinant events in its history.

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.

Three mitovirus strains infecting a single isolate of Fusarium circinatum are the first putative members of the family Narnaviridae detected in a fungus of the genus Fusarium.

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.

Occurrence of two different species of mitoviruses in the European race of Gremmeniella abietina var. abietina, both hosted by the genetically unique Spanish population.

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.

Spanish population of Gremmeniella abietina is genetically unique but related to type A in Europe.

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.