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Purpose of Review: The collective virome of forest trees can be considered to include not only plant viruses, but also viral communities harbored by all tree-associated organisms. In this review, we will concentrate on reviewing recent developments in the two fields of forest tree virology that have received the most research input during the last 5 years: (1) current knowledge of virus diseases affecting forest trees and their causal agents and (2) fungal viruses (mycoviruses) and properties that are required for utilizing them for biocontrol purposes. Recent Findings: The discovery of plant and mycoviruses has been revolutionized during the last few years due to high-throughput sequencing (HTS). This has altered our view of virus diversity and prevalence, but also their host ranges, transmission routes, and host effects. Utilization of HTS has greatly expanded our knowledge of plant virus diversity and disease etiology in forest trees and revealed the commonness of cross-kingdom transmission events between fungi, oomycetes, plants, and arthropods. Research on mycoviruses has also identified several new mycoviruses that restrict the growth or virulence of forest pathogenic fungi. Summary: Gaining knowledge of the collective virome of forest ecosystems is essential not only for understanding virus evolution and diversity but also for improving our understanding on virus impacts, and our ability for biocontrol-based and environmentally friendly management of viral and fungal diseases that affect economically important plants and beneficial insects, and for preventing possible disease outbreaks in the future. Virus infections play a central role in plant health, but viral symptoms on forest trees remain often unrecognized and may be confused with other biotic or abiotic damages. However, recent studies have revealed previously unknown viruses as causes of forest tree symptoms and suggest that viruses are responsible for far greater economic losses than recognized earl

Le champignon pathogène Sclerotinia sclerotiorum s’attaque à une grande variété d’hôtes, dont la laitue. Cette grande variété d’hôtes limite les méthodes de contrôle et de répression du champignon et donc engendre de grandes pertes économiques chaque année en affectant le rendement et la qualité des plantes agricoles. L'utilisation de fongicides synthétiques pour lutter contre cet agent pathogène devient moins efficace en raison du développement de la résistance de S. sclerotiorum, et c’est pourquoi des méthodes de lutte biologiques plus respectueuses de l’environnement sont recherchées. Les travaux de recherche présentés dans ce mémoire s’intéressent à l’utilisation d’agents de biocontrôle, tels que des Pseudomonas spp. phytobénéfiques, pour lutter contre l’infection de la sclérotiniose chez la laitue. Les Pseudomonas spp. ont plusieurs traits qui les rendent performants en tant qu’agents de biocontrôle, tels que la production d’une multitude de métabolites bioactifs comme des antibiotiques et des lipopeptides. Dans ce projet, nous avons donc sélectionné et caractérisé des souches de Pseudomonas spp. capables d’inhiber la croissance du champignon S. sclerotiorum in vitro (objectif 1), validé leur capacité à inhiber le dévelopement de la scérotiniose en conditions in planta, et identifié des gènes ou groupe de gènes potentiellement impliqués dans le biocontrôle observé (objectif 2). Le premier objectif a permis à d’isoler des souches de Pseudomonas spp. prélevées de champs agricoles sous régie biologique situées dans la région de la Montérégie (Québec, Canada). Ces souches ont ensuite été caractérisées, ce qui a permis de construire une phylogénie grâce au séquençage des génomes des souches les plus performantes. Neuf souches de Pseudomonas spp.ont démontré une forte inhibition in vitro dans des essais de confrontation en double culture. Le séquençage du génome entier de ces souches a révélé leur affiliation à quatre sous-groupes phylogénétiques, à savoir P. corruga

Family Genomoviridae was recently established, and only a few mycoviruses have been described and characterized, and almost all of them (Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, Fusarium graminearum gemyptripvirus 1 and Botrytis cinerea gemydayirivirus 1) induced hypovirulence in their host. Botrytis cinerea ssDNA virus 1 (BcssDV1), a tetrasegmented single-stranded DNA virus infecting the fungus Botrytis cinerea, has been molecularly characterized in this work.

Viroids are the smallest known infectious agents that are thought to only infect plants. Here, we reveal that several species of plant pathogenic fungi that were isolated from apple trees infected with apple scar skin viroid (ASSVd) carried ASSVd naturally. This finding indicates the spread of viroids to fungi under natural conditions and further suggests the possible existence of mycoviroids in nature. A total of 117 fungal isolates were isolated from ASSVd-infected apple trees, with the majority (85.5%) being an ascomycete Alternaria alternata and the remaining isolates being other plant-pathogenic or -endophytic fungi. Out of the examined samples, viroids were detected in 81 isolates (69.2%) including A. alternata as well as other fungal species. The phenotypic comparison of ASSVd-free specimens developed by single-spore isolation and ASSVd-infected fungal isogenic lines showed that ASSVd affected the growth and pathogenicity of certain fungal species. ASSVd confers hypovirulence on ascomycete Epicoccum nigrum. The mycobiome analysis of apple tree-associated fungi showed that ASSVd infection did not generally affect the diversity and structure of fungal communities but specifically increased the abundance of Alternaria species. Taken together, these data reveal the occurrence of the natural spread of viroids to plants; additionally, as an integral component of the ecosystem, viroids may affect the abundance of certain fungal species in plants. Moreover, this study provides further evidence that viroid infection could induce symptoms in certain filamentous fungi.

Viroids are the smallest known infectious agents that are thought to only infect plants. Here, we reveal that several species of plant pathogenic fungi that were isolated from apple trees infected with apple scar skin viroid (ASSVd) carried ASSVd naturally. This finding indicates the spread of viroids to fungi under natural conditions and further suggests the possible existence of mycoviroids in nature. A total of 117 fungal isolates were isolated from ASSVd-infected apple trees, with the majority (85.5%) being an ascomycete Alternaria alternata and the remaining isolates being other plant-pathogenic or -endophytic fungi. Out of the examined samples, viroids were detected in 81 isolates (69.2%) including A. alternata as well as other fungal species. The phenotypic comparison of ASSVd-free specimens developed by single-spore isolation and ASSVd-infected fungal isogenic lines showed that ASSVd affected the growth and pathogenicity of certain fungal species. ASSVd confers hypovirulence on ascomycete Epicoccum nigrum. The mycobiome analysis of apple tree-associated fungi showed that ASSVd infection did not generally affect the diversity and structure of fungal communities but specifically increased the abundance of Alternaria species. Taken together, these data reveal the occurrence of the natural spread of viroids to plants; additionally, as an integral component of the ecosystem, viroids may affect the abundance of certain fungal species in plants. Moreover, this study provides further evidence that viroid infection could induce symptoms in certain filamentous fungi.

RNA-based strategies for plant disease management offer an attractive alternative to agrochemicals that negatively impact human and ecosystem health and lead to pathogen resistance. There has been recent interest in using mycoviruses for fungal disease control after it was discovered that some cause hypovirulence in fungal pathogens, which refers to a decline in the ability of a pathogen to cause disease. Cryphonectria parasitica, the causal agent of chestnut blight, has set an ideal model of management through the release of hypovirulent strains. However, mycovirus-based management of plant diseases is still restricted by limited approaches to search for viruses causing hypovirulence and the lack of protocols allowing effective and systemic virus infection in pathogens. RNA interference (RNAi), the eukaryotic cell system that recognizes RNA sequences and specifically degrades them, represents a promising. RNA-based disease management method. The natural occurrence of cross-kingdom RNAi provides a basis for host-induced gene silencing, while the ability of most pathogens to uptake exogenous small RNAs enables the use of spray-induced gene silencing techniques. This review describes the mechanisms behind and the potential of two RNA-based strategies, mycoviruses and RNAi, for plant disease management. Successful applications are discussed, as well as the research gaps and limitations that remain to be addressed.

This study describes a new mycovirus infecting a strain from the Fusarium incarnatum-equiseti species complex. Based on phylogenetic and genomic analyses, this virus belongs to the recently proposed genus “Zetapartitivirus” in the family Partitiviridae. The name “Fusarium equiseti partitivirus 1″ (FePV1) is therefore suggested for this novel viral species. Similar to other partitiviruses, FePV1 genome is composed by two dsRNA segments that exhibit each one large ORF encoding for an RdRp and a CP, respectively. A smaller dsRNA was also detected in infected mycelium and could be a satellite RNA of FePV1. In addition to characterized zetapartitiviruses, other FePV1-related sequences were retrieved from online databases and their significance is discussed. Following conidial isolation, an FePV1-free isogenic line of the fungal host was obtained. In comparison with the original infected strain, this line showed higher growth, biomass production and pathogenicity on tomato, advocating that FePV1 induces hypovirulence on its host.

Mycoviruses are viruses that infect fungi, and hypovirulence-associated mycoviruses have the potential to control fungal diseases. However, it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field, and no mycovirus has been applied for field crop protection. In this study, we found that a previously identified small DNA mycovirus (SsHADV-1) can convert its host, Sclerotinia sclerotiorum, from a typical necrotrophic pathogen to a beneficial endophytic fungus. SsHADV-1 downregulates the expression of key pathogenicity factor genes in S. sclerotiorum during infection. When growing in rapeseed, the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense, hormone signaling, and circadian rhythm pathways. As a result, plant growth is promoted and disease resistance is enhanced. Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6% and improve yield by 14.9%. Moreover, we discovered that SsHADV-1 could also infect other S. sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants. These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism. Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.

Many Mediteranean isolates of Rosellinia necatrix, causing root rot in avocado, have previously been screened for viruses. Among them is isolate Rn459 that has been shown to be infected by at least three viruses such as Rosellinia necatrix partitivirus 10 (RnPV10), Rosellinia necatrix hypovirus 2 (RnHV2), Rosellinia necatrix fusagravirus 1 (RnFGV1), and Rosellinia necatrix virga-like virus (RnVLV). Here, we attempted to eliminate the viruses by hyphal tip cultures to examine their effect on colony growth in vitro and virulence on avocado plants. The obtained fungal strain termed, Rn459_PV10F/VLVF, which was confirmed to be cured of RnPV10 and RnVLV, but still retaining RnHV2, manifested a phenotype different from the original Rn459. Colony growth comparison showed that Rn459_PV10F/VLVF grew faster than the original Rn459 isolate and the virulence on avocado plants of this Rn459_PV10F/VLVF strain was higher than the original Rn459 strain. These combined results suggest that RnPV10 and RnVLV, alone or together, contribute to confer hypovirulence on the R. necatrix isolates.

A novel mycovirus named Fusarium oxysporum f. sp. dianthi mitovirus 1 (FodMV1) has been identified infecting a strain of Fusarium oxysporum f. sp. dianthi from Colombia. The genome of FodMV1 is 2313 nt long, and comprises a 172-nt 5’-UTR, a 2025-nt single ORF encoding an RdRp of 675 amino acid residues, and a 113-nt 3´-UTR. Homology BlastX searches identifies FodMV1 as a novel member of the genus Mitovirus in the family Narnaviridae. As the rest of mitoviruses, the genome of FodMV1 presents a high percentage of A+U (58.8%) and contains a number of UGA codons that encode the amino acid tryptophan rather than acting as stop codons as in the universal genetic code. Another common feature with other mitoviruses is that the 5′- and 3′-UTR regions of FodMV1 can be folded into potentially stable stem-loop structures. Result from phylogenetic analysis place FodMV1 in a different clade than the rest of mitoviruses described in other Fusarium spp. Incidence of FodMV1-infections in the collection of F. oxysporum f. sp. dianthi isolates analyzed is relatively high. Of particular interest is the fact that FodMV1 has been detected infecting isolates from two geographical areas as distant as Spain and Colombia.

Mycoviruses are viruses that infect fungi, and hypovirulence-associated mycoviruses have the potential to control fungal diseases. However, it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field, and no mycovirus has been applied for field crop protection. In this study, we found that a previously identified small DNA mycovirus (SsHADV-1) can convert its host, Sclerotinia sclerotiorum, from a typical necrotrophic pathogen to a beneficial endophytic fungus. SsHADV-1 downregulates the expression of key pathogenicity factor genes in S. sclerotiorum during infection. When growing in rapeseed, the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense, hormone signaling, and circadian rhythm pathways. As a result, plant growth is promoted and disease resistance is enhanced. Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6% and improve yield by 14.9%. Moreover, we discovered that SsHADV-1 could also infect other S. sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants. These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism. Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.

Mycoviruses that induce hypovirulence in phytopathogenic fungi are interesting because their potential use as biological control agents of the plant diseases caused by their fungal hosts. The recently identified chrysovirus Fusarium oxysporum f. sp. dianthi virus 1 (FodV1) has been associated to the induction of hypovirulence in Fusarium oxysporum f. sp. dianthi, the forma specialis of F. oxysporum that causes vascular wilt in carnation (Dianthus caryophyllus). In this work, we have used confocal laser scanner microscopy and two isogenic GFP-labeled strains of F. oxysporum f. sp. dianthi infected (V+) and not infected (V−) with the Fusarium oxysporum f. sp. dianthi virus 1, respectively, to analyze the effect of mycovirus FodV1 on the plant colonization pattern of its fungal host. Results demonstrate that FodV1-viral infection affects the speed and spatial distribution of fungal colonization into the plant. Initial stages of external root colonization were similar for both strains, but the virus-free strain colonized the internal plant tissues faster than the virus-infected strain. In addition, other differences related to the specific zone colonized and the density of colonization were observed between both F. oxysporum f. sp. dianthi strains. The hyphae of both V− and V+ strains progressed up through the xylem vessels but differences in the number of vessels colonized and of hyphae inside them were found. Moreover, as colonization progressed, V− and V+ hyphae propagated horizontally reaching the central medulla but, while the virus-free strain V− densely colonized the interior of the medulla cells, the virus-infected strain V+ appeared mainly in the intercellular spaces and with a lower density of colonization. Finally, the incidence of FodV1-viral infections in a collection of 221 isolates sampled between 2008 and 2012 in the geographic area where the originally infected isolate was obtained has been also analyzed. The very low (<2%) incidence of viral infections

Producción Científica, Pine Pitch Canker disease, caused by the pathogenic fungus Fusarium circinatum, affects conifer species worldwide. However, the virulence of the pathogen may be affected by the presence of mycoviruses. The aim of this laboratory-based study was to investigate the probability and rate of transmission of F. circinatum mitoviruses FcMV1 and FcMV2-2 via microconidia. Ten isolates of mitovirus-infected F. circinatum were subcultured to produce a total of 100 single-spore colonies (ten replicates per isolate). The total RNA and cDNA obtained from each spore isolate (monosporic culture) were amplified by PCR with specific primers for detection of F. circinatum mitoviruses FcMV1 and FcMV2-2. The mitoviruses were detected in a high percentage of the individual spore isolates (between 60% and 100% depending on the fungal isolate). However, the probability of transmission was not statistically significantly associated with either the F. circinatum isolate or the viral strain. A high proportion of transmission via microconidia is critical for development of a biological control program against Pine Pitch Canker (PPC) disease in forests. However, further studies are needed to establish the effect of these mitoviruses on the virulence of F. circinatum., Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (projects AGL2012-39912 / AGL2015-69370-R), European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH)

Producción Científica, Pine Pitch Canker disease, caused by the pathogenic fungus Fusarium circinatum, affects conifer species worldwide. However, the virulence of the pathogen may be affected by the presence of mycoviruses. The aim of this laboratory-based study was to investigate the probability and rate of transmission of F. circinatum mitoviruses FcMV1 and FcMV2-2 via microconidia. Ten isolates of mitovirus-infected F. circinatum were subcultured to produce a total of 100 single-spore colonies (ten replicates per isolate). The total RNA and cDNA obtained from each spore isolate (monosporic culture) were amplified by PCR with specific primers for detection of F. circinatum mitoviruses FcMV1 and FcMV2-2. The mitoviruses were detected in a high percentage of the individual spore isolates (between 60% and 100% depending on the fungal isolate). However, the probability of transmission was not statistically significantly associated with either the F. circinatum isolate or the viral strain. A high proportion of transmission via microconidia is critical for development of a biological control program against Pine Pitch Canker (PPC) disease in forests. However, further studies are needed to establish the effect of these mitoviruses on the virulence of F. circinatum., Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (projects AGL2012-39912 / AGL2015-69370-R), European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH)

Producción Científica, Pine Pitch Canker disease, caused by the pathogenic fungus Fusarium circinatum, affects conifer species worldwide. However, the virulence of the pathogen may be affected by the presence of mycoviruses. The aim of this laboratory-based study was to investigate the probability and rate of transmission of F. circinatum mitoviruses FcMV1 and FcMV2-2 via microconidia. Ten isolates of mitovirus-infected F. circinatum were subcultured to produce a total of 100 single-spore colonies (ten replicates per isolate). The total RNA and cDNA obtained from each spore isolate (monosporic culture) were amplified by PCR with specific primers for detection of F. circinatum mitoviruses FcMV1 and FcMV2-2. The mitoviruses were detected in a high percentage of the individual spore isolates (between 60% and 100% depending on the fungal isolate). However, the probability of transmission was not statistically significantly associated with either the F. circinatum isolate or the viral strain. A high proportion of transmission via microconidia is critical for development of a biological control program against Pine Pitch Canker (PPC) disease in forests. However, further studies are needed to establish the effect of these mitoviruses on the virulence of F. circinatum., Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (projects AGL2012-39912 / AGL2015-69370-R), European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH)

Fusarium oxysporum f. sp. dianthi virus 1 (FodV1) was detected in isolate 116 (116V+) of Fusarium oxysporum f. sp. dianthi, reaching such a high accumulation level that it was clearly visible after agarose gel electrophoresis of total DNA extracts. FodV1 consists of four double-stranded RNA segments that correspond to a new mycovirus in the Chrysoviridae family. We obtained an isolate of F. oxysporum f. sp. dianthi 116 (116V−) with only a residual level of FodV1 RNA accumulation by single-conidia selection. Compared with 116V−, isolate 116V+ showed significant phenotypic alterations in vegetative growth and virulence. Thus, the presence of a high titer of mycovirus FodV1 was associated with a modified morphology and a reduced growth of the colonies on solid medium, and with a diminished conidiation in liquid medium. Inoculation of four susceptible carnation cultivars with either 116V− or 116V+ showed that the presence of a high titer of FodV1 was also correlated with a significantly reduced virulence of its fungal host. All of the results suggest that FodV1 could be associated with hypovirulence, identifying it as a potential biocontrol agent for Fusarium wilt of carnation. This is the first report of a mycovirus potentially associated with the induction of hypovirulence in the species F. oxysporum.

Pine Pitch Canker disease, caused by the pathogenic fungus Fusarium circinatum, affects conifer species worldwide. However, the virulence of the pathogen may be affected by the presence of mycoviruses. The aim of this laboratory-based study was to investigate the probability and rate of transmission of F. circinatum mitoviruses FcMV1 and FcMV2-2 via microconidia. Ten isolates of mitovirus-infected F. circinatum were subcultured to produce a total of 100 single-spore colonies (ten replicates per isolate). The total RNA and cDNA obtained from each spore isolate (monosporic culture) were amplified by PCR with specific primers for detection of F. circinatum mitoviruses FcMV1 and FcMV2-2. The mitoviruses were detected in a high percentage of the individual spore isolates (between 60% and 100% depending on the fungal isolate). However, the probability of transmission was not statistically significantly associated with either the F. circinatum isolate or the viral strain. A high proportion of transmission via microconidia is critical for development of a biological control program against Pine Pitch Canker (PPC) disease in forests. However, further studies are needed to establish the effect of these mitoviruses on the virulence of F. circinatum.

Producción Científica, Aim of the study: To assess the impact on two mycoviruses recently described in F. circinatum mitovirus 1, and 2-2 (FcMV1 and FcMV2-2) on i) mycelial growth, ii) spore germination and iii) relative necrosis. Material and methods: Fourteen monosporic strains of F. circinatum (one of each pair infected with mycoviruses and the other without them) of the pathogen with and without viruses were selected for the assay. The statistical analysis, were a linear mixed model of analysis of variance considering one between-subjects factor (isolate) and one within-subjects factor with four levels (1=without viruses, 2=only virus FcMV1, 3=only virus FcMV2-2 and 4=both viruses). Main results: Colony growth rates of F. circinatum isolates were significantly reduced in presence of mycoviruses (p=0.002). The spore germination was also reduced in the F. circinatum isolates containing mycovirus as compared to mycovirus-free isolates (p<0.001). No significant differences in lesion lengths caused by F. circinatum were found in relation to the presence/absence of mycovirus (p<0.61). Research highlights: Reduction of the percentage of spore germination in the isolates of F. circinatum with mycovirus infections, as compared to free isolates, provides indications of reduction of metabolic activity and plant physiology are discussed. The lack of significant differences found in the length of the lesions caused by F. circinatum with respect to the presence/absence of mycovirus, indicates that further studies with a larger number of variables are required., Ministerio de Economía, Industria y Competitividad (projects AGL2012-39912 and AGL2015- 69370-R), European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH), Portuguese Foundation for Science and Technology (grant SFRH/BPD/122928/2016)

Aim of the study: To assess the impact on two mycoviruses recently described in F. circinatum mitovirus 1, 2-1 and 2-2 (FcMV1, FcMV2-1 and FcMV2-2) on i) mycelial growth, ii) spore germination and iii) relative necrosis.Material and methods: Fourteen monosporic strains of F. circinatum (one of each pair infected with mycoviruses and the other without them) of the pathogen with and without viruses were selected for the assay. The statistical analysis, were a linear mixed model of analysis of variance considering one between-subjects factor (isolate) and one within-subjects factor with four levels (1=without viruses, 2=only virus FcMV1, 3=only virus FcMV2-2 and 4=both viruses).Main results: Colony growth rates of F. circinatum isolates were significantly reduced in presence of mycoviruses (p=0.002). The spore germination was also reduced in the F. circinatum isolates containing mycovirus as compared to mycovirus-free isolates (p<0.001). No significant differences in lesion lengths caused by F. circinatum were found in relation to the presence/absence of mycovirus (p<0.61).Research highlights: Reduction of the percentage of spore germination in the isolates of F. circinatum with mycovirus infections, as compared to free isolates, provides indications of reduction of metabolic activity and plant physiology are discussed. The lack of significant differences found in the length of the lesions caused by F. circinatum with respect to the presence/absence of mycovirus, indicates that further studies with a larger number of variables are required.

Producción Científica, Aim of the study: To assess the impact on two mycoviruses recently described in F. circinatum mitovirus 1, and 2-2 (FcMV1 and FcMV2-2) on i) mycelial growth, ii) spore germination and iii) relative necrosis. Material and methods: Fourteen monosporic strains of F. circinatum (one of each pair infected with mycoviruses and the other without them) of the pathogen with and without viruses were selected for the assay. The statistical analysis, were a linear mixed model of analysis of variance considering one between-subjects factor (isolate) and one within-subjects factor with four levels (1=without viruses, 2=only virus FcMV1, 3=only virus FcMV2-2 and 4=both viruses). Main results: Colony growth rates of F. circinatum isolates were significantly reduced in presence of mycoviruses (p=0.002). The spore germination was also reduced in the F. circinatum isolates containing mycovirus as compared to mycovirus-free isolates (p<0.001). No significant differences in lesion lengths caused by F. circinatum were found in relation to the presence/absence of mycovirus (p<0.61). Research highlights: Reduction of the percentage of spore germination in the isolates of F. circinatum with mycovirus infections, as compared to free isolates, provides indications of reduction of metabolic activity and plant physiology are discussed. The lack of significant differences found in the length of the lesions caused by F. circinatum with respect to the presence/absence of mycovirus, indicates that further studies with a larger number of variables are required., Ministerio de Economía, Industria y Competitividad (projects AGL2012-39912 and AGL2015- 69370-R), European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH), Portuguese Foundation for Science and Technology (grant SFRH/BPD/122928/2016)

Producción Científica, Aim of the study: To assess the impact on two mycoviruses recently described in F. circinatum mitovirus 1, and 2-2 (FcMV1 and FcMV2-2) on i) mycelial growth, ii) spore germination and iii) relative necrosis. Material and methods: Fourteen monosporic strains of F. circinatum (one of each pair infected with mycoviruses and the other without them) of the pathogen with and without viruses were selected for the assay. The statistical analysis, were a linear mixed model of analysis of variance considering one between-subjects factor (isolate) and one within-subjects factor with four levels (1=without viruses, 2=only virus FcMV1, 3=only virus FcMV2-2 and 4=both viruses). Main results: Colony growth rates of F. circinatum isolates were significantly reduced in presence of mycoviruses (p=0.002). The spore germination was also reduced in the F. circinatum isolates containing mycovirus as compared to mycovirus-free isolates (p<0.001). No significant differences in lesion lengths caused by F. circinatum were found in relation to the presence/absence of mycovirus (p<0.61). Research highlights: Reduction of the percentage of spore germination in the isolates of F. circinatum with mycovirus infections, as compared to free isolates, provides indications of reduction of metabolic activity and plant physiology are discussed. The lack of significant differences found in the length of the lesions caused by F. circinatum with respect to the presence/absence of mycovirus, indicates that further studies with a larger number of variables are required., Ministerio de Economía, Industria y Competitividad (projects AGL2012-39912 and AGL2015- 69370-R), European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH), Portuguese Foundation for Science and Technology (grant SFRH/BPD/122928/2016)

Aim of the study: To assess the impact on two mycoviruses recently described in F. circinatum mitovirus 1, 2-1 and 2-2 (FcMV1, FcMV2-1 and FcMV2-2) on i) mycelial growth, ii) spore germination and iii) relative necrosis.Material and methods: Fourteen monosporic strains of F. circinatum (one of each pair infected with mycoviruses and the other without them) of the pathogen with and without viruses were selected for the assay. The statistical analysis, were a linear mixed model of analysis of variance considering one between-subjects factor (isolate) and one within-subjects factor with four levels (1=without viruses, 2=only virus FcMV1, 3=only virus FcMV2-2 and 4=both viruses).Main results: Colony growth rates of F. circinatum isolates were significantly reduced in presence of mycoviruses (p=0.002). The spore germination was also reduced in the F. circinatum isolates containing mycovirus as compared to mycovirus-free isolates (p<0.001). No significant differences in lesion lengths caused by F. circinatum were found in relation to the presence/absence of mycovirus (p<0.61).Research highlights: Reduction of the percentage of spore germination in the isolates of F. circinatum with mycovirus infections, as compared to free isolates, provides indications of reduction of metabolic activity and plant physiology are discussed. The lack of significant differences found in the length of the lesions caused by F. circinatum with respect to the presence/absence of mycovirus, indicates that further studies with a larger number of variables are required.

Following a request from the European Commission, the EFSA Plant Health (PLH) Panel performed a risk assessment for Cryphonectria parasitica in the EU with the aim to assess the current EU phytosanitary requirements and identify the risk reduction options (RROs), which would preserve the protected zone (PZ) status in some parts of the EU, where the pathogen is not known to occur. C. parasitica, a bark-inhabiting fungus causing blight of chestnut trees (Castanea spp.), has a wide distribution in the EU (non-PZs). Three regulatory scenarios were considered for the whole risk assessment (RA) area: the current situation in non-PZs (scenario A0), the situation in the EU without measures (A1) and the current situation in PZs with additional RROs (A2). The Panel considered both the risk of potential spread to PZs of C. parasitica strains currently present in the non-PZs and the risk of introduction from Third Countries and spread in non-PZs of new, virulent strains that would be able to jeopardise the currently effective hypovirulence and cause severe impact. The number of new introductions of C. parasitica into the EU is reduced by approximately a factor 5,000 (median values) in scenario A2 compared to scenario A0. Under the A0, A1 and A2 scenarios, 2, 3.5 and 0.5 (median values) EU Member States, respectively, are expected to be affected in the next 10 years due to spread of C. parasitica strains. The estimated relative impact on ecosystem services, due to the introduction and spread in the EU of new, virulent strains, is higher for scenario A1 compared to scenarios A0 and A2. The current EU requirements and the additional RROs considered in scenario A2 were assessed to be effective in reducing the risk of introduction and spread of C. parasitica, thus preserving the PZ status in some parts of the EU.

The obligate intracellular bacterium Ehrlichia ruminantium (ER) causes heartwater, a fatal tick-borne disease in livestock. In the field, ER strains present different levels of virulence, limiting vaccine efficacy, for which the molecular basis remains unknown. Moreover, there are no genetic tools currently available for ER manipulation, thus limiting the knowledge of the genes/proteins that are essential for ER pathogenesis and biology. As such, to identify proteins and/or mechanisms involved in ER virulence, we performed the first exhaustive comparative proteomic analysis between a virulent strain (ERGvir) and its high-passaged attenuated strain (ERGatt). Despite their different behaviors in vivo and in vitro, our results from 1DE-nanoLC-MS/MS showed that ERGvir and ERGatt share 80% of their proteins; this core proteome includes chaperones, proteins involved in metabolism, protein-DNA-RNA biosynthesis and processing, and bacterial effectors. Conventional 2DE revealed that 85% of the identified proteins are proteoforms, suggesting that post-translational modifications (namely glycosylation) are important in ER biology. Strain-specific proteins were also identified: while ERGatt has an increased number and overexpression of proteins involved in cell division, metabolism, transport and protein processing, ERGvir shows an overexpression of proteins and proteoforms (DIGE experiments) involved in pathogenesis such as Lpd, AnkA, VirB9 and B10, providing molecular evidence for its increased virulence in vivo and in vitro. Overall, our work reveals that ERGvir and ERGatt proteomes are streamlined to fulfill their biological function (maximum virulence for ERGvir and replicative capacity for ERGatt), and we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.

The obligate intracellular bacterium Ehrlichia ruminantium (ER) causes heartwater, a fatal tick-borne disease in livestock. In the field, ER strains present different levels of virulence, limiting vaccine efficacy, for which the molecular basis remains unknown. Moreover, there are no genetic tools currently available for ER manipulation, thus limiting the knowledge of the genes/proteins that are essential for ER pathogenesis and biology. As such, to identify proteins and/or mechanisms involved in ER virulence, we performed the first exhaustive comparative proteomic analysis between a virulent strain (ERGvir) and its high-passaged attenuated strain (ERGatt). Despite their different behaviors in vivo and in vitro, our results from 1DE-nanoLC-MS/MS showed that ERGvir and ERGatt share 80% of their proteins; this core proteome includes chaperones, proteins involved in metabolism, protein-DNA-RNA biosynthesis and processing, and bacterial effectors. Conventional 2DE revealed that 85% of the identified proteins are proteoforms, suggesting that post-translational modifications (namely glycosylation) are important in ER biology. Strain-specific proteins were also identified: while ERGatt has an increased number and overexpression of proteins involved in cell division, metabolism, transport and protein processing, ERGvir shows an overexpression of proteins and proteoforms (DIGE experiments) involved in pathogenesis such as Lpd, AnkA, VirB9 and B10, providing molecular evidence for its increased virulence in vivo and in vitro. Overall, our work reveals that ERGvir and ERGatt proteomes are streamlined to fulfill their biological function (maximum virulence for ERGvir and replicative capacity for ERGatt), and we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.

Colonization and lesion development by virulent, asymptomatic and hypovirulent isolates of Sclerotinia homoeocarpa on nonwounded (NW) and wounded (W) leaves of creeping bentgrass were characterized. Hypovirulent and asymptomatic isolates contain the fungal virus, Ophiostoma mitovirus 3a, and virulent isolates are virus-free. On NW leaves, all isolates infected leaves with appressoria along cell walls and through stomata by 48 hours post-inoculation (hpi). Inter- and intracellular hyphae formed on heavily colonized, NW leaves at 96 hpi. Wound-inoculated grass had a colonization front characterized by inter- and intracellular hyphal colonization within nonsymptomatic tissues at 8 hpi by direct infection of the wound site. The colonization front grew ahead of symptom development for 50 hpi on both NW and W leaves. In contrast to virulent and asymptomatic isolates, the hypovirulent isolate seldom colonized more than 30% of leaf tissue of the NW or W leaves. Callose accumulation under fungal appressoria, oxalate oxidase activity, and hydrogen peroxide accumulation at lesion borders were also demonstrated. The results indicate that S. homoeocarpa is capable of colonizing creeping bentgrass without causing visible symptoms. In addition, the fungus causes disease more rapidly on W than NW grass, and the etiology of virulent, asymptomatic and hypovirulent isolates was similar. The results of this study contribute to our understanding of the basic biology of S. homoeocarpa and may influence integrated management practices. L’apparition et la colonisation de lésions produites par des isolats virulents, asymptomatiques et hypovirulents de Sclerotinia homeocarpa sur des feuilles saines et meurtries d’agrostide rampante ont été caractérisées. Les isolats hypovirulents et asymptomatiques contiennent le virus fongique Ophiostoma mitovirus 3a et les isolats virulents n’en contiennent pas. Tous les isolats ont infecté les feuilles saines en pénétrant les parois cellulaires et les sto

Sixty-two natural cankers on branches and main stems of three 16-year-old grafted American chestnut trees at the Lesesne State Forest were sampled for Cryphonectria parasitica. Cankers were sampled in 1996 and 1997 at various distances from the main stem zone on the grafts (ground to 183 cm) that was inoculated in 1982 and 1983 with a mixture of dsRNA-containing white and pigmented hypovirulent strains. Grafted trees exhibited a high level of blight control, and all bark cores extracted from cankers on the grafted trees showed superficial necrosis. Bark cores extracted from these cankers yielded 156 isolates of C. parasitica. Fifty-three of these isolates were white, and 103 were pigmented. The farthest canker containing a white isolate was located 564 cm from the zone inoculated with hypovirulent strains (H-inoculated zone). The number of white isolates recovered per canker on the grafted trees near the H-inoculated zone (< 0.5 maximum sampling distance) was significantly greater (P=0.0039) than the number of white isolates recovered per canker on the grafted trees far from the H-inoculated zone (>0.5 maximum sampling distance). Lloyd's index of patchiness value for the frequency of white isolates in cankers was 1.36, indicating that white isolates were slightly aggregated in cankers. White isolates of C. parasitica were found in two of seven artificially established cankers 5 months after inoculation with a pigmented virulent strain (WK). Thirteen of 14 pigmented isolates collected from these cankers after 5 months were compatible with WK in vegetative compatibility (VC) tests. Eight of 25 white isolates recovered 5, 11, and 50 months after WK inoculation converted the pigmented WK strain to the white hypovirulent phenotype in vitro. Sixty-five pigmented isolates collected from natural cankers were paired in VC assays, revealing 28 VC groups. All 11 white isolates of C. parasitica assayed contained a 12.7 kb dsRNA in high concentrations. None of 48 pigmented isola