The use of mycoviruses in the control of forest diseases

Producción CientíficaFifteen families of mycoviruses have been described and 80% of these catalogued. However, their evolutionary relationship with fungi is not clear. The mycovirus genome can be formed by single- or double-stranded RNA or single-stranded DNA. The effects of mycoviruses range from the induction of a cryptic state (asymptomatic) to promotion of hyper- or hypovirulence in the host. Horizontal transmission of mycoviruses is determined by the presence of different vegetative compatibility types and mating types. Biocontrol of chestnut blight (Cryphonectria parasitica) has been found to be a successful mycovirus-based treatment and is considered a model in forest disease management. Development of this type of biological control tool for use in other forest pathologies requires a sound knowledge of viral symptomatology and transmission. The present review focuses on the application of mycoviruses and the prospects for future use in the biological control of forest diseases as well as on advances in mycovirus-applied research in forestry, landscape and culture of woody plants.Ministerio de Economía, Industria y Competitividad (Project AGL2012-39912

In vitro antagonism of edible ectomycorrhizal fungi against Fusarium oxysporum and Fusarium verticillioides

Producción CientíficaTwenty-one isolates of edible ectomycorrhizal fungi (ECM) of 15 different species were tested in vitro for mycelial growth and spore germination against two isolates each of Fusarium oxysporum and F. verticillioides. The growth of Fusarium isolates was significantly inhibited when co-cultured with most of the 21 ECM fungi tested. Two ECM fungi (Ba-2 and Xf-2) failed to reduce the growth of Fusariumisolates. In paired cultures, growth of all Fusariumisolates was significantly reduced by Rhizopogon roseolus, Suillus luteus, Tricholoma portentosum, Amanita rubescens, Amanita ovoidea, Boletus fragrans and Laccaria laccata. Spore germination of all Fusariumisolates was strongly inhibited by culture filtrates of R. roseolus and the two S. luteus isolates. Different behavior between ectomycorrhizal fungi and Fusarium species, as well as among isolates of the same species was observed in both assays. Inhibition of Fusarium species suggests that several isolates of edible ectomycorrhizal fungi have a high potential for biological control of damping off. The effect of ECM fungi was less evident in the conidial germination assay than in the growth assay, although inhibition was also observed. The methodology presented here can be used as an effective tool for in vitro selection of ectomycorrhizal fungi and in nursery assays.On a testé in vitro 21 isolats de champignons ectomycorhiziens comestibles (ECM) de 15 espèces différentes sur leur capacité à inhiber la croissance mycélienne et la germination des spores de deux isolats de Fusarium oxysporumet de deux isolats de F. verticillioides. La croissance des isolats de Fusarium a subi une forte diminution lorsqu’ils étaient cultivés en paires avec presque tous les 21 champignons ECM testés. Deux champignons ECM (Ba-2 and Xf-2) n’ont pas réduit la croissance des isolats de Fusarium. Dans les cultures en paires, tous les isolats de Fusarium ont vu leur croissance fortement diminuée par Rhizopogon roseolus, Suillus luteus, Tricholoma portentosum, Amanita rubescens, Amanita ovoidea, Boletus fragrans et Laccaria laccata. La germination des spores de tous les isolats de Fusarium a été considérablement réduite par R. roseolus et des filtrats de culture de deux isolats de S. luteus. Le comportement de chacun des champignons ectomycorhiziens a varié selon l’espèce de Fusarium et l’isolat de chacun. L’effet d’inhibition exercé sur les espèces de Fusarium laisse voir que certains isolats de champignons ectomycorhiziens comestibles pourraient être utilisés dans la lutte contrôle biologique contre la fonte des semis. Les effets des champignons ECM auront moins manifestes lors des essais de germination des conidies que lors des essais de croissance, même s’il y a eu une certaine réduction de la germination. La méthodologie que nous présentons ici pourrait aussi servir pour la sélection in vitro de champignons ectomycorhiziens et pour diverses expériences en pépinière.Ministerio de Ciencia y Tecnología - Fondo Europeo de Desarrollo Regional (project AGL2001-1771

Aggressiveness of Fusarium oxysporum and F. verticillioides isolates on stone and scots pine under greenhouse conditions

Producción CientíficaScots pine (Pinus sylvestris) and Stone pine (Pinus pinea) are two important species used in re-forestation that are subject to damage by damping-off fungi in forest nurs-eries. Twenty-two isolates of Fusarium oxysporum and F. verticillioides from diseased seedlings of eight different hosts were tested for aggressiveness on seeds and seed-lings of both pine species, including the effects on seedling emergence and mortal-ity. Scots pine was more susceptible to damping-off than Stone pine, as indicated both by reduced seedling emergence and elevated seedling mortality. The impact of F. oxysporum and F. verticillioides on seeds and seedlings did not differ significantly for either pine species. Our findings support previous studies that found that these are damping-off pathogens on the studied pines. Whereas most isolates proved to be pathogenic, some isolates of both Fusarium species showed to be non- pathogenic.Ministerio de Ciencia e Innovación projects (PID2019-110459RB-I00) and (PLEC2021-008076

New insights on the integrated management of plant diseases by RNA strategies: Mycoviruses and RNA interference

Producción CientíficaRNA-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.Ministerio de Ciencia e Innovación y Next Generation EU - (grant PID2019-110459RB-I00; PLEC2021-008076)Junta de Castilla y León - (Orden EDU/601/2020

Susceptibility of Several Northeastern Conifers to Fusarium circinatum and Strategies for Biocontrol

Producción CientíficaFusarium circinatum, the causal of pine pitch canker disease (PPC), is now considered among the most important pathogens of Pinaceae in the world. Although in Europe PPC is only established in the Iberian Peninsula, the potential endangered areas cover over 10 million hectares under the current host distribution and climatic conditions. It is therefore a priority to test the susceptibility of those species and their provenances, within Central and Northern Europe and find biological control agents (BCAs) against the disease. In this study, the susceptibility of Pinus sylvestris, P. mugo and Picea abies Romanian provenances to F. circinatum was tested using three inoculum doses. In parallel, the potential use of Trichoderma atroviride and Trichoderma viride as BCAs against F. circinatum was also tested. This study has demonstrated, for the first time, the susceptibility of P. mugo to F. circinatum. Likewise, the susceptibility of P. abies was also confirmed. The fact that the Romanian provenance of P. sylvestris has not been susceptible to F. circinatum suggests genetic resistance as a potential tool to manage the disease. This, together with the apparent effectiveness of Trichoderma species as BCAs, seems to indicate that an integrated management of the disease might be feasible.European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH)Ministerio de Economía, Industria y Competitividad (projects AGL2012-39912 / AGL2015-69370-R)Portuguese Foundation for Science and Technology (grant SFRH/BPD/122928/2016

Potential control of forest diseases by solutions of chitosan oligomers, propolis and nanosilver

Producción CientíficaThere is a growing necessity to replace chemical agents with ecofriendly materials, arising from the impact on the environment and/or human health, which calls for the design of new broad-spectrum fungicides. In this work, chitosan oligomers (COs), propolis (Ps) and silver nanoparticles (AgNPs) mixtures in solution were assessed to control the growth of different phytopathogenic fungi and oomycetes in vitro. Binary solutions of COs-Ps and COs-AgNPs evinced the highest antifungal effect against Fusarium circinatum and Diplodia pinea fungi, respectively, with a ca. 80% reduction in their mycelial growth. The COs solution by itself also proved to be greatly effective against Gremmeniella abietina, Cryphonectria parasitica and Heterobasidion annosum fungi, causing a reduction of 78%, 86% and 93% in their growth rate, respectively. Likewise, COs also attained a 100% growth inhibition on the oomycete Phytophthora cambivora. On the other hand, Ps inhibited totally the growth of Phytophthora ×alni and Phytophthora plurivora. The application of AgNPs reduced the mycelial growth of F. circinatum and D. pinea. However, the AgNPs in some binary and ternary mixtures had a counter-productive effect on the anti-fungal/oomycete activity. In spite of the fact that the anti-fungal/oomycete activity of the different treatments showed a dependence on the particular type of microorganism, these solutions based on natural compounds can be deemed as a promising tool for control of tree diseases.European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH)Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (project AGL2015-69370-R

Effect of mycoviruses on growth, spore germination and pathogenicity of the fungus Fusarium circinatum

Producción CientíficaAim 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

Effect of mycoviruses on the virulence of Fusarium circinatum and laccase activity

Producción CientíficaLaccase enzymes (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) play a major role in the degradation of phenolic compounds such as lignin. They are common in fungi and have been suggested to participate in host colonization by pathogenic fungi. Putative mycoviruses have recently been isolated from the causal agent of pine pitch canker disease, Fusarium circinatum Nirenberg & O'Donell. In this study, the effects of single and double mycoviral infections on laccase activity, growth rate and pathogenicity were investigated in fourteen F. circinatum strains. Extracellular laccase activity was analyzed by the Bavendamm test, image processing and a spectrophotometric method. Mycelial growth, in vivo pathogenicity and seedling survival probability were also determined in Monterrey pine (Pinus radiata D. Don) seedlings. The findings showed that (i) mycelial growth of isolates from the same fungal population was homogeneous, (ii) the presence of mycovirus appears to increase the virulence of fungal isolates, (iii) co-infection (with two mycoviruses) caused cryptic effects in fungal isolates, and (iv) laccases embody a possible auxiliary tool in fungal infection. The prospects for biocontrol, the adaptive role of F. circinatum mycoviruses and the importance of laccase enzymes in host colonization are discussed.Ministerio de Economía, Industria y Competitividad (Project AGL2012-39912

Residual effects caused by a past mycovirus infection in Fusarium circinatum

Producción CientíficaMycoviruses are known to be difficult to cure in fungi but their spontaneous loss occurs commonly. The unexpected disappearance of mycoviruses can be explained by diverse reasons, from methodological procedures to biological events such as posttranscriptional silencing machinery. The long-term effects of a virus infection on the host organism have been well studied in the case of human viruses; however, the possible residual effect on a fungus after the degradation of a mycovirus is unknown. For that, this study analyses a possible residual effect on the transcriptome of the pathogenic fungus Fusarium circinatum after the loss of the mitovirus FcMV1. The mycovirus that previously infected the fungal isolate was not recovered after a 4-year storage period. Only 14 genes were determined as differentially expressed and were related to cell cycle regulation and amino acid metabolism. The results showed a slight acceleration in the metabolism of the host that had lost the mycovirus by the upregulation of the genes involved in essential functions for fungal development. The analysis also revealed a weak expression in the annotated genes of the RNA silencing machinery. To our knowledge, this is the first time that a potential residual effect on the host transcriptome caused by the past infection of a mycovirus is reported.Ministerio de Ciencia e Innovación - Fondo Europeo de Desarrollo Regional - (Proyecto PID2019-110459RB-I00)Junta de Castilla y León (Project VA208P20

Relationships between rhizosphere microbiota and forest health conditions in Pinus pinaster stands at the Iberian Peninsula

Producción CientíficaMaritime pine (Pinus pinaster) is a Mediterranean forest tree species of great ecological importance within the European Union and the Iberian Peninsula in particular, whose presence is seriously threatened by forest decline. Knowledge of the diversity, abundance, and functionality of rhizospheric microorganisms can provide important information in the design of new strategies for sustainable forest management against forest decline. In this work, rhizospheric samples were collected from representative pine forests in the region of Castilla y León (North-western Spain) (in 10 municipalities of 5 different provinces), analyzing different physicochemical parameters and bacterial and fungal biodiversity (by metabarcoding). In addition, different variables of tree health and climatic conditions were analyzed. The main phylum of microorganisms found in the rhizosphere of P. pinaster were the Ascomycota (54.5 %) and Acidobacteria (16.4 %) in fungi and bacteria, respectively. A clear relationship was found between the presence/absence of certain bacterial and fungal groups (taxonomic and functional) and the presence/absence of healthy/sick trees. Specifically, the fungal genus Umbelopsis and the bacterial genus Paenibacillus were thought as possible control agents of decline, as their presence was related to the absence of disease. Understanding the relationships between rhizosphere microbiota and forest health parameters in Pinus pinaster can be only achieved by exploring the complex ‘ecosystem microbiome’ and its functioning using focused, integrative microbiological and ecological research performed across multiple habitats