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

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

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

Pine Pitch Canker and Insects: Relationships and Implications for Disease Spread in Europe

Producción CientíficaThe fungal pathogen Fusarium circinatum (Nirenberg and O’ Donnell) is the causal agent of pine pitch canker (PPC) disease, which seriously affects conifer species in forests and nurseries worldwide. In Europe, PPC is only established in the Iberian Peninsula; however, it is presumed that its range could expand through the continent in the near future. Infection caused by this fungus requires open wounds on the tree, including physical damage caused by insects. Therefore, a relationship probably occurs between PPC and a wide variety of insects. The aim of this review is to outline the taxonomic and ecological diversity of insect species with high potential association with F. circinatum in Europe and elsewhere. The insects were classified as vectors, carriers and wounding agents according to the association level with the PPC disease. In addition, we discuss the insect-mediated spreading of PPC disease in relation to the different phases of forest stand development, from seeds and seedlings in nurseries to mature stands. Lastly, to improve our predictive capacities and to design appropriate intervention measures and strategies for controlling disease dissemination by insects, variables such as geographic location, time of the year and host species should be considered,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)Centro de Estudios do Ambiente e do Mar (CESAM) (project UID/AMB/50017/2019)Portuguese Foundation for Science and Technology (projects PTDC/AGR-FOR/2768/2014 , POCI-01-0145-FEDER-016785 , SFRH/BPD/122928/2016)Russian Foundation for Basic Research (grant 17-04-01486)Saint Petersburg State Polytechnical University (project 2019-0420

Sydowia polyspora dominates fungal communities carried by two Tomicus species in pine plantations threatened by Fusarium circinatum

Producción CientíficaBark beetles (Coleoptera, Scolytinae) carry a diverse filamentous fungal community sometimes acting as vectors or carriers of phytopathogens. In this study, mycobiota carried by two Tomicus species (Tomicus piniperda and Tomicus destruens) were investigated through (i) morphological and molecular identification of taxa; (ii) taxonomic richness, diversity, evenness, dominance and phoresy indices; (iii) ecological network analysis and (iv) statistical co-occurrence analysis. The studied mycobiota were formed by eleven taxa and showed a moderate fungal diversity with low evenness. The fungus Sydowia polyspora was significantly abundant and dominated the community. All the fungal taxa were randomly associated. Both insect species (T. piniperda and T. destruens) were collected from plantations of Pinus radiata infected by Fusarium circinatum. The ecological factors that could drive community ecology and phoretic links between fungi and bark beetles are discussed.Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (project AGL2015-69370-R)Ministerio de Economía, Industria y Competitividad (project AGL2012-39912)Junta de Castilla y León - Fondo Europeo de Desarrollo Regional (grant ORDEN EDU/1083/2013

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

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

Evaluación de la susceptibilidad de procedencias europeas de Pinus sylvestris frente al chancro resinoso del pino (Fusarium circinatum)

El hongo Fusarium circinatum (teleomorfo Gibberella circinatum) causante del chancro resinoso del pino, es actualmente la enfermedad más importante de masas de coniferas silvestres y comerciales a nivel mundial, ha tenido una expansión global en los últimos años amenazando a países donde aun no se conoce su presencia. En esta investigación se pretende identificar la acción patogénica pre-emergencia y post-emergencia en busca de resistencia a la enfermedad por medio de pruebas de patogenicidad de con veinte procedencias europeas de Pinus sylvestris y Pinus radiata las cuales fueron inoculadas vía sustrato con dos concentraciones esporales (1x103 y 1x106 esporas/ml) midiendo el porcentaje de germinación y de mortalidad, el tiempo de supervivencia por cada procedencia. Aplicando la pruebas de X2 (corrección de Yates) y el estimador de sobrevivencia de Kaplan-Meier para analizar los datos con un 95% de confiabilidad estadística. Demostramos que indistintamente de la concentración esporal utilizada de Fusarium circinatum, este hongo mata la totalidad las plantas de cada procedencia, lo que, indica que la diversidad geográfica no es un limitante a la expansión de la enfermedad al no existir resistencia y/o tolerancia a la enfermedad con una acción altamente virulenta. Demostramos la elevada susceptibilidad de Pinus radiata y Pinus sylvestris en relación a las demás por su velocidad e porcentaje mortalidad. Deben seguirse investigando con otras procedencias y especies de Pinus combinadas con agentes antagónicos y endófitos para el control de la enfermedad. Identificamos el elevado riesgo de las coniferas de los países donde no se ha detectado el hongo.Departamento de Producción Vegetal y Recursos ForestalesMáster en Investigación en Ingeniería para la Conservación y Uso Sostenible de Sistemas Forestale

Tecnologías transcriptómicas y su potencial en la gestión del chancro resinoso del pino

Pine pitch canker (PPC) is a serious disease of Pinus spp. and Pseudotsuga menziesii globally. The infection of its causal agent, Fusarium circinatum, causes pitch or resin-soaked cankers on trunks and lateral branches of mature hosts, which may eventually die due to girdling or stem breakage. In nurseries, the main symptoms are damping-off and tip dieback of seedlings. However, the pathogen, with a hemibiotrophic nature, can remain endophytic in pine seedlings that do not show symptoms of infection and even inconspicuous in some herbaceous species. Since the first report in 1945 in North America, the presence of F. circinatum has been notified in 14 countries in America, Asia, Africa and Europe. Several factors have contributed to the spread of the disease to all these continents, the most important being globalization in terms of trade in reproductive plant material. Wind, raindrops and forest insects associated with pines contribute to the local dispersion of the pathogen. Despite its importance, no effective measures are available to eradicate or control PPC disease either in nurseries or in the field. The main objective of this doctoral thesis was to shed light on effective regulatory mechanisms for the control of PPC disease. For this purpose, firstly, a review focused on collecting current information on pathways of pathogen spread and proposing preventive mechanisms to avoid its introduction into disease-free areas was elaborated. The multiple pathways of spread make F. circinatum challenging to prevent, exacerbated by the recent discovery of its endophytic colonization of non-reported host species that illustrates the importance of the biological and ecological knowledge for the design of effective intervention strategies. In addition, eradication of the disease may be feasible only if its entry is detected at a very early stage. In this regard, new methods for detection and diagnosis for the prompt detection of F. circinatum in seeds, plants, and vector insects are urgently needed. For that, it is essential the collaboration between phytosanitary authorities and researchers through interdisciplinary networks that allows increasing knowledge of the disease and raising awareness of the risks and mitigation measures among crucial target groups. The review also identified weak points in current regulations and provided suggestions for implementation.El chancro resinoso del pino (PPC, por sus siglas en inglés) es una grave enfermedad que afecta a Pinus spp. y Pseudotsuga menziesii en todo el mundo. La infección por su agente causal, Fusarium circinatum, provoca chancros de resina en los troncos y ramas laterales de los huéspedes maduros, que pueden acabar muriendo por anillamiento o rotura del tronco. En los viveros, los principales síntomas son el puntisecado, marchitamiento y muerte de las plántulas. Sin embargo, el patógeno, de naturaleza hemibiotrófica, puede permanecer endofítico en plántulas de pino sin mostrar síntomas de infección e incluso ser inadvertido en algunas especies herbáceas. Desde el primer registro en 1945 en Norteamérica, se ha notificado la presencia de F. circinatum en 14 países de América, Asia, África y Europa. Varios factores han contribuido a la propagación de la enfermedad a todos estos continentes, siendo el más importante la globalización en lo que respecta al comercio de material vegetal de reproducción. El viento, las gotas de lluvia y los insectos forestales asociados a los pinos contribuyen a la dispersión local del patógeno. A pesar de su importancia, no se dispone de medidas eficaces para erradicar o controlar la enfermedad del PPC ni en los viveros ni en el monte. El objetivo principal de esta tesis doctoral fue esclarecer los mecanismos de regulación eficaces para el control de la enfermedad PPC. Para ello, en primer lugar, se llevó a cabo una recopilación de información actual sobre las vías de propagación del patógeno, proponiendo mecanismos preventivos para evitar su introducción en zonas libres de la enfermedad. Las múltiples vías de propagación hacen de F. circinatum un reto a prevenir, agravado por el reciente descubrimiento de su colonización endofítica de especies no contampladas previamente como hospedantes que ilustra la importancia del conocimiento biológico y ecológico para el diseño de estrategias de intervención eficaces. Además, la erradicación de la enfermedad puede ser factible sólo si se detecta de forma inmediata a su introducción. En este sentido, se necesitan urgentemente nuevos métodos de detección y diagnóstico para la pronta detección de F. circinatum en semillas, plantas e insectos vectores. Para ello, es fundamental la colaboración entre las autoridades fitosanitarias y los investigadores a través de redes interdisciplinarias que permitan aumentar el conocimiento de la enfermedad y sensibilizar a los agentes implicados sobre los riesgos y las medidas de mitigación. En este trabajo también se identificaron los puntos débiles de la normativa actual y se aportaron sugerencias para su aplicación.Departamento de Producción Vegetal y Recursos ForestalesDoctorado en Conservación y Uso Sostenible de Sistemas Forestale

Global Geographic Distribution and Host Range of Fusarium circinatum, the Causal Agent of Pine Pitch Canker

Fusarium circinatum, the causal agent of pine pitch canker (PPC), is currently one of the most important threats of Pinus spp. globally. This pathogen is known in many pine-growing regions, including natural and planted forests, and can affect all life stages of trees, from emerging seedlings to mature trees. Despite the importance of PPC, the global distribution of F. circinatum is poorly documented, and this problem is also true of the hosts within countries that are affected. The aim of this study was to review the global distribution of F. circinatum, with a particular focus on Europe. We considered (1) the current and historical pathogen records, both positive and negative, based on confirmed reports from Europe and globally; (2) the genetic diversity and population structure of the pathogen; (3) the current distribution of PPC in Europe, comparing published models of predicted disease distribution; and (4) host susceptibility by reviewing literature and generating a comprehensive list of known hosts for the fungus. These data were collated from 41 countries and used to compile a specially constructed geo-database. A review of 6297 observation records showed that F. circinatum and the symptoms it causes on conifers occurred in 14 countries, including four in Europe, and is absent in 28 countries. Field observations and experimental data from 138 host species revealed 106 susceptible host species including 85 Pinus species, 6 non-pine tree species and 15 grass and herb species. Our data confirm that susceptibility to F. circinatum varies between different host species, tree ages and environmental characteristics. Knowledge on the geographic distribution, host range and the relative susceptibility of different hosts is essential for disease management, mitigation and containment strategies. The findings reported in this review will support countries that are currently free of F. circinatum in implementing effective procedures and restrictions and prevent further spread of the pathogen