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

Fluctuación estacional del inóculo de Hymenoscyphus fraxineus en la República Checa: (Seasonal fluctuations of airborne inoculum of Hymenoscyphus fraxineus in Czech Republic)

El decaimiento del fresno causado por el hongo ascomicete Hymenoscyphus fraxineus (anamorfo: Chalara fraxinea) es considerado una enfermedad muy seria surgida durante los últimos 20 años. Polonia fue el primer lugar donde se observó y se ha extendido rápidamente por la mayor parte del área de distribución de Fraxinus excelsior en Europa. Hymenoscyphus fraxineus es un patógeno invasivo y letal procedente del este de Asia que ataca brotes, hojas y, después, ramas, troncos y raíces de fresnos de todas las edades. Después vive como saprófito en peciolos caídos. El único medio de propagación e infección parece ser sus ascosporas, que son producidas por apotecios que se forman en la hojarasca principalmente en verano. La información de la abundancia de ascosporas es importante para llegar a la epidemiología de las enfermedades de plantas, y por tanto, encontrar adecuados métodos de control los cuales pueden ser útiles para el manejo de la enfermedad. Los objetivos del estudio fueron (i) describir la fluctuación estacional del inóculo de H. fraxineus en un bosque infectado en el sureste de la República Checa y (ii) determinar la influencia de las condiciones meteorológicas en su biología. Para ello una trampa automática de esporas volumétrica de 7 días fue utilizada junto a una estación meteorológica. El período de estudio fue desde mediados de mayo hasta finales de octubre del año 2015. El DNA fue extraído de las muestras de la trampa de esporas y, posteriormente, fue cuantificado mediante una PCR a tiempo real. El análisis de la qPCR se llevó a cabo mediante una sonda fluorogénica TaqMan probe, TaqMan Universal PCR master mix y primers específicos. Los resultados muestran ocurrencia del patógeno durante todo el período de muestreo, con los mayores niveles de copias de ADN en agosto. La variable de humedad relativa mínima del aire mostró influencia significativa en la cantidad de ADN de H. fraxineus. Esta correlación resultó ser inversa, esto podría ser por la elevada humedad relativa que presentó la zona, pudendo haber inhibido de algún moda la liberación de ascosporas.Máster en Ingeniería de Monte

Genome-wide identification and characterization of Fusarium circinatum-responsive lncRNAs in Pinus radiata

[EN] Background: One of the most promising strategies of Pine Pitch Canker (PPC) management is the use of reproductive plant material resistant to the disease. Understanding the complexity of plant transcriptome that underlies the defence to the causal agent Fusarium circinatum, would greatly facilitate the development of an accurate breeding program. Long non-coding RNAs (lncRNAs) are emerging as important transcriptional regulators under biotic stresses in plants. However, to date, characterization of lncRNAs in conifer trees has not been reported. In this study, transcriptomic identification of lncRNAs was carried out using strand-specific paired-end RNA sequencing, from Pinus radiata samples inoculated with F. circinatum at an early stage of infection. Results: Overall, 13,312 lncRNAs were predicted through a bioinformatics approach, including long intergenic non-coding RNAs (92.3%), antisense lncRNAs (3.3%) and intronic lncRNAs (2.9%). Compared with protein-coding RNAs, pine lncRNAs are shorter, have lower expression, lower GC content and harbour fewer and shorter exons. A total of 164 differentially expressed (DE) lncRNAs were identified in response to F. circinatum infection in the inoculated versus mock-inoculated P. radiata seedlings. The predicted cis-regulated target genes of these pathogen-responsive lncRNAs were related to defence mechanisms such as kinase activity, phytohormone regulation, and cell wall reinforcement. Co-expression network analysis of DE lncRNAs, DE protein-coding RNAs and lncRNA target genes also indicated a potential network regulating pectinesterase activity and cell wall remodelling. Conclusions: This study presents the first comprehensive genome-wide analysis of P. radiata lncRNAs and provides the basis for future functional characterizations of lncRNAs in relation to pine defence responses against F. circinatumSIThis work was supported by project PID2019-110459RB-I00 funded by MICINN (Spain) as well as the project VA208P20 funded by JCYL (Spain), both co-financed by FEDER (EU) budget. University of Valladolid supported CZ-B (Rector’s Resolution of November 11, 2016

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

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

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

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

Funding: This study was financially supported by COST Action FP1406 (PINESTRENGTH), the Estonian Science Foundation grant PSG136, the Forestry Commission, United Kingdom, the Phytophthora Research Centre Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000453, a project co-financed by the European Regional Development Fund. ANSES is supported by a grant managed by the French National Research Agency (ANR) as part of the “Investissements d’Avenir” programme (ANR-11-LABX-0002-01, Laboratory of ExcellenceARBRE). SW was partly supported by BBSRC Grant reference BB/L012251/1 “Promoting resilience of UK tree species to novel pests & pathogens: ecological & evolutionary solutions (PROTREE)” jointly funded by BBSRC, Defra, ESRC, the Forestry Commission, NERC and the Scottish Government, under the Tree Health and Plant Biosecurity Initiative. Annual surveys in Switzerland were financially supported by the Swiss Federal Office for the Environment FOEN. Acknowledgments: Andrea Kunova and Cristina Pizzatti are acknowledged for the assistance in the sampling. Thanks are due to Dina Ribeiro and Helena Marques from ICNF-Portuguese Forest Authority for providing location coordinates. We thank three anonymous reviwers for valuable corrections and suggestions.Peer reviewedPublisher PD

The Fourteenth Data Release of the Sloan Digital Sky Survey: First Spectroscopic Data from the extended Baryon Oscillation Spectroscopic Survey and from the second phase of the Apache Point Observatory Galactic Evolution Experiment

The fourth generation of the Sloan Digital Sky Survey (SDSS-IV) has been in operation since July 2014. This paper describes the second data release from this phase, and the fourteenth from SDSS overall (making this, Data Release Fourteen or DR14). This release makes public data taken by SDSS-IV in its first two years of operation (July 2014-2016). Like all previous SDSS releases, DR14 is cumulative, including the most recent reductions and calibrations of all data taken by SDSS since the first phase began operations in 2000. New in DR14 is the first public release of data from the extended Baryon Oscillation Spectroscopic Survey (eBOSS); the first data from the second phase of the Apache Point Observatory (APO) Galactic Evolution Experiment (APOGEE-2), including stellar parameter estimates from an innovative data driven machine learning algorithm known as "The Cannon"; and almost twice as many data cubes from the Mapping Nearby Galaxies at APO (MaNGA) survey as were in the previous release (N = 2812 in total). This paper describes the location and format of the publicly available data from SDSS-IV surveys. We provide references to the important technical papers describing how these data have been taken (both targeting and observation details) and processed for scientific use. The SDSS website (www.sdss.org) has been updated for this release, and provides links to data downloads, as well as tutorials and examples of data use. SDSS-IV is planning to continue to collect astronomical data until 2020, and will be followed by SDSS-V.Comment: SDSS-IV collaboration alphabetical author data release paper. DR14 happened on 31st July 2017. 19 pages, 5 figures. Accepted by ApJS on 28th Nov 2017 (this is the "post-print" and "post-proofs" version; minor corrections only from v1, and most of errors found in proofs corrected

The 13th Data Release of the Sloan Digital Sky Survey: First Spectroscopic Data from the SDSS-IV Survey Mapping Nearby Galaxies at Apache Point Observatory

The fourth generation of the Sloan Digital Sky Survey (SDSS-IV) began observations in July 2014. It pursues three core programs: APOGEE-2,MaNGA, and eBOSS. In addition, eBOSS contains two major subprograms: TDSS and SPIDERS. This paper describes the first data release from SDSS-IV, Data Release 13 (DR13), which contains new data, reanalysis of existing data sets and, like all SDSS data releases, is inclusive of previously released data. DR13 makes publicly available 1390 spatially resolved integral field unit observations of nearby galaxies from MaNGA,the first data released from this survey. It includes new observations from eBOSS, completing SEQUELS. In addition to targeting galaxies and quasars, SEQUELS also targeted variability-selected objects from TDSS and X-ray selected objects from SPIDERS. DR13 includes new reductions ofthe SDSS-III BOSS data, improving the spectrophotometric calibration and redshift classification. DR13 releases new reductions of the APOGEE-1data from SDSS-III, with abundances of elements not previously included and improved stellar parameters for dwarf stars and cooler stars. For the SDSS imaging data, DR13 provides new, more robust and precise photometric calibrations. Several value-added catalogs are being released in tandem with DR13, in particular target catalogs relevant for eBOSS, TDSS, and SPIDERS, and an updated red-clump catalog for APOGEE.This paper describes the location and format of the data now publicly available, as well as providing references to the important technical papers that describe the targeting, observing, and data reduction. The SDSS website, http://www.sdss.org, provides links to the data, tutorials and examples of data access, and extensive documentation of the reduction and analysis procedures. DR13 is the first of a scheduled set that will contain new data and analyses from the planned ~6-year operations of SDSS-IV.PostprintPeer reviewe

Sloan Digital Sky Survey IV: mapping the Milky Way, nearby galaxies, and the distant universe

We describe the Sloan Digital Sky Survey IV (SDSS-IV), a project encompassing three major spectroscopic programs. The Apache Point Observatory Galactic Evolution Experiment 2 (APOGEE-2) is observing hundreds of thousands of Milky Way stars at high resolution and high signal-to-noise ratios in the near-infrared. The Mapping Nearby Galaxies at Apache Point Observatory (MaNGA) survey is obtaining spatially resolved spectroscopy for thousands of nearby galaxies (median ). The extended Baryon Oscillation Spectroscopic Survey (eBOSS) is mapping the galaxy, quasar, and neutral gas distributions between and 3.5 to constrain cosmology using baryon acoustic oscillations, redshift space distortions, and the shape of the power spectrum. Within eBOSS, we are conducting two major subprograms: the SPectroscopic IDentification of eROSITA Sources (SPIDERS), investigating X-ray AGNs and galaxies in X-ray clusters, and the Time Domain Spectroscopic Survey (TDSS), obtaining spectra of variable sources. All programs use the 2.5 m Sloan Foundation Telescope at the Apache Point Observatory; observations there began in Summer 2014. APOGEE-2 also operates a second near-infrared spectrograph at the 2.5 m du Pont Telescope at Las Campanas Observatory, with observations beginning in early 2017. Observations at both facilities are scheduled to continue through 2020. In keeping with previous SDSS policy, SDSS-IV provides regularly scheduled public data releases; the first one, Data Release 13, was made available in 2016 July