Summary Experiments were conducted to test whether Fourier transform‐infrared (FT‐IR) spectroscopy, coupled with chemometric methods, can distinguish healthy xylem tissues collected from elms known to differ significantly in their susceptibility to Ophiostoma novo‐ulmi Brasier. Twig samples from resistant Ulmus pumila L., susceptible U. minor Mill. and resistant U. minor clones were harvested on 1 May, 15 May, 1 June, 1 July and 1 September 2004, and subjected to FT‐IR analysis. The application of principal component analysis to the spectral data, followed by discriminant function analysis, discriminated between the three groups of samples in all harvesting dates. The examination of the DF‐loading plots allowed the identification of key regions within the spectra for the separation between clusters. The chemical assignments of these key regions allowed the following interpretations to be made: (i) U. pumila tissues contained enhanced levels of starch, cellulose and lignin with respect to U. minor tissues and (ii) resistant U. minor tissues contained enhanced levels of starch, cellulose and pectic polysaccharides with respect to susceptible U. minor tissues. The possible roles of the compositional differences in disease resistance, as well as the potential use of FT‐IR spectroscopy and chemometrics as a tool for screening resistant elms are discussed.

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ResumenSe presentan, revisan y discuten los resultados de una serie de trabajos encaminados al estudio de una olmeda situada en el término municipal de Rivas-Vaciamadrid (Comunidad de Madrid). Desde el año 2000, se han analizado la historia, estado sanitario, taxonomía, dendrocronología, y características reproductivas y genéticas de los olmos que la componen. Esta olmeda puede tener un origen parcialmente antrópico, dada la coexistencia en ella de Ulmus minor s.l., de origen ibérico, con Ulmus minor var. vulgaris, de origen italiano. Existen dos categorías reproductivas en la olmeda, con pies cosexuales y pies funcionalmente masculinos, siendo más frecuentes estos últimos, aunque con una variabilidad genética menor. Desde el punto de vista sanitario, se observa un lento pero progresivo decaimiento de la olmeda a lo largo de la segunda mitad del siglo XX, probablemente como consecuencia de la grafiosis. Se proponen una serie de medidas para su mejor conservación.Palabras clave: conservación, historia forestal, biología reproductiva, taxonomía, sanidad forestal, Ulmus minor, Ophiostoma novo-ulmi. Abstract Historical, ecological, taxonomic and health characterization of the relict elm stand of Rivas-Vaciamadrid (Madrid)A series of studies focused on the characterization of an elm stand located at Rivas-Vaciamadrid (Comunidad de Madrid, Central Spain) are presented, revised and discussed. Since 2000, the elm stand history, its health, taxonomy, dendrochronology, and the reproductive and genetic characteristics of the elms have been analysed. The elm stand could have a partially anthropic origin, as indicated by the coexistence of Ulmus minor s.l. of Iberian origin with Ulmus minor var. vulgaris of Italian origin. The presence of cosexual and functionally-male trees indicates the existence of a gender differentiation. Functionally-male trees are numerically more frequent, but they have less genetic variability than the cosexual trees. A low but progressive decline during the second half of 20 th century has been observed, probably as a consequence of Dutch elm disease. In order to help towards the elm stand conservation, some actions are proposed.

One century after the first report of Dutch Elm Disease (DED), there is still no practical solution for this problem threatening European and American elms (Ulmus spp.). The long breeding cycles needed to select resistant genotypes and the lack of efficient treatments keep disease incidence at high levels. In the present work, the expression of defense-related genes to the causal agent of DED, Ophiostoma novo-ulmi, were analyzed in in vitro clonal plantlets from two DED-resistant and two DED-susceptible U. minor trees. In addition, the effect of the inoculation of an endophytic pink-pigmented yeast (Cystobasidium sp.) on the plant’s defense system was tested both individually and in combination with O. novo-ulmi. The multifactorial nature of the resistance to DED was confirmed, as no common molecular response was found in the two resistant genotypes. However, the in vitro experimental system allowed to discriminate the susceptible from the resistant genotypes, showing higher levels of oxidative damage and phenolic compounds in the susceptible genotypes after pathogen inoculation. Inoculation of the endophyte before O. novo-ulmi attenuated the plant molecular response induced by the pathogen and moderated oxidative stress levels. Niche competition, endophyte-pathogen antagonism, and molecular crosstalk between the host and the endophyte are discussed as possible mechanisms of stress reduction. In sum, our results confirm the multifactorial nature of DED resistance mechanisms and highlight the possibility of using certain endophytic yeasts as biological tools to improve tree resilience against biotic stress.

Certain fungal endophytes are known to improve plant resistance to biotic stresses in forest trees. In this study, three stem fungal endophytes belonging to classes Cystobasidiomycetes, Eurotiomycetes and Dothideomycetes were selected among 210 isolates for their potential as enhancers of Ulmus minor resistance to Ophiostoma novo-ulmi. We evaluated phenotypic traits of these endophytes that could be beneficial for O. novo-ulmi inhibition in the host plant. Under in vitro conditions, the Dothideomycetous isolate YCB36 strongly inhibited O. novo-ulmi growth, released antipathogen VOCs, chitinases and siderophores, and overlapped with the pathogen in nutrient utilization patterns. These functional traits could explain the 40% reduction in leaf wilting due to O. novo-ulmi in elm trees pre-inoculated with this endophyte. Ulmus minor trees responded to endophyte inoculation with increased stomatal conductance and higher amounts of flavonoids and total phenolic compounds in xylem tissues, suggesting induction of defence metabolism.