The saprophytic phase

As soon as a beetle vector contaminated with sticky O. ulmi s.l. spores starts to excavate a brood tunnel in a suitable host tree, the fungal conidia can be deposited. In an elm tree previously infected with DED, two distinct sources of inoculum may contribute to the subsequent colonization of elm bark, i.e., beetle-introduced O. ulmi s.l. and xylem-derived O. ulmi s.l. In the latter case, the fungus grows into the cambium through the pits of intact xylem vessels. The outward movement of O. ulmi s.l. from the xylem into the bark tissue represents a fungal transition from the pathogenic to the saprophytic phase, enabling recombination with the beetle-introduced O. ulmi s.l. Most of this recombination probably occurs in midwinter. The strength of this feedback event varies from one season to another (and probably from tree to tree), and appears to be critical for the maintenance of pathogenic fitness. During the pathogenic phase in the xylem, a strong selection in favor of the most pathogenic phenotypes will take place {[371],[548]}.

Within a few days after the onset of tunnel construction, oval brown lesions are found around the maternal galleries of the bark beetle. Initially, the elm bark appears to restrict the fungal attack. However, after a brief period the host tissue starts to decay and the elm phloem gradually dies within 3-4 weeks. At this time, the saprophytic phase has truly begun. Enzymes and toxins released by O. ulmi s.l. may be involved in colonization of the living phloem tissue (see Metabolites of O. ulmi s.l.). In comparison to O. novo-ulmi, O. ulmi is a poor colonizer of elm bark {[548]}.

The fungal lesions around the beetle galleries progressively extend and confluence to form a complex mosaic of O. ulmi s.l. genotypes colonizing the inner elm bark. Webber et al. {[548]} showed that within a small piece of elm bark (11 x 16 cm), 39 different genotypes of the DED fungus can be present. Inevitably, numerous incompatible interactions will occur between the different genotypes during the saprophytic phase. Competition between O. ulmi s.l. individuals results in domination by genotypes that are strongly invasive and which expand their territory at the cost of less invasive O. ulmi s.l. genotypes (penetration effect). Stretches of these dominant genotypes are found over large elongated areas of phloem tissue (lateral spread is hampered by the vertical orientation of the phloem cells). However, the penetration effect favors sexual reproduction. Since the latter event primarily occurs between O. ulmi s.l. genotypes with the greatest dissimilarity, genetic diversity of the fungal population is promoted. Eventually, in addition to the inner bark tissues, even the dead phelloderm arcs of the outer bark are colonized by the pathogen {[593]}.

Lea and Brasier {[540]} reported a distinct succession of O. ulmi fruiting structures within beetle breeding galleries in the bark of diseased elms in Britain. After the beetles have colonized the bark in summer, spaces excavated by female egg-laying beetles and feeding larvae initially become filled with O. ulmi s.l. mycelium bearing Sporothrix stage conidia. Subsequently, synnemata are formed in this high-nutrient environment {[371]}. The Sporothrix conidia and Graphium-type spores predominate in the warm period following beetle colonization. During the long overwintering saprophytic phase, both types of spores can be observed soon after beetles enter the tree to breed {[548]}. Brasier {[513]} showed that incubation of pieces of diseased elm bark in a damp chamber at about 20°C increases the frequency of synnemata and conidia formation. Depletion of nutrients and the onset of winter (November/December) result in a decreased formation of Sporothrix-type and Graphium-type spores. Antagonistic microorganisms and arthropods feed on the degraded mycelium and synnemata {[371]}. Notably, the number of synnematal fruiting bodies tends to decline at this time. In correlation with the fall in temperature, abundant formation of perithecia occurs from November to February {[540]}. In vitro incubation of diseased elm bark at 8-10 °C may induce perithecia formation {[513],[535]}. By the end of winter, the perithecia are destroyed by degradation and predation {[371]}. Since perithecia are also produced during the short saprophytic phase of O. ulmi s.l. in summer, these fruiting structures do not function solely as an overwintering stage of the fungus.

When the temperature starts to rise (March), prior to beetle emergence a resurgence of mycelial Sporothrix conidia can be observed. Therefore, besides a limited number of ascospores, the inoculum carried by the new adult beetles most frequently occurs in the form of conidia. Webber {[371]} examined the production of O. ulmi fruiting bodies in a number of pupal chambers in Britain. In May, 50 % of the 97 pupal chambers contained sporulating O. ulmi (primarily Sporothrix conidia). In June, this percentage had increased to 64%. In this month, the synnematal conidia predominate. By July/August, mycelial conidia, Graphium-type spores, and some ascospores were detected in the pupal chambers.

Although influenced by temperature changes, O. ulmi s.l. fruiting bodies appear to be synthesized in a predetermined order: first the Sporothrix stage, then the synnemata, and finally the perithecia. The timing of this reproductive sequence may depend on the availability of a suitable environment for fructification. The first flush of fruiting bodies found in the beetle galleries is followed slightly later by a series of sporulation in the intersurface between the inner and the outer bark. The pupal chambers provide the DED fungus with a final opportunity to fruit. Recombination events between genetically different O. ulmi s.l. individuals during the first fructification events can contribute to the inoculum acquired by the young adult beetles before their emergence from the elm bark. Eventually, the inoculum of one adult beetle can consist of a mixture of O. ulmi s.l. genotypes {[548]}.

The location of the pupal chamber in the bark is important for the vigor of O. ulmi s.l. sporulation. Pupal chambers in the inner bark – which provide an environment with a high moisture content and a high concentration of nutrients – are preferred by the fungus. In comparison to bark, pupal cells in the sapwood contain fewer nutrients and have a lower moisture content. Formation of O. ulmi s.l. fruiting bodies is likely to be less abundant in the sapwood tissue.