Brown spot needle blight (BSNB), a disease of pine trees caused by the fungus Lecanosticta acicola, has been known in Slovenia since 2008 and in Croatia since 1975. Recent outbreaks in Slovenia prompted this study to compare L. acicola populations in these two neighbouring European countries. Sixty-nine isolates collected from three pine species (Pinus mugo, P. halepensis and P. nigra) were used to determine the phylogenetic relationships, genetic structure, and reproductive strategy of the pathogen. EF1-a sequences showed that Slovenian and Croatian isolates share a common ancestry with individuals from central and northern Europe. Population structure analysis revealed four distinct population clusters of L. acicola in these two countries, generally corresponding to their respective geographic location and host. An unequal ratio of mating types and a low overall genetic diversity in the population indicated a strong influence of asexual reproduction. Although some of the oldest recorded European occurrences of BSNB are from Croatia, this study provided no evidence that the population studied in Croatia was the source of the sampled outbreaks in Slovenia. Recent outbreaks of L. acicola in Slovenia are most likely due to introductions from other, yet to be identified, sources.

Abstract:The research on incidence of mistletoe (Viscum album ssp. abietis) on silver fir (Abies alba) was carried out in natural fir stands in Croatia. In the area of Gorski Kotar 32.8 % of the examined dominant fir trees were infected with mistletoe. The mistletoe incidence was presented according to the damage degrees of silver fir from 0 (healthy trees) to 4 (dead trees), rated by the crown defoliation and needle discoloration. With the increase of incidence, mistletoe spreads more onto more vital, i.e. less damaged trees. In the Dinaric Alps 28.6% of the examined fir trees were infected, this percentage amounting to 27.1% for the mountainous regions between the Sava and Drava rivers. The site and stand parameters (exposure, elevation, site quality, forest community and crown closure) were analysed in order to establish whether there was a correlation between these parameters and mistletoe incidence. There was a negative correlation between the elevation and mistletoe incidence. Among the compartments with a closed stands there were considerably less compartments with higher mistletoe incidence than among compartments with a sparsely closed and understocked stands. The other analysed site and stand parameters individually had no significant influence on mistletoe incidence. The correlation of silver fir mortality in 2004 and mistletoe incidence in 2002/03 was analysed, and their strong correlation was established. Mistletoe could be considered as a bioindicator of silver fir decline, and probably a significant contributor to that decline. In the areas where mistletoe incidence is great it can be presumed that silver fir is significantly damaged.

When natural regeneration of Quercus robur stands is hampered by an insufficient acorn yield, human assisted sowing of acorns collected in non-affected stands and stored for some period of time is performed. To inhibit the development of fungi and acorn deterioration during storage, thermotherapy is usually applied by submerging acorns for 2.5 h in water heated to 41 °C. This research aimed to test the effect of four thermotherapy treatments of different durations and/or applied temperatures as well as short-term storage at −1 °C or 3 °C on acorn internal mycobiota and germination. Fungal presence in cotyledons was analyzed in 450 acorns by isolation of mycelia on artificial media, followed by a DNA-based identification. Germination of 2000 acorns was monitored in an open field trial. Thermotherapy significantly decreased fungal diversity, while storage at 3 °C increased the isolation frequency of several fungi, mainly Penicillium spp. The most frequently isolated fungi did not show a negative impact on acorn germination after short-term storage. The study confirmed the efficiency of thermotherapy in the eradication of a part of acorn internal mycobiota, but also its effect on the proliferation of fast-colonizing fungi during storage. However, the latter showed to be more stimulated by storage conditions, specifically by storage at 3 °C.

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Sequoiadendron giganteum Lindl. [Buchholz] is a long-lived tree species endemic to the Sierra Nevada Mountains in California. Due to its massive size and beauty, S. giganteum is a popular ornamental tree planted in many parts of the world, including Europe. Since 2017, scattered branch die-back has been observed on S. giganteum trees in Zagreb, Croatia. Other symptoms included resinous branch cankers, reddish-brown discoloration of the sapwood and, in severe cases, crown die-back. Branches showing symptoms of die-back and cankers were collected from six S. giganteum trees in Zagreb and the aim of this study was to identify the causal agent of the disease. The constantly isolated fungi were identified using morphology and phylogenetic analyses based on the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA), and partial sequencing of two housekeeping genes, i.e., translation elongation factor 1-α (TEF 1-α), and β tubulin 2 (TUB2). The fungi were identified as Botryosphaeria dothidea (Moug.) Ces. and De Not. and Neofusicoccum yunnanense G.Q. Li & S.F. Chen. The pathogenicity test was conducted in a plant growth chamber on S. giganteum seedlings and revealed that N. yunnanense was more aggressive compared to B. dothidea. N. yunnanense was able to reproduce symptoms of canker and die-back and kill plants seven weeks after inoculation whereas B. dothidea produced cankers. To the best of our knowledge, this is the first report of B. dothidea and N. yunnanense causing canker and die-back disease of S. giganteum in Croatia. It is also the first record on the identity and pathogenicity of any fungal species associated with S. giganteum in this country. The study expended the known host range of N. yunnanense to include S. giganteum, which is a valuable ornamental tree in Croatian landscapes. Disease management strategies should be developed to mitigate or reduce the impact of the disease.

Large-scale ash (Fraxinus spp.) dieback caused by the fungus Hymenoscyphus fraxineus has been a major concern throughout Europe for more than two decades. Most of the related research has been focused on Fraxinus excelsior L., and there is still little information on fungal involvement in the dieback of Fraxinus angustifolia Vahl, especially in roots and stem bases, which play an important role in decline progress and tree stability. The objectives of this study were to identify fungi present in visually healthy and symptomatic wood tissues in basal parts of narrow-leaved ash trees in different decline phases, in order to determine the possible role of these fungi and their importance in the dieback process. The stem bases and roots of 90 trees in three different health categories, determined based on crown defoliation, were sampled in natural stands affected by ash dieback. Isolated fungal cultures were identified based on the rDNA ITS (Internal transcribed spacer) region and their association with tree health status was analyzed. In total, 68 different fungal taxa were confirmed, including Hymenoscyphus fraxineus and Armillaria spp., which were mainly present in roots, although overall in lower frequencies than on common ash in other studies. Most frequently isolated fungal taxa, which encompassed 51% of all obtained isolates, were Trichoderma spp., Ilyonectria robusta, Fusarium solani, Cladosporium cladosporioides, and Diaporthe cotoneastri. Their associations with tree health categories and presence in both symptomatic and visually healthy wood indicate that they act as opportunistic pathogens and early colonizers of weakened ash tissues. Research also revealed that, although the extent of crown defoliation and presence of root and stem necroses were associated, basal symptoms occurred on a number of trees with healthy looking crowns, meaning they can develop independently.