DIGITALNA ARHIVA ŠUMARSKOG LISTA
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necessary for nursery production of seedlings, majority of which are further used for forest stand regeneration or afforestation. For this purpose seeds are collected from adult trees in existing natural stands selected and registered as seed sources, forest stands which are phenotypically above average and specially managed for the purpose of seed collection and thus registered as seed stands and seed orchards established also for the purpose of seed collecting, from the genetically superior individual trees (Anon 2009, 2011, 2013, 2014). Fungal presence in the seeds of forest tree species in general is considered to be a significant cause of shortened seed longevity during storage (Sutherland et al. 2002), reduced seed germination due to embryo or endosperm deterioration and potential cause of diseases that affect other developmental stages of plants, such as increased damping-off, shoot dieback, cankers and dieback of older seedlings (Cram 2009), although number of species just act as endophytes or saprotrophs and do not adversely affect the performance of seeds sown in nurseries (Mittal and Wang 1987). In Croatian narrow-leaved ash forest stands there was a recorded case of seedlings delivered from a nursery Zalužje, Forestry Office (FO) Vinkovci, which expressed symptoms of Hymenoscyphus fraxineus dieback approximately one month after being planted in the field, FO Vinkovci, Management Unit (MU) Vrbanske šume, Subcompartment (SC) 91b, although the pathogen wasn’t confirmed on older ash trees sampled in the area nearby SC 132a (MU Vrbanske šume) and SC 49a (MU Kusare) (FO Vinkovci) (Anon 2015). This finding raised a question of infection origin and possibility that pathogen spread from seeds into the plant tissue, eventually causing visible dieback symptoms in grown seedlings. The objective of this research was to screen narrow-leaved ash seeds for the presence of pathogenic fungus Hymenoscyphus fraxineus and simultaneously detect other possible seed-borne pathogens in order to estimate the health status and suitability of seeds collected from registered seed sources and seed stands for further nursery seedling production. MATERIALS AND METHODS MATERIJALI I METODE Fraxinus angustifolia seeds were collected in period from August to November 2017 from visually healthy trees in four natural forest stands registered as narrow-leaved ash seed sources and one registered narrow-leaved ash seed stand (Table 1). Seeds were examined for fungal presence after one to two months of storage at room temperature, using both classical method of mycelia isolation on artificial media and a nested PCR method to analyse DNA directly from seeds. Seeds were additionally screened for presence of pathogenic fungus Hymenoscyphus fraxineus using species specific primers (Johansson et al. 2010). Isolation of fungi from seeds – Izolacija gljiva iz sjemena Twelve seeds from each of five locations were used for fungal isolation on malt extract agar medium (MEA, Oxoid, Basingstoke, UK) supplemented with streptomycin sulphate (200 mg l-1, Sigma-Aldrich, St. Louis, USA). Seeds were surface sterilized in a solution of sodium hypochlorite (approx. 4% active chlorine) for one minute and then rinsed three times in sterile distilled water. Seeds cut in half were plated on medium in Petri dishes (9 cm diameter) and incubated in dark at 20 °C for four weeks (Bulovec 2018). Petri dishes were checked weekly for fungal growth and emerging mycelia were subcultured to MEA medium. Pure cultures were grouped into morphotypes and at least one isolate of each morphotype group was used for molecular identification. Extraction of DNA was performed according to Allemann et al. (1999) with modifications (Kranjec et al. 2017) and PCR amplification was conducted with primers ITS 1 and ITS 4 (White et al. 1990) in 25 µl reactions containing 200 µM deoxyribonucleoside triphosphates, 0.4 µM of each primer, 0.5 U of Taq DNA polymerase with reaction buffer (Sigma-Aldrich, St. Louis, USA), 1.5 mM MgCl2 and 1 µl of 100-fold diluted DNA template. Cycling conditions were as follows: an initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 45 s, extension at 72 °C for 90 s and a final |