DIGITALNA ARHIVA ŠUMARSKOG LISTA
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| ŠUMARSKI LIST 7-8/2019 str. 35 <-- 35 --> PDF |
of meridic wheat germ diet that had been poured into 250 ml plastic cups. L. dispar were obtained from egg masses provided by the USDA-APHIS Otis Method Development Center, Massachusetts, USA, and reared on meridic wheat germ diet in 250-ml plastic cups at 24°C, 16h light/8 h dark. Twenty larvae in 2nd day of 3rd instar were transferred into each of the diet cups treated with microsporidia and were reared for 20 days. At the end of the incubation period, all larvae were dissected individually and inspected under light microscope for the presence of microsporidia to confirm infection. c) Individual infections – Individualne infekcije The spore viability in liquid nitrogen was studied in more detail for Nosema portugal and Nosema sp. (Ebergassing); spores of these isolates had been stored in liquid nitrogen for 19 years. Spores were prepared as described above and adjusted to three different concentrations – 100 sp/µl, 1000 sp/µl and 10 000 sp/µl. Blocks of the wheat germ diet cut to 4 mm3 were placed individually into the wells of tissue cultures 24 wells plates and 1 µl of spore suspension was applied to the surface of each block. Second day third instar larvae that starved for 24 h before infection were placed individually into each well. Each larva that consumed the whole diet block within 24 h was used in the experiments. Controls were treated in the same manner; however, the diet blocks were inoculated with distilled water. Totally 336 L. dispar larvae were used in this experiment, 48 larvae in each treatment. Larvae were reared individually in 30 ml diet cups at 24°C until 25th day post infection. At the end of the incubation period, all larvae were dissected individually and inspected under light microscope for the presence of microsporidia to confirm infection. Results REZULTATI a) Surface contamination experiment – Test kontaminacije hranjivog supstrata From the 8 microsporidian isolates used for surface contamination, only 4 isolates - Vairimorpha disparis, Nosema lymantriae, Nosema sp. (Ebergassing), and Nosema sp. (Poland) - produced infections in the challenged L. dispar larvae (Table 1). While all test larvae (100%) were infected with the first three mentioned isolates, only 21.1% of the test larvae exposed to spores of Nosema sp. (Poland) were infected. At the end of the incubation period, all dissected larvae infected with the four isolates showed signs of a heavy infection; tissues were filled with environmental spores. b) Individual infection – Individualne infekcije Microscopic evaluations of the L. dispar larvae individually infected with Nosema portugal using three different dosages showed that the spores of N. portugal had lost infectivity after storage in liquid nitrogen for 18.75 years; nonе of the tested larvae was infected. In contrast, spores of Nosema sp. (Ebergassing) had retained infectivity after 18.75 years of |