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ŠUMARSKI LIST 11-12/2020 str. 45     <-- 45 -->        PDF

Materials and Methods
Materijali i metode
Acorns of two families of pedunculate oak (Quercus robur L.) were collected from two single trees (Nos. 2 and 7) in a seed stand located at the territory of Public Enterprise Vojvodinašume; Forest Estate Sremska Mitrovica; Forest Management Unit Morović; during autumn 2017 and stored in a cooling chamber until March, when seeds were sown in 0.5-liter pots with soil medium consisting of a peat:sand:soil mixture of equal volumes (1:1:1). Prior to sowing, seeds were soaked in water for 24 hours in order to enhance germination. Plants were grown in the laboratory under constant light conditions until the beginning of May and then placed outside in insect cages where they were exposed to treatments. Before the start of the treatments, plants developed 5 to 10 leaves with height ranging from 15 to 35 cm.
The treatments included drought (D), damage caused by gypsy moth (Lymanthria dispar L.) (GM), and a combination of both stress factors (D+GM). Drought treatment (D) was preceded by a 15-day consecutive decrease of soil moisture to reach value of 30% of field capacity prior to the beginning of the treatment. Treatment with gypsy moth (GM) included 3 pieces of instar 2 caterpillars which were added to the plants in each pot. Both stress factors (D+GM) included addition of caterpillars to the drought stressed plants, while control treatment used well-watered plants without damages caused by the insects. Treatment lasted for 15 days followed by a 7-day recovery period. During recovery period, drought treated plants were irrigated to obtain optimal field capacity between 70 and 90%, while caterpillars were removed from the affected plants. Measurements of physiological factors were performed at the end of the 15-day treatment period and repeated after the 7-day recovery period. Measured parameters included gas exchange, pigments content and nitrate reductase activity (NRA). Each family had 10 pots per treatment from which four plants were selected for gas exchange measurements. Gas exchange measurements were made with a portable photosynthesis system (LCPro+, ADC Bioscientific, UK) under controlled constant light conditions of 1000 μmol m-2 s-1 and constant ambient air supply of 100 μmol s-1, with ambient levels of humidity and temperature. Measurements were performed on a fully developed leaf from the first set of leaves. For the gas exchanged measurement on insect damaged plants, selected leaves had a minimum of 50% damage with sufficient leaf blade area remaining for gas exchange measurements. Instantaneous water use efficiency (WUE [μmol mmol-1]) was computed as the ratio of net photosynthesis to transpiration (A/E) (Farquhar et al., 1989). Chlorophyll content was determined with the use of a portable chlorophyll meter (Minolta SPAD-502, Tokyo, Japan) on the same leaves where gas exchange measurements were performed. The SPAD values were converted to chlorophyll content (μg cm-2) according to Cerovic et al. (2012): Chl = (99 × SPAD value) (144 - SPAD value)-1. The in vivo NRA in leaves was assayed using the spectrophotometrical method of Hageman and Reed (1980) and expressed as µmol NO2- g-1FW h-1.
All statistical analyses were performed by STATISTICA software, version 13 (TIBCO Software Inc, 2017). Nested analysis of variance (ANOVA) was computed for each trait in order to evaluate relative importance of the following sources of phenotypic variation: date of measurement (the difference between performances of stressed and recovered seedlings), treatment nested within date (treatment effect during stress and recovery), family nested within date*treatment (genetic variation for seedlings response to stress and recovery). Significant differences were determined at p ≤ 0.05.
Response of physiological parameters to stress
Induced stress disturbed nitrogen metabolism in both investigated oak families. However, significant decrease of NRA was recorded in family 2, especially during drought treatment. During treatment, in family 2 values decreased by 55.1, 59.2 and 80.5% in plants exposed to GM, D+GM and D, respectively (Chart 1), compared to control. On the other hand, family 7 did not show significant decreases in NRA in all tested treatments although there were even increases in enzymatic activity in treatments D and GM. Net photosynthesis (A) decreased in both families (Chart 1). Significant decrease of A ranging from 42.9 to 61.2% compared to controls was recorded in both families in all treatments. Even though GM treatment had lowest values in both families, it did not significantly decrease compared to other stress treatments. Sub-stomatal concentration of CO2 (Ci) showed significant increases in GM- and D+GM-treated plants from family 2 (295.4 and 281.1 µmol µmol-1, respectively) and GM and D treated plants from family 7 (340.7 and 315.9 µmol µmol-1, respectively) (Chart 1). Gypsy moth attack decreased transpiration rate of both oak families during the treatment period (Chart 1), regardless if it was sole treatment (GM) or in combination with drought (D+GM). However, the decrease of stomatal conductance of family 2 D+GM plants was not significant (0.092 mol m-2 s-1). Within families, the change in WUE values (Chart 1) was not significant between the treatments, although decreases were recorded in plants of family 7 under separate effects of gypsy moth and drought (34.7% and