Investigated species and tree selection
We chose the widespread shrub species Corylus avellana (hazel) and tree species Alnus glutinosa (common alder) and Betula pendula (silver birch), which are characterized by a high allergenicity of their pollen and the availability of common allergens (cross-reactive Bet v1) (Biedermann et al. 2019; Niederberger et al. 1998; Weber 2003). These species are monoecious with typical long catkins containing only male flowers (Filbrandt-Czaja and Adamska 2018). Hazel and alder flower early in January to March, whereas birch flowers later in March to June (only in northern latitudes).
The sampling took place on the specimen in the 3 km surroundings of the campus of the Technical University of Munich at Freising/Weihenstephan (48.400292 N, 11.716874 E). Care was taken to ensure that the selected trees had a minimum distance of 15 m from buildings and varied in age, diameter, and height. From this selection, 3 hazel, 2 alder, and 2 birch tree specimen were chosen. Heights were measured with a relascope, diameters were measured with a tape measure, and ages were estimated. The selected hazels were approximately of the same age (20 years old), height (8 m), crown length (6 m), and diameter (20 cm) and had similar densities to male catkins. The two alder trees were likely of the same age (25 years old), height (15 m), crown length (13 m), and diameter (30 cm) and had about the same number of male catkins. The two birch specimens were of different estimated age (30 and 50 years old), height (15 and 25 m), crown length (13 and 23 m), and diameter (30 and 40 cm).
Harvest and treatments of twigs
The investigation period was from the 5th of November 2018 to the 18th of April 2019. Twigs of the respective species and specimens were harvested in weekly intervals before the actual flowering (Table 1). It was assured that the harvested twigs (length 40 to 50 cm) had the required minimum number of five male inflorescences. The sampling height was 2–3 m above the ground level. After cutting, the twigs were disinfected with 90% ethanol and their basis was recut underwater to avoid disruption of water uptake. The twigs were then stored in glass bottles (capacity 100 ml) which were filled with tap water.
Previous experiments suggested that the usage of different fertilizers as an additive in irrigation may have influence on development of the inflorescences (AL-Kahtani and Ahmed 2012; Maksoud 2000). Therefore, hazel was treated respectively with pure water, tissue fertilizer (B5 medium—100 ml/liter), and plant fertilizer (nitrogen (N) 8%, phosphorus (P2O5) 8%, potassium (K2O) 6%—1 ml/liter) (Table 1). Since the first sampling series on hazel twigs did not show any difference in the development of inflorescences between the fertilizers, twigs of alder and birch were treated with pure water and tissue fertilizer only. Additionally, to each substrate, Chrysal Clear was added as remedy against mold growth.
The twig samples were then stored in the climate chamber which was running under a day-night cycle of 15 and 9 h, with 20 °C and 15 °C air temperature, respectively (Fig. 1). As backup in case of technical failures, a second set of samples was kept under room conditions on a window sill within the university building. Those conditions did not reflect any climate scenario; they were merely used to achieve flowering and pollen shedding.
Comparison of cut and uncut twigs on donor trees
For hazel, alder, and birch, on-tree comparisons between cut and uncut twigs under outdoor conditions were conducted. Single twigs with a minimum of five inflorescences were cut from the donor trees at different time intervals before natural flowering (Table 1) and directly put into plastic water containers (capacity 25 ml) which were afterwards attached vertically with wires to their original position. As a control, on the respective cutting dates, the same number of uncut twigs was selected on the donor trees in the surrounding of the cut twigs. Shortly before flowering, the cut and the selected uncut branches were covered with glassine bags for the in situ collection of pollen. After the pollen emission was completed, the twigs with their glassine bags were cut.
Before separating the pollen from the anthers, the glassine bags had to be dried for approximately 1 week. Since the bags were only made of a material similar to paper, the outdoor in situ pollen collection was heavily impacted by precipitation (rain and snow) as well as by condensed water. The pollen already emitted into the bags got affected by humidity when the bags were soaked through. The inherent problem was that pollen partly burst into small pieces and after drying could not be separated from the paper anymore. To overcome this issue, we tried to cover the glassine bags additionally with plastic bags filled with a desiccant. Unfortunately, after a short time, condensation water had accumulated in the plastic bags causing similar issues as before. Still, part of the samples could be used for further laboratory analysis.
Phenological observations and climatic parameters
During the study period from the 5th of November, 2018, to the 18th of April, 2019, we recorded phenological stages once or twice a week according to the BBCH code (Meier 2001), with a particular focus on the period with sporadically first flowers open (BBCH 60). The BBCH was recorded indoors (climate chamber and window sill) and outdoors on the sampling trees. Temperature and air humidity were continuously measured in 30 min temporal resolution by Hobo Loggers (type U23 Pro v2), always at the height of the samples.
After completion of flowering, the bags were inverted and gently shaken, resulting in accumulation of pollen at the bottom of the bags. After removal of the branches, the catkins/anthers were manually separated from the pollen using tweezers. The remaining pure pollen was finally weighed. For each twig, the average pollen amount per catkin and the average catkin length were determined. The weighted pollen was stored at −20 °C.
The subsequent laboratory analysis comprised the measurement of mean pollen weight per grain, protein content per grain, and allergen content. 5 mg pollen was weighed in, and then 1 ml buffer was added, and 20 μl of this mixture was given onto a slide which was then inserted into a cell counter (Bio-Rad; type TC10) for automatic counting. The average pollen grain weight was derived from the initial weight (5 mg) and the count from the instrument.
Before protein and allergen quantification, the protein had to be extracted from the pollen. For this purpose, 10 mg pollen given in 1 ml buffer was shaken continuously for 3 h. The supernatant was stored at −20 °C. For protein quantification, we tested the BCA (Pierce BCA™ Protein Assay) and Bradford (Bio-Rad Protein Assay Dye Reagent Concentrate) methods in a pre-test with purification of the extract and gel electrophorese. The results of the Bradford test for protein content were in good agreement with other related studies (Ozler et al. 2009; Schäppi et al. 1997), while the results obtained with the BCA method showed much higher values. Therefore, protein quantification as protein content per pollen grain was performed using the Bradford test only. To determine allergen content, the Western blot technique was applied, which uses three different antibodies: human antibody (sera mixture of 29 patients who are allergic to Birch), monoclonal anti-human IgE antibody produced in mouse (Sigma-Aldrich), and rat anti-mouse IgG2b (–HRP conjugated, produced by The Monoclonal Antibody Core Facility at the Helmholtz Center ). After the application of all three antibodies, the allergens were detected in the last step under chemiluminescence (ECL™ select Western blotting detection reagent) (Fig. 2). Among the allergens visualized, the allergen Bet v1 showed the clearest signal as it is highly abundant. Bet v1 is cross-reactive in hazel, alder, and birch and can therefore be detected in all three species. For the calculation of Bet v1 allergen content, the signal intensity of chemiluminescence was extracted using the Fiji package from ImageJ (Rueden et al. 2017; Schindelin et al. 2012) and the protein content was determined via Ponceau S staining during Western blotting. The allergen content was then calculated by dividing signal intensity and protein content, thus it is a relative value without unit. All laboratory analyses were conducted in cooperation with the Helmholtz Center.
The statistical analysis was conducted in RStudio (version 3.5.1/2018-07-02). The per treatment distributions of the parameters average catkin length (cm), average pollen weight per catkin (mg), pollen weight per grain (ng), protein content per grain (ng), and allergen content (unitless) were tested by the Shapiro-Wilk test for normality. Since the parameters were not normally distributed (p-value of Shapiro-Wilk test < 0.05), non-parametric tests were further used: Student’s t-test for comparisons between two groups, the univariate ANOVA (Kruskal-Wallis test) for comparisons with more than two groups, and subsequently using pair-wise Wilcoxon tests for individual group comparison. p-values less than 0.05 were considered statistically significant. Due to the limited number of samples per treatment which could be analyzed for the pollen weight per grain (ng) and allergen content, no statistical test could be performed and thus, only differences are shown in the results.