The following chapters discuss the European spruce, the communication between spruce trees through volatile organic compounds (VOCs) and their detection using GC-MS.
The European spruce (Picea abies) is an evergreen, shallow-rooted conifer that can grow to a height of around 50 meters and live for 120 years. It is monoecious, which means male and female cones are found on the same tree. The soft, stable and resin-rich wood can be utilized in many ways [1]. Due to its shallow roots, the spruce has a limited resistance to drought and is susceptible to windthrow, which makes it vulnerable to beetle infestations. When attacked, it usually protects itself by increasing resin flow, however, this mechanism is hindered by a lack of water supply. Heat, drought or heavy rainfall trigger stress in plants, which manifests itself in the emission of volatile organic compounds (VOCs), amongst other symptoms. This is largely due to man-made climate change, but monocultures also weaken the natural balance within the forest and thus its defenses [2]. Some of these VOCs are attractive to pests, as they indicate weakened defense mechanisms [3]. Terpenes, however, are in most cases toxic to predators [4].
Forests can be understood as social communities. In addition to symbioses between plants and fungi, from which both sides derive benefits [5], other connections are formed. It has been shown that trees in a forest community support each other in emergencies by exchanging nutrients, since the entire forest ecosystem benefits from healthy and strong individuals. Healthy trees, especially those at the forest margins, protect the entire population from wind damage [6]. Many tree species live in symbiosis with fungi, which enables them to expand their root network, which in turn helps with water uptake. In return, the fungus receives nutrients from the tree. Trees also exchange signals or nutrients with each other via this extended root system [7]. However, a faster and more effective form of communication takes place via VOCs, i.e. volatile organic hydrocarbons. These are emitted via the leaves or needles and the bark and are used to transmit signals between trees or within individual specimens [8]. Green leaf volatiles, e.g. 𝛼-pinene, 𝛽-pinene, camphene and D-limonene, are mainly emitted via the needles of the spruce [9]. Spruce trees also emit the alcohols ethanol, methanol [10] and hexanol [11] as well as acetone, isoprene, monoterpenes [10] and hexanal [11] via the wood, the bark and again via the needles.
The bark beetle infestations resulting from drought and heat in recent years, along with the crucial role the spruce plays in forestry in Austria, are reasons why this tree species was chosen for this research. The aim of this project is to establish the differences in volatile organic hydrocarbons emitted by healthy and stressed spruce trees, and to determine the stress level based on the VOC composition (fingerprint). Subsequently, a mobile device will be developed to detect stressed individuals at an early stage using chemical and optical signals.
20 spruce trees, 7 to 10 years old, were taken from a forest in the northern Innviertel (Engelhartszell, Austria). The lighting conditions in the laboratory were set to 27 𝜇mol s -1 m -2 (measured with a PAR meter CaTEC type 060501, PAR probe LI-COR Quantum Q49404) and to a day-night rhythm of 12:12 hours using daylight lamps (SYLVANIA, LUXLINE PLUS, F18W, 865). During the initial measurements, the laboratory maintained an average temperature of 25 °C. After an acclimatization period of two weeks, the experiments began. 4 trees each were selected for drought stress and waterlogging, 8 remained as an indoor control group and 4 trees were placed outdoors not far from the laboratory. Stress was induced by either not watering the trees or placing them in a sealed pot and watering excessively, which caused the roots to grow mold. Measurements were taken at regular intervals to determine how prolonged stress affected the VOC emissions of the spruce trees.
A display box made of acrylic glass (40 cm * 40 cm * 110 cm) with hose connections was made, in which the spruces (including pot) were placed 15 minutes before the start of the experiment to adjust the vapor equilibrium of the volatile substances. An activated carbon filter (self-made, DARCO, Mesh 4-12) for adsorbing the VOCs of the room air and a commercially available vacuum diaphragm pump (VWR, type PM204005-86.18) were placed before the display box. Previous experiments had shown that the push/pull method, i.e. with additional purge air, is more suitable than the simpler pull-method for measurements of this type due to the lower CO 2 concentration in the display case and thus higher VOC concentrations [12]. The air was drawn at a flow rate of 6 L min -1 through inert PTFE tubes, through the display box and then through activated carbon sorbent tubes (ORBO32, Supelco, Mesh 60-80). An identical vacuum pump was used for this purpose. The flow rate was regulated with a variable area anemometer and hose clamps. The pot and soil were sealed with PET roasting foil (Toppits® roasting tube, 3 m), which was baked in advance at 120 °C for 2 h [13], to avoid contamination. The sample taking set up is visualized in Figure 1 below.
After the bound VOCs were eluted from the activated carbon by elution with solvent (petroleum ether (40-60 a.r., CHEM-LAB), this eluate was transferred to a GC vial (1,5 ml Rollrandflasche, 32x11,6 mm, Brucker Analysentechnik) [14]. In the gas chromatograph (Shimadzu GC 2010 Plus), 1 𝜇L of each sample was injected in splitless mode with the autosampler (Thermo Scientific, TriPlus RSH) at 200 °C. The temperature gradient of the column (Agilent DB 5 MS 30 m * 0.25 mm * 0.25 𝜇m) started at 40 °C and was held for 5 min. Subsequently, it was first heated to 130 °C at 6 °C min -1 and then to 240 °C at 15 °C min -1 . Helium (He 5.0) was used as the mobile phase with which the eluate was passed over the column at 3 mL min -1 purge flow and a volume flow of 1.51 mL min -1 . The temperature in the transfer line to the mass spectrometer (Shimadzu, GCMS-QP2020) was 200 °C and 240 °C for the ionization source. The detection masses were set from 41 m/z to 300 m/z.
The chromatograms of the stressed spruces were compared to those of healthy spruces to determine which peaks differed in height and area. The detection limit was set at H = 19905. Blank samples were also included to exclude distortions caused by the sampling setting, the adsorbent or the solvent. The values of the areas in TIC (total ion current) of the GC-MS measurements were transferred to Microsoft Excel tables and the percentage distribution was calculated. 18 relevant substances were determined, and the arithmetic mean values of the peak areas were calculated over three measurement rounds with healthy spruce trees and three measurement rounds with stressed spruce trees. The 95 % confidence interval was then calculated according to Formula 1 [15] and a two-sided t-test with unequal variances was carried out to prove that the emission of VOCs during the stress tests differed significantly from the normal state.
𝑥 = arithmetic mean value 1.96 = z-value for the 95 % confidence interval 𝜎 = standard deviation n = number of samples
First, the VOCs emitted by healthy spruce trees in their normal state were measured by adsorption on activated carbon and subsequent elution with petroleum ether using GC-MS. In the next step, the trees were exposed to stress situations such as drought or waterlogging. They were examined to see whether these emitted substances differed in quantity and distribution depending on the level of stress.
18 chemical compounds were identified that are thought to be associated with stress in spruce trees and are therefore relevant to this research. They are listed in table 1.
4 spruce trees were exposed to dry stress or waterlogging by no or excessive water supply and VOC emissions were measured in 3 rounds (n = 24). Mean, standard deviation and standard error were calculated for all substances to determine the 95% confidence intervals and additional t-tests were performed. The results were then compared to those of the same trees during the initial measurements to determine whether individual substances differ in percentage. These differences were presented in 3 diagrams for better understanding.
Figure 2 shows chemical compounds whose percentage share of VOC emissions decreased during the stress tests. The percentages of the substances in figure 3, on the other hand, increased slightly. The largest and most significant differences in terms of VOC emissions can be seen in figure 4. Figure 2 displays the differences in VOC emissions between healthy and stressed spruce trees for 9 of the 18 substances, along with 95% confidence intervals. The values of the standard measurements were set at 1, and the stress results are presented in relation to these standard measurements. The drop in the percentage share cannot be proven with certainty if the values are within the fluctuation range. It was also possible to identify substances whose percentage share decreased.They can be seen in Figure 3 below. The most significant differences were found in the emissions of 1-dodecanol, 2,4-dimethyl-1-heptene, 3 ethyl-3-methylheptane, 4,6-dimethyldodecane and 3,7-dimethyloctan1-ol, all of which increased by several hundred percentage points. This can be seen in Figure 4. It was therefore possible to determine that the values of 5 out of a total of 18 substances deviated significantly from the values of the normal measurements during the stress tests. These were identified as the alcohols 1-dodecanol and 3,7-dimethyloctan1-ol, the alkanes 3 ethyl-3-methylheptane and 4,6-dimethyldodecane and the alkene 2,4-dimethyl-1-heptene.
The measurements took place between 22.05.2023 and 17.07.2023. As the temperatures both indoors and outdoors rose continuously due to the season, the trees reacted accordingly with increased emissions of VOCs. At the beginning of the measurement series, the temperatures were around 20 °C, whereas they approached 30 °C over the course of the experiment. The vapor pressure of VOCs increases with temperature [16], which could be an explanation for the increased emission of volatiles. To truly verify these peaks, comparisons with standards and retention indices are sought in the future. Currently, this set of experiments is being repeated under more controlled conditions. The trees are kept in grow tents, which are isolated, ventilated and illuminated and the temperatures are being kept steady at 25 °C (except for the trees under drought stress). Stress caused by drought and waterlogging can also be determined by measuring the rate of photosynthesis. The aim of this research is to scientifically prove the increased VOC emissions of spruce trees under stress once more. Additional information about the gathered data (e.g. data distributions) will be generated, and a machine learning aspect will be incorporated. The continuation of this research can be found in "Enhancing accuracy and efficiency of a digital nose system with sensor technology for early detection of changes in the forest" by authors Leo Biljesko, Georg Roman Schneider, Claudia Probst.