EDUCATIONAL NETWORK ON SOIL AND PLANT ECOLOGY AND MANAGEMENT (EduSapMan) Summer School Soil & Water 2017 Practical exercises Content: 1. Stomatal kinetics in response to CO2 and its relation to stomatal size and density 2 2. Soil compaction and oxygen in soil 12 3. Soil zoology 17 4. Land properties influenced by land use and fertilization 25 5. Fast Plant test with various substrates and composts 49 6. Allelopathy experiment with Estonian trees 60 1 1. Stomatal kinetics in response to CO2 and its relation to stomatal size and density Markus Hügel, Kateřina Kabeláčová, Thomas Link-Hessing, Claude-Eric Marquet and Patrick Waldhelm Supervisors: Tiina Tosens, Linda-Liisa Veromann 1 Introduction Water use efficiency (WUE) is very often topic lately. The level of greenhouse gas CO2 is rising. That means, inorganic C is more available for plants to assimilate, but for photosynthesis is important water also. So on the other hand there is problem with water sufficiency, because of greenhouse gases, the average temperature of the planet is rising and dry areas are expanding. Plants are under a constant stress, because of drought. 2 Materials and methods 2.1 Measuring stomatal conductance and photosynthesis Materials: - Two plants Platanus orientalis (normal and stressed) - Fern Microsorum diversifolium - Two instruments GFS-3000 Portable Photosynthesis System Procedure: There were used two plants Platanus orientalis and one fern Microsorum diversifolium. One P. orientalis was not watered for seven days and the second P. orientalis was well watered. Plants were kept under artificial conditions. Plane trees were kept under the light intensity of 1000 µmol/m²s, 22 °C and 65% humidity. The M. diversifolium was kept under artificial light at 600 µmol/m²s, 22 °C and 65% humidity. Experiments were done in the plant physiology laboratory. The stomatal conductance and the photosynthesis were measured with two instruments GFS- 3000 Portable Photosynthesis System. At first chambers of the machines were adjusted to 2 these settings: temperature 25 °C and humidity 65%. The light settings differed: For plane trees it was set to 1000 µmol/m²s and 600 µmol/m²s for M. diversifolium. The CO2 levels in chambers were changed during measurements. At first the experiment begun with 400 ppm of CO2, then 100 ppm and at the end 800 ppm. Changes of CO2 levels were made after a stabilization of assimilation and stomatal conductance. There was also measured the time of a stabilization. Figure 1: GFS-3000 Portable Photosynthesis System Figure 2: P. orientalis leaf in measurement chamber 3 2.2 Stomatal size and density measurement Materials: - Transparent nail polish, transparent tape, slides - Light microscope, PC, Image-J software Procedure: The “varnish”-method was used. The leaf which was previously used for measuring, was coated with clear nail polish on the bottom of the leaf. Leaf-veins were avoided. After the nail polish was dry, transparent tape was sticked on it and pulled off. The tape with the dry varnish was sticked on the slides and analysed under the light microscope. Photos of the leaf bottom imprint were taken in a magnification of 100 and 200. To calculate the stomatal size the length and width of 10 stomata for each plant were measured with the computer program ImageJ. The stomatal size was calculated by multiplying the length with the width. To obtain the stomatal density the amount of stomata on three different parts of the leaf bottom were counted. To acquire average density the formula: number of stomata / area. Figure 3: Leaf bottom surface imprint on slides 4 Figure 4: Stomata of M. diversifolium at 200 magnification 5 Figure 5: Stomata of P. orientalis at 200 magnification 3 Results During the first experiment gained data about the stomatal conductance and the photosynthesis are in tables 1 and 2. Graphs in figure 6 and 7 were constructed from these data and from CO2 levels. We calculated values of WUE according to this formula: 퐴푠푚푙푎푡표푛 푊푈퐸 = 푝ℎ표푡표푠푛푡푒푠푠 푠푡표푚푎푡푎 푐표푛푑푢푐푡푎푛푐푒 Table 1: CO2 Assimilation of the three test plants CO2 P. orientalis (normal) P. orientalis (drought) M. diversifolium [ppm] 100 1,07 0,02 0,51 400 6,86 0,97 3,5 800 12,26 2,37 6,84 6 Figure 6: CO2 Assimilation of the three test plants Table 2: Stomata conductance of the three test plants CO2 P. orientalis (normal) P. orientalis (drought) M. diversifolium [ppm] 100 103,4 9,1 52,2 400 58,6 9,9 33,7 800 42,2 9,6 33,8 7 Figure 7: Stomata conductance of the three test plants WUE values were transferred into the table 3 and figure 8. Stabilization periods of plants was inserted into the table 4. Table 3: WUE of the three test plants CO2 P. orientalis (normal) P. orientalis (drought) M. diversifolium [ppm] 100 10 2 9 400 117 97 103 800 290 246 202 8 Figure 8: WUE of the three test plants Table 4: Stabilization period of the three test plants Measurement P. orientalis (normal) P. orientalis (drought) M. diversifolium 1. 40 min 8 min 62 min 2. 13 min 4 min 13 min 3. 22 min 5 min 17 min Summary 75 min 17 min 92 min The stomatal size and density are shown in figure 9 and 10. 9 Figure 9: Stomatal size of the three test plants Figure 10: Stomatal density of the three test plants 10 4 Discussion We observed reactions of stomata to different levels of CO2. The plant which was stressed by drought had lower stomata conductance than other plants. It could be related to the physiological mechanism which tend to protect the plant from complete dryness. The plant was stressed even more when there was low CO2 level. At the level 100 ppm well-watered plants managed to open the stomata more than the plant which suffered by drought. At the level 800 ppm plants had enough of CO2 in the chamber so they could close stomata. Even though the difference between plane trees is very visible, for better significance and good strength of the experiment, it would be good to do the experiment with more plants and different species. The varnish method showed us how simple it is to observe stomata structures and compare them between different plant families as gymnosperm and angiosperm. According to the evolution, we can suggest that angiosperm plants have smaller stomata structure and higher density compared to ancient gymnosperm family. On the other hand the habitat of the fern is more humid so it can manage to have bigger stomata than the plane tree. We realized that the fern needs more time to open stomata and have slower reaction to the changes of CO2 levels. For better resolution, there would be needed to have wide range of gymnosperm and angiosperm species to compare. 5 Conclusion The plane tree which suffered by drought had low stomata conductance during the whole experiment. The measuring took for 17 minutes in total and gs values were 9,1; 9,9 and 9,6 -1 mmolH2O. WUE values were 2; 97 and 248 µmolCO2∙mmolH2O . Well-watered plane tree had higher values. Stomatal conductance values were 103,4; 58,6 and 42,2 mmolH2O. WUE values -1 for well-watered plane tree were 10; 117 and 290 µmolCO2∙mmolH2O . The measuring took for 75 minutes. The fern had slightly lower values in comparison to well-watered plane tree. Values of stomatal conductance were 52,2; 33,7 and 33,8 mmolH2O. WUE values were 9; 103 -1 and 202 µmolCO2∙mmolH2O . The measuring of the fern took for 92 minutes. 11 Summer School Soil & Water Tartu 2017 Supervisor: Prof. Dr. Marian Kazda, Ahmed Sharif 2. Soil compaction and oxygen in soil Linda Ahner Milan Varsadiya Laurie-May Gonzales Bernhard Glocker Contents 1 Introduction 2 Materials and Methods 3 Results 4 Discussion 5 Conclusion 6 References 12 1 Introduction Nowadays drip irrigation has gained more important in agriculture and has been used very intensively on the fields. Although this is a more sustained way to irrigate the plants, it limits oxygen availability for plant roots by creating a nearly saturated condition. Furthermore, high soil compaction also affects the amount of oxygen content in soil which changes gas exchange. In general, gas exchange is also affecting growth and activity of roots and soil organisms, and leading to an alteration of chemical processes (Ampoorter et al., 2010). For instance, N fertilization has significant effect on microbial CO2 respiration and communities functioning. This was also proved by laboratory incubation experiments (Kowalenka et al., 1978). In Summary soil compaction involves the compression of pores, which leads to decreased porosity, increase in dry bulk density and reduce hydraulic conductivity. The questions of this short experiment were “Soil with organic manure has more oxygen depletion rate than soil without manure” and “Soil managed with organic fertilizer contains lower bulk density”. 2 Materials and Methods Six samples were collected on 26th of June 2017 from the long-term fertilization experiment-site IOSDV in Tartu. Two samples were collected from the same field without organic fertilizers and without manure, two from the same field but with manure. And the last two samples were collected from the field with an alternative organic fertilizer. The samples were collected in metal cylinders from five centimetres under the surface and these were put in plastic cups. The second part was in the laboratory. First, the samples were weighted and after that all samples were flooded with double distilled water (ddH2O) until the complete soil was saturated. All samples got an oxygen sensor through the plastic lid. For the measurement, the “FIBOX LCD” was used.