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«(落葉樹の根圏動態に対する高CO2とO3及び高窒素負荷の影響に関する研究) Wang Xiaona 王 晓娜 Division of ...»

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Therefore, greater ECM species richness may increase the effectiveness of nutrient acquisition from different locations and/or soil substrates (Jonsson et al. 2001; Leake 2001).

In contrast, O3 usually has negative effects on tree growth. For instance, it impairs the physiological and biochemical processes in leaves and accelerates leaf senescence (Agathokleous et al. 2015; Matyssek and Sandermann 2003; Watanabe et al. 2010a; Zhang et al. 2002). These negative effects lead to belowground responses (Agathokleous et al. 2015; Blum and Tingey 1977). As a result of these negative

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effects on aboveground, carbon assimilation is reduced by O3, limiting the allocation to belowground (Grantz and Farrar 2000; King et al. 2005) and reducing standing fine-root mass (Kasurinen et al. 2005). These effects are expected to decrease ECM colonization and to affect species-host compatibility.

A study of silver birch (Betula pendula) in Open-Top-Chambers (OTCs) revealed that double ambient O3 clearly decreased the proportion of black and liver-brown mycorrhizae after three growing seasons (Kasurinen et al. 2005). Haberer et al. (2007) measured N uptake and symbioses of ECM in adult beech trees (Fagus sylvatica) using radioactive isotopes (delta 15N). They found that the number of fine roots, which were all mycorrhizal, increased markedly with long-term O3 fumigation. Additional research on a 70-year-old mixed spruce-beech forest stand revealed that after two-year of O3 fumigation, the number of vital ECM root tips increased, and the ECMF community changed significantly (Grebenc and Kraigher 2007). However, this result was not always found, and Zeleznik et al. (2007) reported ambiguous results for two-year-old beech seedlings exposed to elevated O3 for two years. In this specific case, the mycorrhization of seedlings was very low, and the ECM types were lower in O3 fumigated plants than in control plants. Predominantly, the number of vital mycorrhizal root tips decreased with O3 treatment (Zeleznik et al. 2007).

Previous researchers found that ECM colonization under O3 might decrease (Adams and Oneill, 1991; Edwards and Kelly, 1992) or increase (Gorissen et al. 1991;

Kasurinen et al. 1999; Wollmer and Kottke 1990). Therefore, the effects of O3 on ECM symbiosis are inconsistent, and the outcome depends on the age of the material plants and the length of fumigation.

External stressors, such as drought and high O3, may regulate stomatal conductance. Water-soluble elements potentially are selectively absorbed by ECM as

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a defense against the harmful effects of these stressors (Jourand et al. 2014). It has also been reported that aquaporin expression is enhanced in ECM seedlings (Marjanovic et al. 2005a), and this enhancement could be particularly important in conditions of water stress (Marjanovic et al. 2005b). Hence, as a defense to O3 stress, element absorption and uptake ability might be adjusted. For example, Haberer et al.

(2007) found that they were reduced in the fine-roots of European beech under enhanced O3.

Despite the extent of ECM colonization, elevated CO2 and/or O3 alter the ECM community composition by affecting specific ECM species. A study of silver birch in OTCs showed that elevated CO2 impaired light brown/orange mycorrhizae (Kasurinen et al. 2005). In a long-term exposure experiment with elevated CO2 and/or O3 concentrations, elevated CO2 alone induced an increase in the proportion of Sistotrema spp. in the ECM community colonizing aspen/birch trees (Edwards and Zak 2011). The responses of ECM to combinations of elevated CO2 and O3 may not be a simple sum of the effects of each. According to Volin et al. (1998), the belowground responses to O3 were variable, and elevated CO2 typically mitigated the negative effects of O3 (e.g., Karnosky et al. 2003c; Watanabe et al. 2010b). For instance, the total level of mycorrhizal colonization of silver birch clones was stimulated by enhanced CO2 and elevated O3 separately, but not when combined (Kasurinen et al. 2005).

The effects of combined elevated CO2 and O3 on ECM species richness are not known in detail (Grebenc and Kraigher 2007; Matyssek et al. 2012b). I expected the species richness of ECM to depend on the photosynthetic activity of the host plants.

Photosynthesis in F1 seedlings increased under elevated CO2 for a short period, but was reduced under elevated O3 (Koike et al. 2012). Therefore, I anticipate that

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colonization and species richness of F1 ECM should increase in elevated CO2 and decrease in elevated O3.

The present work seeks to answer the following questions regarding hybrid larch F1 afforestation in the coming decades: (1) which ECM species colonize F1 under elevated CO2 and/or O3, (2) how do the gas treatments influence the ECM species community structure, and (3) what are the effects of these gases on the growth of the F1 plant?

3.2 MATERIALS AND METHODS 3.2.1 Experimental site and plant materials This study was conducted at Sapporo Experimental Forest of Hokkaido University, in northern Japan (43°07′ N, 141°38′ E, 15 m a.s.l., annual mean temperature and precipitation in 2011 were 13.5°C and 1254 mm). Two-year-old seedlings of the hybrid larch F1 (L. gmelinii var. japonica × L. kaempferi) were provided by the Hokkaido Research Organization, Forestry Research Institute, near Sapporo. The height and diameter of each seedling was determined before planting, and the initial mean height and mean diameter of the seedlings were 38.6 ± 0.3 cm and 5.2 ± 0.2 mm, respectively.

The ECM colonization was also determined before planting (see Table 3.2). The soil at the study site was well-homogenized brown forest soil with no previous planting of tree species. All seedlings were planted in May 2011, and they were periodically irrigated with tap water to prevent desiccation. Gas treatments began one month later when all seedlings were established in the site, and the seedlings were dug

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out after two growing seasons (in late October 2012).

3.2.2 CO2 and O3 treatment I set up the OTC system at the experimental forest site of Hokkaido University. The 16 chambers (volume, W × W × H = 1.2 × 1.2 × 1.5 m; H = 2.2 m after Sep. 2012) were made of steel frames with polyvinyl chloride film (Noh-bi, Sapporo, Japan) that had a transmittance of 88% of full sunlight (simply cutting UV-B). I set up four gas treatment regimes: 1) control (CO2 = about 380 μmol/mol; O3 6 nmol/mol), 2) elevated O3 (60 nmol/mol: 7 hours, 10:00–17:00), 3) elevated CO2 (600 μmol/mol during daytime), and 4) their combination (elevated O3 + CO2). Charcoal-filtered ambient air was introduced and tanked CO2 was supplied either as ambient or elevated CO2 treatments. The CO2 concentration in chambers was regulated by a control unit (GMM222, Vaisala, Helsinki, Finland). Regarding the O3 fumigation, O3 was generated from ambient air using an electrical-discharge O3 generator (PZ-1B, Kofloc-Kojima, Kyoto, Japan).

In the growth chambers, O3 was continuously monitored with an ultraviolet (UV) absorption O3 analyzer (EG-3000F, Ebara, Tokyo, Japan and model 202-EPA, 2Btechnologies, Boulder, CO, U.S.A.). The O3 concentration was lower than 6 nmol/mol in the untreated O3 chambers. There were four replications of each treatment, and four larch seedlings were planted in each chamber (64 seedlings in total). A proportional-integrative-differential (PID) control algorithm was applied to maintain the desired concentration of O3. The monthly data are shown in Table 3.1.

3.2.3 ECM identification

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After the two-year fumigation, I analyzed ECM on the roots of F1. The roots extended widely after two growing seasons, trying to get accurate biomass, I removed the chambers and dug out the roots manually by shovel. After removal from the soil, all roots were covered with wet paper tissue, stored in a plastic bag, and immediately transferred to the laboratory where they were refrigerated at 4°C. Within a maximum of two days, the harvested roots were first washed until no large clods remained, and the fine roots were then gently cleaned using a small paintbrush. A microscope (Olympus szx-ILLK100, Japan) was used to observe the extent of ECM colonization (as was conducted before planting the seedlings). In total, 500 root tips were randomly counted for each replicate following the method of Shinano et al. (2007), and Fig.3.1 shows the sampling process. A classification of ECM morphological types was estimated, and final ECM identification was carried out using molecular analyses (Table 3.2).

I first extracted ribosomal DNA (rDNA) from the root tips using a DNeasyTM Plant Mini Kit (Qiagen). Then I conducted PCR amplification using primer 1F/4 to determine the sequences of the ITS-region (Bellemain et al. 2010; Gardes and Bruns 1993). Sequencing reactions were performed using the BigDye Terminator v3.1/1.1 Cycle Sequencing Kit (Applied Biosystems, USA). Finally, ECM sequences were compared with the GenBank database at the DNA Data Bank of Japan using the basic

local alignment search tool (BLAST) program as follows:

(http://blast.ddbj.nig.ac.jp/blast/blastn? lang=en).

The colonization rate of ECM (CRE) was calculated using the following formula:

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ECM type (Choi et al. 2005; Shinano et al. 2007).

The ECM diversity (H') was calculated as Shannon's diversity index (Keylock,

2005) according to the following formula:

′ = − � PlogP, =1 where S denotes the total number of ECM types, and Pi is the proportion of the ith ECM type (Pielou 1966).

3.2.4 Measurement of seedling growth and nutrient concentration The diameter and height of the seedlings were measured in July and November 2011 and in September 2012. All seedlings were harvested on October 20, 2012 to estimate the dry mass of plant organs. The seedlings were then separated into needles, branches, stems, and roots. The plant organs were dried in an oven at 80°C for one week and then weighed. The needle and root samples were crushed into powder by mills and digested with HNO3, HCl, and H2O2, after which an inductively coupled plasma-atomic emission spectrometer (ICP-AES, IRIS/IRIS Advantage ICAP, Thermo Fisher Scientific Inc., Massachusetts, U.S.A.) was used to determine the concentration of phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), aluminum (Al), iron (Fe), manganese (Mn), and molybdenum (Mo). The N concentration was determined by the combustion method using an NC analyzer (NC-900, Sumica-Shimadzu, Kyoto, Japan).

3.2.5 Measurement of the leaf gas exchange rate

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The leaf gas exchange rates of seedlings were measured on September 21–25, 2011 and September 11–15, 2012 using an infrared gas analyzer system (LI-6400, Li-Cor Inc., Lincoln, NE, U.S.A.). Two seedlings in each chamber were randomly selected for measurement of their leaf gas exchange rates (eight measurements per treatment).

The measurements were conducted on the same seedlings throughout the experiment.

The net photosynthetic rate (A) and stomatal diffusive conductance to H2O (Gs) were determined at 24 ± 0.1°C leaf temperature, 380 μmol/mol CO2, 60 ± 5% relative air humidity, and a photosynthetically active photon flux (PPF) of 1500 μmol m–2s–1 per the method described by Watanabe et al. (2012). Finally, the A and the stomatal conductance values at the growth concentration [CO2] (denoted Agrowth and Gs) were determined.

3.2.6 Statistical analysis Statistical analyses were undertaken using R and SPSS (version 16.0) software. All data were distributed normally, as verified by the Kolmogorov-Smirnov Test. The statistical unit was single OTC, two-way analysis of variance (ANOVA) was used to test the independent effects of elevated CO2 and O3 as well as their interaction.

Tukey’s HSD test was applied to identify significant differences among the four treatments. Distance based redundancy analyses (db-RDA) were performed to determine the species abundance of ECM community variation according to the gas treatment regimes.


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3.3.1 ECM types colonizing F1 I found six types of ECM colonizing F1 after CO2 and/or O3 treatments compared with the three types identified before the treatments. Suillus grevillei was the only one species existed before and after the treatments. According to mycorrhizae taxonomy, all eight ECM types belong to either the classes Basidiomycetes (Type A, C, D, F, G and H) or Ascomycetes (Type B, E). Typical morphology of each ECM species was attached as appendix. The detailed morphological description for each type of ECM and their similarly matched sequence are all listed in Table 3.2.

3.3.2 Extent of colonization and diversity of ECM The ECM colonization rate was significantly increased by elevated CO2, whereas it was sharply reduced by O3 compared to the control (Fig. 3.2). However, there was no interactive effect of elevated CO2 and O3 on the ECM colonization rate, and the ECM colonies varied in diversity among the four treatments (Fig. 3.2). ECM diversity reduced significantly by O3 exposure, and it also decreased under elevated CO2 + O3 treatment relative to the control. The diversity between control and elevated CO2 did not differ significantly (P = 0.49).

3.3.3 Abundance of ECM by species The six ECM types were found in different amounts under the four gas treatments.

According to the integrated estimation of ECM colonization and species diversity,

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types A, C, D, and F were the major ECM colonizers of F1 (Fig. 3.3b). The ECM abundance under elevated O3 and the mixed fumigation differed significantly from control and elevated CO2 treatments (63.7 % of the variance was explained along the axis-1 direction, P ≤ 0.01). Based on the distance of the four ellipses in Fig. 3.3a, the ECM abundance was similar to that of the control at elevated CO2.

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