«（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 ...»
0.5µL of taq DNA polymerase, 1 µL each of 10 µM forward and reverse primer, 2 µL of a genomic DNA template (40 ng µL-1) and 31.5µL of PCR grade water. The PCR thermal profile included an initial denaturation and enzyme activation step of 95 °C for 2 min, followed by 40 cycles of 95 °C for 20 sec, 50 °C for 40 sec and 72 °C for 30 sec. PCR products were evaluated for amplification and their lengths by agarose gel electrophoresis, and purified with the PCR purification Kit (Labopass Cosmogenetech co, Ltd). Amplicons were sequenced by a BigDye Terminator v3.1/1.1Cycle Sequencing Kit (Applied Biosystems, USA). Finally the ECM sequences were then compared with the GenBank database of NCBI via basic local alignment search tool (BLAST). All the sequences were deposited at NCBI, the alignment similarity and accession number was list in Table 4.3.
The colonization rate of ECM (CRE) was determined based on the formula:
CREi (%) = ER/(ER+NR) × 100, where ER and NR denote the number of ectomycorrhizal and non-ectomycorrhizal root tips, i is an ECM type (Choi et al. 2005;
Shinano et al. 2007). The diversity of ECM was calculated as Shannon's diversity
index (Keylock 2005): H’ = -∑Si=1 PilogPi, where S denotes the total number of types of ECM, and Pi is the proportion of the ith ECM type (Pielou 1966).
4.2.4 Plant growth and concentration of P in needles To determine the total dry mass of each organ of seedlings, all the harvested seedlings were separated into needles, branches and roots, and then put into an oven at 80 °C for 48h (root for one week). The dry mass of different organs was weighted.
P uptake capacity to distal parts of seedlings were checked, the dried needles were ground to fine powder digesting by HNO3, HCl and H2O2, after which an inductively coupled plasma-atomic emission spectrometer (ICP-AES, IRIS/IRIS Advantage ICAP, Thermo Fisher Scientific Inc., Massachusetts, USA) was used to determine the concentration of P in the needles.
4.2.5 Statistical analysis All data were following normal distribution, two-way ANOVA was performed for the fertilizer effects on colonization rates, ECM diversity and element concentration. I ran distance-based redundancy analyses for ECM composition with R software (version 2.15.0), and each calculation carried out with six individual seedlings.
4.3 RESULTS 4.3.1 Taxonomic identification
Four ECM types Suillus laricinus, S. grevillei, Inocybe sp. and Thelephora sp. (Type A, B, D and F) were observed before nutrient treatment (Table 4.2). After two growing seasons, three more ECM species appeared with three larch species. These were Russula sp. (C), Hebeloma sp. (E) and Tomentella sp. (G) (Table 4.3). According to taxonomy, all seven types were Hymenomycetes of Basidiomycotina, and were divided into two orders, Agaricales and Aphyllophrales. Agaricales contained three families: Boletaceae (Type A, B), Russulaceae (Type C) and Cortinariaceae (Type D, E). Aphyllophrales involved one family, Thelephoraceae (Type F, G).
4.3.2 ECM colonization and species diversity The extent of ECM colonization in response to the N and P treatments were different in the three larch species (Fig. 4.2). Individual factor of N and P did not impact the ECM colonization rate for DL, the species diversity was significantly affected with increasing trend over N and P loading (R2 = 0.94) (Fig.4.2 a, d). No pronounced effects of N and P were found on colonization rate and species diversity of JL except an interaction effect of N and P on colonization rate (Fig.4.2 b, e). F1 showed same response with JL under different N and P treatments, and species diversity appeared a positive trend with increased nutrient level (R2 = 0.96) (Fig. 4.2 c, f).
With N loading, colonization rate was reduced by 19.76% for DL, and increased by 39.44% and 33.59% for JL and F1. Phosphorous application positively affected the ECM colonization for all three larch species. Phosphorous application positively affected the ECM colonization for all three larch species.
4.3.3 Species composition of ECM
The ECM composition responded to the N and P treatments with all of the three larch species (Fig. 4.1). After two-year treatment, type B was replaced by new types (C, D, E, F and G) for the DL. For the JL, the number of ECM types increased from one to five (A, C, E, F, and G) and for F1, type D was replaced by types A, C, E and G (Fig.
4.1). In control conditions, the JL and F1 were dominated by ECM types A and E, and the DL was dominated by type A. Increasing N loading to the high N (P0N100) level, DL was shifted from type D to type C, whereas type C replaced types E and F for JL.
As for F1, type G out-competed type B. With high P (P50N0) and high P+N (P50N100) applications, the dominant ECM types were type G for DL and type A for JL and F1.
With increased P and N loading together (to P50N100), type F appeared on DL, and types C and E replaced type A on JL, whereas for F1, two new ECM types appeared (C and E), and type E became the dominant species (Fig. 4.1).
4.3.4 Community structure of ECM species The ECM community structure differed within four nutrient treatments of each larch species (Fig.4.3). For the JL, there was a significant difference in the ECM community structure among nutrient treatments (P = 0.01) (Fig. 4.3 b), but no distinct differences in the DL and F1 (Fig. 4.3 a, c). The asterisk shows the significance along the axis 1 direction, which explains 50.5 % of the variance of the JL, and revealing a significant difference between P50N0 and P50N100 (P=0.02). F1 had properties intermediate between its parents, exhibited no significant difference among the four nutrient regimes; only 69.9 % of the variance was explained along axis 1, having marginal significance (P=0.06; see Fig. 4.3 c).
Under the same nutrient treatment, the ECM community structure differed significantly of three larch species. A significant difference was found among the three larch species in the control (P0N0) and high P (P50N0) treatments (Fig. 4.4 a, b).
Distinction was obvious along axis 1(explaining 74.9 % and 80.1 % of the variance, respectively), with P values of 0.03 and 0.02 in each case (Fig. 4.4 a, b). This implies that, in the controls (P0N0), the community structure of ECM species differed significantly between the DL and other two species (Fig. 4.4 a), whereas in the high P (P50N0) regime a significant difference had opened between the DL and F1 (Fig. 4.4 b). The community structure of ECM species did not differ significantly with high N (P0N100) and high P+N (P50N100) nutrient loading among the three larches (Fig. 4.4 c, d).
4.3.5 Biomass of seedlings F1 showed higher biomass (shoots and roots) in comparing with their parents (Table 4.4). P significantly affected the root biomass of DL, and JL was unaffected by any nutrient factor, F1 showed marked effect of P and the interaction effect with N on root and shoot biomass.
4.3.6 Concentration of P in distal parts of seedlings The concentration of P was determined in needles. No significant effect of N and P was found on F1. However N greatly decreased the P concentration in needles of its parents. In high N (P0N100) condition, the needle P concentration was decreased by 54.4 % for DL and 35.5 % for JL comparing with control. And with high P+N
3(P50N100) treatment, 23.2 % decrease for Dahurian larch and 28.9 % decrease for Japanese larch were detected respectively (Fig. 4.5).
4.4 DISCUSSION I estimated the ECM colonization and diversity in symbiosis with larch seedlings grown in immature volcanic ash undergoing N and P application, which had a statistically significant impact on all three larches. Especially the community structure of ECM differed among three host larch species and four nutrient regimes.
The ECM species and colonization rate changed from their initial values after two-year treatment. It has been shown that larch species are mostly associated with Suillus spp. under natural conditions (Zhou et al. 2000; Zhou and Hogetsu 2002).The DL was colonized with Suillus spp. as the dominant ECM species at the beginning. In the end of experiment, it hosted up to six species of ECM. Thelephora spp. was the only one ECM species colonized JL before treatment, finally the species number increased to six with JL. This greater shift indicates a higher variety of ECM community structure among different treatments of JL (Fig.4.2 b). F1 colonized more ECM species than their parents before the treatment, while S. grevillei only survived symbiosis with F1 after two growing seasons. It has been found that larch species-Larix laricina seedlings were colonized rapidly and extensively by S. grevillei with inoculum of 109 fungal isolates (Samson and Fortin, 1986). This intensive symbiosis was also reported by Qu et al. (2003b), they detected that S. grevillei colonizes larch seedlings faster than S. laricinus, especially for F1. However, for JL, Suillus spp. did not appear firstly, probably due to a common sequence of succession of symbiotic ECM (Nara et al. 2003b, 2006b; Yamakawa 2012). Additionally, ECM
succession was investigated in the coniferous species-Scots pine (Pinus sylvestris), the results showed that Suillus spp. was not the earliest pioneer species but developed after Paxillus involutus (Shaw et al. 2003). Thelephora spp. has also been found in naturally regenerated European larch seedlings (Larix decidua), and as a less abundant species with other ECMs (Hydnotrya tulasnei, Pseudotomentella tristis, Tomentella sublilacina and Russula puelaris) (Leski and Rudawska 2012). After the treatment, Thelephora spp. was found in all three larch species, probably because Thelephora spp. is a universal type in woody plants with wide age range. Ma et al.
(2010) reported seedlings of the Red Pine (Pinus densiflora) aged from 1 to 5 years in a naturally regenerating area of a mature forest have also been colonized by Thelephora spp. Thus, in my case, all three host larches were likely to be colonized by Thelephora spp. after two-year treatment.
The ECM colonization and diversity were significantly affected by nutrient treatment, which led to the distinct difference of ECM community structure of three larch species. The colonization enhanced with elevated N and also with elevated P, and there was an interaction effect of N and P for JL and F1. Some studies has been indicated that the ECM colonization rate or EMM production increase with high P availability (Jentschke et al. 2001; Bakker et al. 2009). It is possible that high N and high P treatment lead to nutrient imbalance, and ECM assistance was required for nutrient uptake. Furthermore, plants grown under high nutrient level tend to have more dichotomous architecture, readily generate more root tips. It is therefore with greater root tips, potentially colonized with greater amount of ECM (Fitter and Stickland 1991; Taub and Goldberg 1996). In addition, the JL and F1 have the same pattern of root growth (Sato, 1995), perhaps resulted in the same trend of colonization rate as well. In the deep rhizosphere, especially beyond the inorganic phosphate
(Pi)-depletion zone, their shallow root system requires greater colonization by ECM for efficient nutrient-absorption. Another possibility may be related to inheritance (Fujimoto et al. 2006; 2008), there are many similar patterns of character expression between Japanese larch and F1. One typical character is the chloroplast DNA, it predicts the genus Larix exhibits paternal inheritance (Szmidt et al. 1987), thus showing the same colonization rate of ECM for JL and F1. There was no significant effect on ECM colonization rate for the DL, the value was lower in high N condition than control. Probably because the first-order roots colonized by ECM were 17 % reduced under high N (100 kg N ha-1yr-1) condition (Sun et al. 2010). The greatly affected diversity of DL by individual N and P factor showed an increasing trend with nutrient loading, but with lower changes of community structure among four nutrient regimes. This indicated less flexibility of DL for adapting soil nutrient stress, and this probably leading to inefficient nutrient absorption, especially P uptake. Overall, with the trend of unaffected colonization, the diversity showed increasing trend for JL and F1 (Fig. 4.2). It revealed a markedly difference of community structure under different nutrient condition (Fig. 4.3).
The difference of ECM community structure was diminished with high N loading.
This revealed the trend of similar ECM composition of three larches. However, the N loading reduced P content in needles for the parents not F1, demonstrated F1 had a high capacity and efficient uptake of P under high N condition (Goldstein 2013).
Since there was no difference of community structure for three larches under P0N100 and P50N100, the benefit for unaffected P uptake of F1 may from some dominate ECM species, perhaps type A, B and E, particular type B (S. grevillei) which only colonized F1 in P50N100 condition. Strictly, it was not comprehensive to explain the P uptake by community structure or colonization rate and diversity, the plasticity of
ECM short root can affect the P uptake significantly, such as specific root length (SRL), specific root area (SRA) and root tissue density (Lambers et al. 2006).
Changes in these morphological root traits are related to species-specific impact of ECM fungi (Ostonen et al. 2009).
Additionally, P and its interaction with N affected the above- and belowground biomass of F1, reduced biomass was found with high P loading, especially without N application (P50N0). Based on the photosynthetic parameters, the growth of F1 was not enhanced by P application up to 50 kg hr-1yr-1 (Ryu et al. 2009; Mao et al. 2014).