Thesis or Dissertation ROLE OF ARBUSCULAR MYCORRHIZAL FUNGI ON THE PERFORMANCE OF FLOODPLAIN PLANTS UNDER NUTRIENT-LIMITED AND HEAVY METAL STRESS CONDITIONS

Animesh SARKAR

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指導教員 : 王青躍
List of Figures ivList of Tables viAcknowledgments viiiAbstract 11 General Introduction 41.1 Arbuscular mycorrhiza in flood plain . . . . . . . . . . . . . . . . . 41.2 Arbuscular mycorrhiza for Zn uptake . . . . . . . . . . . . . . . . . 61.3 Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.4 General objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.5 Thesis outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Arbuscular mycorrhizal influences on growth, nutrient uptake, and use efficiency of Miscanthus sacchariflorus growing on nutrient-deficient river bank soil 112.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 132.2.1 Soil and plant propagate collection from study sites . . . . . 132.2.2 Spore isolation from collected soil . . . . . . . . . . . . . . . 142.2.3 Experimental procedure and design . . . . . . . . . . . . . . 142.2.4 Chlorophyll content measurement . . . . . . . . . . . . . . . 152.2.5 Harvesting and processing . . . . . . . . . . . . . . . . . . . 152.2.6 AM colonization determination and nutrient analyses from soil and plant samples . . . . . . . . . . . . . . . . . . . . . 152.2.7 Calculation of inoculation effectiveness . . . . . . . . . . . . 162.2.8 N and P budget calculation . . . . . . . . . . . . . . . . . . 162.2.9 Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . 172.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.3.1 Root colonization . . . . . . . . . . . . . . . . . . . . . . . 172.3.2 Chlorophyll content in leaves . . . . . . . . . . . . . . . . . . 172.3.3 Changes in dry mass production due to AM fungi . . . . . . 172.3.4 Total phosphorus (TP) and total nitrogen (TN) concentrations in plants . . . . . . . . . . . . . . . . . . . . . . . . . . 192.3.5 Total phosphorus, available phosphorus and total nitrogen content in post-harvest soils . . . . . . . . . . . . . . . . . . 212.3.6 Nitrogen and phosphorus budgeting . . . . . . . . . . . . . . 232.3.7 Major and trace elements content in plants . . . . . . . . . 232.3.8 Inoculation effectiveness . . . . . . . . . . . . . . . . . . . . 232.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252.4.1 Percent colonization of root, chlorophyll content, and dry mass production . . . . . . . . . . . . . . . . . . . . . . . . 252.4.2 Nutrient assimilation and budgeting . . . . . . . . . . . . . . 272.4.3 Efficiency of AMF after inoculation . . . . . . . . . . . . . . 312.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Role of arbuscular mycorrhizal fungi on the performance of floodplain Phragmites japonica under nutrient stress condition 333.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 353.2.1 Plant propagule collection from study sites . . . . . . . . . . 353.2.2 Soil analysis and spore isolation from collected soil . . . . . 353.2.3 Experimental procedure and design . . . . . . . . . . . . . . 363.2.4 Harvesting and processing . . . . . . . . . . . . . . . . . . . 373.2.5 Chlorophyll content measurement . . . . . . . . . . . . . . 373.2.6 AM colonization determination and nutrient analyses from plant samples . . . . . . . . . . . . . . . . . . . . . . . . . . 373.2.7 Calculation of inoculation effectiveness . . . . . . . . . . . . 383.2.8 N and P budget calculation . . . . . . . . . . . . . . . . . . 393.2.9 Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . 393.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393.3.1 Root colonization . . . . . . . . . . . . . . . . . . . . . . . 393.3.2 Leaf chlorophyll concentration . . . . . . . . . . . . . . . . 393.3.3 Plant dry weight . . . . . . . . . . . . . . . . . . . . . . . . 413.3.4 Nutrient concentration . . . . . . . . . . . . . . . . . . . . . 413.3.5 Nitrogen and phosphorus budgeting . . . . . . . . . . . . . . 423.3.6 Inoculation effectiveness . . . . . . . . . . . . . . . . . . . . 433.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433.4.1 Nutrient uptake and budgeting . . . . . . . . . . . . . . . . 443.4.2 Effectiveness of AMF after inoculation . . . . . . . . . . . . 493.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504 Arbuscular mycorrhizal association for growth and nutrients assimilation of Pharagmites japonica and Polygonum cuspidatum plants growing on river bank soil 514.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 534.2.1 Soil and plant propagate collection from study sites . . . . . 534.2.2 Spore isolation from collected soil . . . . . . . . . . . . . . . 534.2.3 Experimental procedure and design . . . . . . . . . . . . . . 544.2.4 Harvesting and processing . . . . . . . . . . . . . . . . . . . 544.2.5 AM colonization determination and nutrient analyses from soil and plant samples . . . . . . . . . . . . . . . . . . . . . 554.2.6 Calculation of inoculation effectiveness . . . . . . . . . . . . 554.2.7 N and P budgeting . . . . . . . . . . . . . . . . . . . . . . . 564.2.8 Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . 564.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574.3.1 Root Colonization . . . . . . . . . . . . . . . . . . . . . . . 574.3.2 Changes of dry mass production due to AM fungi . . . . . . 574.3.3 Total phosphorus content in plants . . . . . . . . . . . . . . 574.3.4 Total nitrogen content in plants . . . . . . . . . . . . . . . . 574.3.5 Total phosphorus and available phosphorus content in postharvest soils . . . . . . . . . . . . . . . . . . . . . . . . . . 604.3.6 Total nitrogen content in soils after harvesting of plants . . . 604.3.7 Nitrogen and phosphorus budgeting . . . . . . . . . . . . . . 604.3.8 Inoculation effectiveness . . . . . . . . . . . . . . . . . . . . 604.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634.4.1 Nutrients (N, P) assimilation and budgeting . . . . . . . . . 674.4.2 Efficiency of AMF after inoculation . . . . . . . . . . . . . . 694.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705 Response of Miscanthus sacchariflorus to zinc stress mediated by arbuscular mycorrhizal fungi 715.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . 735.2.1 Collection of soil and plant propagate from study sites . . . 735.2.2 Experimental set-up . . . . . . . . . . . . . . . . . . . . . . 755.2.3 Chlorophyll content and chlorophyll fluorescence . . . . . . . 755.2.4 Hormone and enzyme analysis . . . . . . . . . . . . . . . . . 765.2.5 Harvesting and processing . . . . . . . . . . . . . . . . . . . 765.2.6 AM colonization determination and nutrient analyses from soil and plant samples . . . . . . . . . . . . . . . . . . . . . 765.2.7 Scanning electron micrograph (SEM) analyses . . . . . . . . 775.2.8 Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . 775.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775.3.1 Root colonization . . . . . . . . . . . . . . . . . . . . . . . . 775.3.2 Chlorophyll content and fluorescence in leaves . . . . . . . . 775.3.3 IAA concentration and IAA catabolism . . . . . . . . . . . . 795.3.4 ROS production and POD activity . . . . . . . . . . . . . . 815.3.5 Above and below ground dry mass (DM) production . . . . 815.3.6 Total phosphorus (TP) and total nitrogen (TN) concentration in plants . . . . . . . . . . . . . . . . . . . . . . . . . . 835.3.7 Zinc content in plants . . . . . . . . . . . . . . . . . . . . . 845.3.8 Zinc crystal in mycorrhizal infected root . . . . . . . . . . . 855.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855.4.1 Root colonization, growth, hormone and enzyme activity of plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855.4.2 Nutrients and Zinc uptake . . . . . . . . . . . . . . . . . . . 865.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 886 Conclusion & scope of study 89Bibliography 91Appendices I
Mycorrhizal fungi are species of fungi that intimately associate with plant roots forming a symbiotic relationship, with the plant providing sugars for the fungi and the fungi providing nutrients to the plants. A vesicular arbuscular mycorrhiza (VA mycorrhiza), now known as arbuscular mycorrhiza (AM), plays a very important role in enhancing plant growth and yield due to an increased supply of nutrients to the host plant. Arbuscular mycorrhizal fungi (AMF) are ubiquitous components of terrestrial ecosystems across the world with multiple functions from the level of individual plants to the ecosystem. Mycorrhizal fungi occur in riparian areas but their functions in promoting plant growth are unknown across floodplain chronosequences. Mycorrhizal fungi can absorb, accumulate, and transport large quantities of nutrients within their hyphae which are then release and translocate to the host plant cells in root tissue. Arbuscular mycorrhiza (AM) is the predominant mycorrhizal type found in early stages of primary succession; for example, in sand dunes and river floodplains though distribution and functions of AMF at transitional zones between aquatic and terrestrial ecosystems, such as flood plains, remain less studied. In such areas, however, the level of colonized plant species has a wide range. Some species, such as Miscanthus sacchariflorus or Phragmites japonica, grow in stony sterile as well as low nutrient conditions, while other species prefer relatively fine sediment with nutrient rich conditions. Response to mycorrhizal inoculation is linked to the level of soil fertility, and it is well documented that P is the most influential element in mycorrhizal development and efficiency. In nutrient-deficient soils, the yields of horticultural and field crops were found to be largely dependent on their mycorrhizal status under field and greenhouse conditions. Since soils in the early stages of primary succession generally have low nutrient contents, it is possible that AM fungi play an important role in the growth and establishment of pioneer plants.A preliminary survey was conducted on the banks of the Ara River, Saitama, Japan (36°4'58.02" N and 139°26'28.85" E) to identify suitable, experimental plant materials for this study. Five meter × four meter transects (henceforth referred as S1~S5, respectively) were randomly selected along the river and marked with poles and rods. The distance between these transects ranged between 10 and 30m. All plant species within a transect were identified, and their percent coverage was calculated. Though the species composition varied between the transects, M. sacchariflorus and P. japonica appeared to be the most common species and covered a higher percentage of the transects areas whereas P. cuspidatum covered a lower percentage. Therefore, based on the survey and previous literature, we chose M. sacchariflorus, P. japonica and P. cuspidatum as our experimental plants.The potential effects of arbuscular mycorrhizal fungi (AMF) on growth, nutrient uptake, and inoculation effectiveness of AMF on the dominant pioneer plants Miscanthus sacchariflorus(C4), Phragmites japonica (moderately C4) and Polygonum cuspidatum (C3) were evaluated.Spores of AMF strains (Gigaspora margarita Becker & Hall) were collected from the commercial product ‘Serakinkon’.Four treatments such as natural soil, natural soil inoculated by AM fungi, sterilized soil inoculated by AM fungi, and sterilized soil without AM fungi inoculation were selected to determine the effects of applied and indigenous AMF by performing pot experiments in greenhouse Saitama University, Japan. The average colonization level of M. sacchariflorus was 23–28%, P. japonica was 24-33% and P. cuspidatum was 0.2%-0.5% whereas no colonization was found in sterilized soil. AMF colonization increased the chlorophyll content, plant dry mass, N, P, K, Mg, Fe, Cu, and Zn concentration of the M. sacchariflorus plant’s roots, stems, and leaves when AMF was applied with natural and sterilized soil. Mn concentration decreased in M. sacchariflorus roots and stems but increased in leaves in natural soil, and AMF with natural soil treatment. AMF colonization also increased the chlorophyll content (r= 0.84, p<0.01), plant dry mass (r=0.89, p<0.01), and N, P, K, Mg, and Fe concentration of the P. japonica plant’s roots, stems, and leaves with natural and sterilized soil. Mn concentration decreased in the P. japonica roots but increased in the leaves. Cu concentration was not significantly affected by treatments. In all cases, maximum values showed when both plants were applied with natural soil in combination with AMF, but Ca concentration decreased as colonization level increased. N loss minimization from the soil was significant when colonization level was high. Therefore, AMF have some potential effects for growth of the M. sacchariflorus and P. japonica. Nitrogen retention from the soil was also significant when the colonization level was high in aquatic-terrestrial inter-faces (river banks) whereas P. cuspidatum showed very less or a negative response to AMF colonization in all cases.In addition, the unfavorable oxidative effects adversely influence plant growth under heavy metal stress. However, AM are able to enhance production of antioxidant enzymes, which can alleviate the stress of heavy metals. Zinc is an essential micronutrient for plant growth. Conversely, Zn is also an important environmental contaminant in some situations, often reaching phytotoxic concentrations. The feasibility of employing AM in soil re-vegetation and remediation has elicited great interest, and numerous studies have focused on the functions of AM fungi in metal-contaminated soils. Although AM are most often considered important for uptake of immobile nutrients, they also play an important role in reducing uptake of heavy metals, including Zn, where soil concentrations are high. Thus, AMF have various roles in terms of plant-Zn interactions. To understand these roles, there is a need to study responses of AM across a range of soil types, Zn concentrations and plant species. Therefore, we studied the effects of arbuscular mycorrhizal association on growth and survival capabilities of Miscanthus sacchariflorus under different Zn concentrations in soil. Considering the type of mycorrhizal inoculation and addition of Zn, six treatments were taken, viz. (1) soil inoculated by AM fungi (2) soil inoculated by AM fungi and addition of zinc (Zn) 100 mg Kg−1 (3) soil inoculated by AM fungi and addition of zinc (Zn) 1000 mg Kg−1 (4) soil without AM fungi inoculation (5) soil without AM fungi inoculation but addition of zinc (Zn) 100 mg Kg−1 and (6) soil without AM fungi inoculation but addition of zinc (Zn) 1000 mg Kg−1 . The Zn0 treatment received no additional Zn, while the Zn100 and Zn1000 treatments were established by adding ZnSO4.7H2O to give Zn additions of 100 mg Zn Kg−1 and 1000 mg Zn Kg−1.The Zn addition treatments were selected on the basis of the reported existence of soil zinc.The experiment was conducted in the form of pot cultures of M. sacchariflorus in a greenhouse at Saitama University, Japan. The pots were laid out in complete randomized design (CRD) in the greenhouse, with four replicates per treatment.In our experiment, addition of Zn did not show significant effect of mycorrhizal colonization. Even, when the Zn addition level was as high as 1000 mg kg−1, the mycorrhizal infection rate slightly decrease compared to the control receiving no Zn but not statistically significant different. This may imply that Zn has no or little effect on spore germination and AM colonization. Inoculation of AMF (Gigaspora margariata) with Zn (100mg kg−1) increased chlorophyll content, Fv/Fm, total dry mass, IAA, TN, TP and Zn concentration and H2O2 level, IAAO activity, POD activity was low compare to other two treatments whereas with Zn (1000mg kg−1) induces lower concentrations of these metals in the aerial part of the plant and consequently a beneficial effect on plant growth. In addition, AMF can able to accumulate Zn in plant root. When approaching the inner part of the root, heavy metals are located in the parenchyma cells. Accordingly, it can be stated that AM are able to keep heavy metals out of plant or reduce concentration of areal parts of plants especially for Zn.Lead (Pb) is a heavy metal that is present in the soil in very small amounts, but anthropogenic activities have increased its content in some locations, which can make these areas unproductive or inappropriate for crop production. Under heavy metal stress, the unfavorable oxidative effects adversely influence plant growth. However, AM are able to enhance production of antioxidant enzymes, which can alleviate the stress of heavy metals and finally plant growth. In the present experiment, we studied the effects of arbuscular mycorrhizal fungal(AMF) association on growth, survival capabilities, nutrients and Pb uptake of Miscanthus sacchariflorus under different Pb concentrations in soil.The experiment was conducted in the form of pot cultures of M. sacchariflorus. The pots were laid out in complete randomized design (CRD) with three replicates per treatment. The treatments were composed of the inoculation or no inoculation of the AM fungus, Gigaspora margarita, and the addition of three Pb concentrations in the soil (0, 100 and 1,000 mg kg−1). Addition of Pb significantly decreased mycorrhizal colonization. Inoculation of AMF with Pb increased chlorophyll content, Fv/Fm, total dry mass, IAA, TN, and TP whereas H2O2 level, IAAO activity, POD activity was low compare to non-inoculated treatments. Moreover, application of AMF with Pb doses induces concentrations of Pb in the plant where at higher dose Pb (1000mg −1) induces lower content of Pb in the aerial part of the plant but higher content in root. AMF enhanced the tolerance of M. sacchariflorus against Pb toxic condition and accumulate Pb in plant root whereas translocation to the shoots was inhibited in higher dose Pb (1000mg kg−1).
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