Soil-plant-microbial interactions at the nano-scale CAS-UWA Soil Systems Biology Joint Laboratory Soil-plant-microbial interactions at the nano-scale Professor Daniel Murphy Chair in Soil Science (土壤学科带头人) Australian Research Council - Fellow The University of Western Australia Guest Professor (客座外聘教授) CAS Visiting Professor CAAS Visiting Professor Email: daniel.murphy@uwa.edu.au

Daniel V. Murphy Professor, Chair in Soil Science School of Earth and Environment The University of Western Australia 35 Stirling Highway， Crawley WA 6009， Australia 西澳大利亚大学地球与环境学院教授 专长：土壤有机质、土壤氮素、农作制度、 微生物生态及土壤生物化学 Areas of Expertise Soil organic matter, soil nitrogen, farming systems, isotopes, NanoSIMS, microbial ecology, and soil biochemistry 农民培训 样品采集 田间观测

The University of Western Australia is located on the River near Perth City, Western Australia

The other University priority is in Minerals and Energy Achieving International Excellence Plants, Animals, Agriculture and Environment are priority research areas at The University of Western Australia 植物,动物,农业和环境 为西澳大利亚大学优先研究领域 The other University priority is in Minerals and Energy 矿产和能源 为西澳大学其他优先研究领域

Within the Faculty of Science we train students in a range of science disciplines using computer, laboratory, glasshouse and field experience.

Well equipped research facilities are able to be used by postgraduates and visiting researchers. For example NanoSIMS.

Micro-scale interactions Our research interest is to: 植物-土壤-微生物相互关系 Describe spatial arrangement and interaction of soil minerals, plant roots, organic matter and microbial cells. 描述土壤矿物质、植物根系、土壤有机质以及微生物细胞的空间布局 Quantify carbon and nutrient flow and competition in the rhizosphere. 定量研究根际碳、氮和磷元素的流动 Understand soil-plant-microbial function ecosystem services. 理解“土壤-植物-微生物”功能 Micro scale Plot scale Catchment scale

Micro-scale interactions However: Soil-plant-microbial interactions occur at a scale smaller (micro- to nano-meters) than many methods of assessment. “土壤-植物-微生物”的相互作用在较小的尺度（纳米到微米范围）进行，常规方法很难观测 Many methods destroy the spatial arrangement in soil between biology, chemistry & physics. 很多实验方法会破坏土壤中生物、化学和物理性质的空间布局 Quantifying while simultaneously visualizing these interactions is therefore challenging. 定量并且可视化“土壤-植物-微生物”的相互作用是一项挑战 plant root bacteria fungi nematode archaea

Biochemical processes occur at a scale smaller than many methods Why use NanoSIMS? (为何使用纳米质谱仪) Biochemical processes occur at a scale smaller than many methods Process Physical scale Method Gas fluxes m3 or hectares Greenhouse gas monitoring cm3 or grams soil SOM fractionation Microbial biomass Suction cups mm or <1 gram soil Inorganic nutrients Roots Micro-samplers Aggregates Molecular assays Faunal grazing Sand µm CAT scanning Rhizosphere Silt Mass spectrometer Clay Microbial growth Imaging 14C nm UWA’s Centre for Microscopy, Characterisation and Analysis Microscopy Organic matter turnover Nutrient cycling NanoSIMS Herrmann et al. (2007) Soil Biology & Biochemistry 39: 1835-1850.

Nano-Secondary Ion Mass Spectrometry (纳米质谱仪简介) NanoSIMS is an Ion Microprobe linked to a Mass Spectrometer. We are using this to study Soil-Plant-Microbe Interactions at 100 nm resolution. NanoSIMS技术通过将离子微探针连接到质谱仪，来研究在100纳米解析度上的“土壤-植物-微生物”相互作用。

Ion microprobe linked to a mass spectrometer. NanoSIMS (纳米质谱仪简介) Ion microprobe linked to a mass spectrometer. Isotopic mapping at the ~100 nm scale Solid materials including biological samples Imaging isotopic tracers (e.g., 13C, 15N) in biological materials at sub-cellular level Stable isotope tracer measurements E.g., 13C, 15N, 18O, 34S, 2D Uptake and assimilation in biological systems, in situ. Hybridisation experiments, microbial tagging. Elemental mapping Co-location of elements in complex systems, e.g., soil, plant roots, biological systems. Isotopic mapping at parts per million 13C/12C in organic materials. 15N/14N uptake in microbial and plant cells. 18O tracing for P18O4 Herrmann et al. (2007) Nano-scale secondary ion mass spectrometry – A new analytical tool in biogeochemistry and soil ecology: A review article. Soil Biology & Biochemistry 39: 1835-1850.

Why use NanoSIMS? Mass spectrometer data is from each pixel 可从每一质谱像素中获取数据 Colour bar = 15N/14N Within the NanoSIMS image each pixel contains mass spectrometer data (256 x 256 pixels). Red = 15N/14N ratio in microbial cells Blue = Silica (mass 28) = Quartz soil Green = Carbon (mass 12) = organic matter = same location on the 2 images: Top image = 15/14N ratio in bacteria Bottom image illustrates the location of the bacteria in the soil matrix alongside Silica (28Si) to identify quartz and 12C to identify carbon. 256 x 256 pixels = 65,536 data points per image 在纳米质谱仪图像中，每一像素都包括质谱数据（256×256像素）

纳米质谱图像 Mass spectrometer data is read from each pixel 可从每一质谱像素中获取数据 Superimposed NanoSIMS images where Blue = 28Si-, Green = 12C14N- (represents organic matter) and Red = 15/14N ratio images (distribution of 15N enriched P. fluorescens) 蓝： 28Si- 红 ： 15N /14N 绿： 12C14N-（表示有机质） Herrmann et al (2007) Soil Biology & Biochemistry 39, 1835-1850

Membrane and air-gap. Only mycorrhizal hyphae can cross the membrane. Example: Plant-fungal C and N transfer Wheat plants grown in 13CO2 enriched air. 15N labeled sources added to soil. Membrane separated 15N from plant. 15N transfer via mycorrhizal fungi. Root Side No Root Membrane and air-gap. Only mycorrhizal hyphae can cross the membrane. 13CO2 15N Kaiser et al. (2015). New Phytologist 205: 1537-1551.

No direct access to 15N by root Example: Plant-fungal C and N transfer Tracing 13C via plant photosynthate (13CO2). Tracing 15N via mycorrhiza (AMF). 15N No direct access to 15N by root 13CO2 13C 31P 15N Xylem cell Endodermis Phloem cells Cortex with Mycorrhiza Kaiser et al. (2015). New Phytologist 205: 1537-1551.

No direct access to 15N by root Example: Plant-fungal C and N transfer Tracing 13C via plant photosynthate (13CO2). Tracing 15N via mycorrhiza (AMF). 15N No direct access to 15N by root 13CO2 Phloem cell is highly enriched in 13C from photosynthesis. Xylem cell Endodermis Phloem cells Cortex with Mycorrhiza 13C/12C Xylem cell is highly enriched in 15N from mycorrhiza. 15N/14N Kaiser et al. (2015). New Phytologist 205: 1537-1551.

合作前景 – Conclusions NanoSIMS enables high spatial resolution of isotopic data between plant, bacterial and fungal cells. This is not possible with traditional mass spectrometry where soil+plant+microbes = one isotopic measurement result. NanoSIMS offers exciting opportunities to study soil-plant-microbe-fungal interactions at a previously unconsidered scale.