The use of PET/CT scanning technique for 3D visualization and quantification of real-time soil/plant interactions

Aims

Conventional methodology using destructive sampling, which is laborious and has poor spatial and temporal resolution, has limited our understanding of soil-plant interactions. New non-invasive tomographic techniques have the potential to significantly improve our knowledge. In this study we demonstrated the simultaneous use of PET (positron emission tomography) and CT (X-ray computed tomography) to (a) non-destructively image a whole plant growing in sand, and (b) to link the observed morphology with recently assimilated C. The PET scanner was used to detect and visualize the location of the short-lived radioisotope ¹¹C (with a half-life of 20.4?min) taken up by the plant through ¹¹C-labelled CO₂. This provided information on carbon translocation and the metabolism of photo-assimilates in the plant as well as root structure. The CT scanners yielded data on soil and root structure.

Methods

A medical PET/CT scanner was used to scan a fodder radish plant growing in a pot with test soil composed of homogenous sand. We constructed an air-plant-soil controller system (APS) to control the environmental conditions, such as CO₂, temperature and light during the experiment. The plant was allowed to assimilate ¹¹CO₂ for 90?min before PET scanning was initiated. We carried out PET scanning for 60?min. Subsequently, the aerial parts of the plant was cut off and the pot was rescanned using a micro-CT scanner to obtain more detailed information on structure of the root system and the growth medium structure.

Results

The acquired PET and CT images gave images clearly visualizing the architecture and morphology of root and soil. Using a CT scanner, we were able to detect the main taproot located at 0 to 30?mm depth. With the PET scanner, we were able to measure a signal down to 82?mm below the surface of the sand. We found the highest concentration of ¹¹C at the position of the main root. The PET images, at different time intervals, showed the translocation and metabolisation of photo-assimilates from top to root. Using the micro-CT scanner (voxel size of 90?μm), we were able to detect roots down to 100?mm depth. These findings correlated the PET signals measured down to 82?mm depth.

Conclusions

We conclude that the simultaneous use of PET and CT technologies was successfully applied for soil-plant studies. The combined PET/CT technology has potential to provide new fundamental insight into soil-plant interactions and especially into the effect of abiotic stresses in spite of the limitation due to spatial resolution.     

红壤坡地不同土地利用方式土壤侵蚀的时空分布规律研究

应用定位土芯Eu(Europium)示踪新方法 ,研究红壤坡地不同土地利用方式下土壤侵蚀的时空分布规律 .结果表明 ,新方法对以片蚀和细沟侵蚀为主的红壤坡地是适用的 ;土壤侵蚀的时间分布与降雨量的年时间分布相一致 ,过程性暴雨期表现为全年土壤侵蚀的高峰期 ;在复合坡面 ,随坡面的陡、缓、凹 ,土壤侵蚀表现强、弱、沉积 ;相同坡度和坡长条件下 ,幼龄板栗园的土壤侵蚀速率 >雷竹园 >稀疏马尾松林地 >茶园 .     

Development of an eco-friendly agar extraction technique from the red seaweed Gracilaria lemaneiformis

The red seaweed, Gracilaria lemaneiformis growing as an aquaculture bioremediator along the coasts of Liaodong Peninsula, China, was investigated for the agar production. An eco-friendly method called agar photobleaching extraction process was developed for the benefit of workers' health and safety of the environment. The native agar (NA), alkali-modified agar (AA), chemical-bleached agar (CA) and photobleached agar (PA), which were extracted using different processes, were evaluated for their physical and chemical properties. The PA showed most desirable performances in terms of gel strength, gelling temperature, sulfate content and 3,6-anhydro-l-galactose content. Among the different processed agars, PA gel strength was 1913 g/cm2, the highest among the different processed agars, which increased 8.6% on the basis of the AA. Further we applied this new technique to extract agars from Gracilaria asiatica, and similar results were obtained with that of G. lemaneiformis. This indicates that the agar photobleaching extraction process is a feasible method for Gracilaria species and has a potential application. During the whole agar photobleaching extraction process the pigment content of G. lemaneiformis declined gradually and the TOC concentration in photobleaching solution increased along with the increase in the irradiation time. The mechanism of agar photobleaching could be elucidated by the photolysis theory.     

A note on the use of microwaves in an improved non-radioactive DNA hybridization procedure for the detection of bacteria in foodstuffs

Foodstuffs were artificially contaminated with Escherichia coli carrying plasmid pBR322, dot blotted onto nylon membranes and briefly subjected to microwaves in the presence of 1·5 mol/l NaC1/0·5 mol/l NaOH. Subsequent hybridization with a biotin-labelled probe specific for pBR322 enabled the detection of cell concentrations > 10⁴ cells/dot blot, equivalent to 2 × 10⁷ cells/g food tested. This shortened and simplified method was effective for all ten foods tested, generated low background levels and should be applicable to a wide range of bacteria.     

Broad-scale approaches to the determination of soil microbial community structure: Application of the community DNA hybridization technique

Broad-scale approaches seek to integrate information on whole microbial communities. It is widely recognized that culture techniques are too selective and unrepresentative to allow a realistic assessment of the overall structure of microbial communities. Techniques based on fatty acid or metabolic profiles determine the phenotypic composition of the community. Complementary information about the genotypic structure of soil microbial communities necessitates analysis of community DNA. To determine broad-scale differences in soil microbial community structure (i.e., differences at the whole community level, rather than specific differences in species composition), we have applied a community hybridization technique to determine the similarity and relative diversity of two samples by cross hybridization. In previous studies this assay failed with whole-soil community DNA. Usable hybridization signals were obtained using whole-soil DNA, in this study, by digesting the DNA with restriction enzymes before the labeling with a random-primer reaction. The community hybridization technique was tested using a graded series of microbial fractions, increasing in complexity, all isolated from the same soil sample. This demonstrated that single bacterial species and a mixture of cultivable bacteria were less complex and only 5% similar to whole-community DNA or bacteria directly extracted from the soil. Extracted bacterial and whole-community DNA were 75% similar to each other and equally complex. When DNA was extracted from four different agricultural soils, their similarities ranged from 35 to 75%. The potential usefulness of community hybridization applied to soil microbial communities is discussed.     

Development of an automatic machine for in situ hybridization and immunohistochemistry.

An instrument for the automation of in situ hybridization and immunohistochemistry has been developed. This machine is capable of analyzing 20 microscope glass slides via all of the steps required for colorimetric in situ hybridization or immunohistochemistry. The slides are placed specimen-side down on a specialized Teflon slide-holder set in the reaction chamber of the machine. The system uses a unique type of capillary action between the slide and the holder. The holder has two small holes and is designed to apply, incubate and sequentially add and remove reagents from the slide surface. The system performs the complete processes of in situ hybridization and immunohistochemistry from dewaxing to colorization. Some applications were carried out using this instrument. Cultured cells infected with cytomegalovirus, adenovirus, or herpes simplex virus were hybridized with homologous biotinylated probes, and showed strong purple signals with alkaline phosphatase in the presence of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Automatic in situ hybridization using other colorimetric detection systems (e.g., peroxidase-labeled probes/diaminobenzidine/H2O2) was also examined in cells infected with Chlamydia trachomatis and in paraffin-embedded hepatic tissue sections from patients with hepatitis. For conventional immunohistochemical staining, formalin-fixed and paraffin-embedded tissues were used. Glial fibrillary acidic protein and gamma-immunoglobulins were detected automatically in human brain white matter and tonsillar tissues, respectively, as peroxidase-based reddish signals. The intensity of staining was equal to that achieved by manual methods.     

杏树吐伦球坚蚧空间分布型及抽样技术研究

应用聚集度指标和回归分析,对杏园吐伦球坚蚧Rhodococcus turanicus Arch.空间分布型进行研究,结果表明:在中度受害杏园,吐伦球坚蚧越冬若虫和初孵若虫呈聚集分布,聚集度随虫口密度增大而减小;聚集原因与吐伦球坚蚧本身的生活习性有关;确定了不同种群密度下、不同允许误差下的最适抽样数.通过对不同方位虫口密度进行数据分析,结果表明,吐伦球坚蚧若虫在杏树各方位的聚集是随机的,各方位对整体抽样均无代表性.     

Fine-scale spatial distribution of plants and resources on a sandy soil in the Sahel

Three species, wheat, maize and cotton, were grown in pots and subjected to high (85–100% field capacity, _F), medium (65–85% _F) and low (45–65% _F) soil moisture treatments and high (700 l l^–1) and low (350 l l^–1) CO₂ concentrations. Biomass production, photosynthesis, evapotranspiration and crop water use efficiency were investigated. Results showed that the daily photosynthesis rate was increased more in wheat and cotton at high [CO₂] than in maize. In addition, differences were more substantial at low soil water treatment than at high soil water treatment. The daily leaf transpiration was reduced significantly in the three crops at the high CO₂ concentration. The decrease at low soil water was smaller than at high soil water. Crop biomass production responses showed a pattern similar to photosynthesis, but the CO₂-induced increase was more pronounced in root production than shoot production under all soil water treatments. Low soil water treatment led to more root biomass under high [CO₂] than high soil water treatment. CO₂ enrichment caused a higher leaf water use efficiency (WUE) of three crops and the increase was more significant in low than in high soil water treatment. Crop community WUE was also increased by CO₂ enrichment, but the increase in wheat and cotton was much greater than in maize. We conclude that at least in the short-term, C₃ plants such as wheat and cotton may benefit from CO₂ enrichment especially under water shortage condition.     

Nitric oxide spatial distribution in single cultured hippocampus neurons: investigation by projection of reconstructed 3-D image and visualization technique

Recent studies have revealed a non-homogeneous distribution of nitric oxide synthase (NOS) in neurons. However, it is not yet clear whether the intracellular distribution of NOS represents the intracellular nitric oxide (NO) distribution. In the present study, software developed in our laboratory was applied to the reconstructed image obtained from confocal slice images in order to project the 3-D reconstructed images in any direction and to cut the neuron in different sections. This enabled the spatial distribution of NO to be visualized in any direction and section. In single neurons, NO distribution was seen to be heterogeneous. After stimulation with glutamate, the spatial changes in different areas of the neuron were different. These findings are consistent with immunocytochemical data on the intracellular localization of nNOS in hippocampus neurons, and will help to elucidate the specificity of nitric oxide signaling. Finally, the administration of SNAP and L-NAME was used to examine DAF-2 distribution in the neurons. The results showed this distribution to be homogenous; therefore, it did not account for the NO distribution results.     

Effects of vegetation coverage on the spatial distribution of soil nematode trophic groups

The spatial variability of total soil nematodes and trophic groups in bare and fallow plots in Shenyang Experi-mental Station of Ecology, Chinese Academy of Sciences was examined using geostatistics combined with classic statistics. Results showed that the soil pH value had a negative effect on plant-parasites in both bare and fallow plots; the mean number of total nematodes was significantly higher in fallow plots than in bare plots, which was 1485.3 and 464.0 individuals per 100 g dry soil in fallow and bare plots, respectively; the nugget (C0)/sill (C0+C) ratio of total nematodes, plant-parasites and bacterivores were lower in fallow plots (27.3%-45.6%) than in bare plots (49.5%-100%); the spatial distribution of total nematodes and trophic groups was found to be different between fallow and bare plots, which indicated that vegetation coverage had an effect on soil nematodes.     

Effects of vegetation coverage on the spatial distribution of soil nematode trophic groups

The spatial variability of total soil nematodes and trophic groups in bare and fallow plots in Shenyang Experimental Station of Ecology, Chinese Academy of Sciences was examined using geostatistics combined with classic statistics. Results showed that the soil pH value had a negative effect on plant-parasites in both bare and fallow plots; the mean number of total nematodes was significantly higher in fallow plots than in bare plots, which was 1485.3 and 464.0 individuals per 100 g dry soil in fallow and bare plots, respectively; the nugget (C ₀)/sill (C ₀+C) ratio of total nematodes, plant-parasites and bacterivores were lower in fallow plots (27.3%–45.6%) than in bare plots (49.5%–100%); the spatial distribution of total nematodes and trophic groups was found to be different between fallow and bare plots, which indicated that vegetation coverage had an effect on soil nematodes. __________ Translated from Chinese Journal of Applied Ecology, 2006, 17(2): 295–299 [译自: 应用生态学报]     

Systematic position of the genusCicer L. (Fabaceae) from data on DNA/DNA hybridization

This investigation was carried out in an attempt to determine the systematic position ofCicer via DNA/DNA hybridization. The data showed that members of the tribeVicieae were related toCicer than toTrifolium andOnonis. It is also showed thatPisum was the nearest species toCicer. Thus, the data presented in this work recommend the classification ofCicer underVicieae rather than a separate tribeCicerideae.     

Direct eye visualization of Cy5 fluorescence for immunocytochemistry and in situ hybridization.

Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red range, usually regarded as unsuitable for direct observation by the human eye. We describe here the optimization of a direct visualization approach to Cy5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in situ hybridization. Crucial factors were (a) an excitation path in the microscope not absorbing light in the orange-red range, up to 640 nm, (b) a 588-640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650-700-nm emission filter range, transmitting the low-wavelength portion of Cy5 emission, and (d) high-efficiency filter set components allowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path.     

Development of an immunofluorescence technique for detecting Pfiesteria piscicida

While several DNA-based methods have been developed for the putatively toxic dinoflagellate Pfiesteria piscicida Burkholder et Steidinger, an independent detection method such as immunofluorescence can be a useful alternative. In this study, P. piscicida-specific antisera were developed, and an immunofluorescence (IF) procedure was optimized. A total of six antisera were raised using whole cells (WCA) and the insoluble cellular fraction (ICF) as antigens, respectively, and their titer and specificity were examined using dot blot analysis and whole cell IF. Results showed that the two antisera produced from the ICF antigen had a markedly higher titer (1500) than the other four yielded from the WCA (200). In addition, the two ICF-derived antisera exhibited much higher species specificity, showing no cross-reaction with P. shumwayae, Cryptoperidiniopsis sp., Karlodinium micrum, and other more distant algae tested, and very low background for field collected samples. In evaluation of the IF technique using a P. piscicida-specific polymerase chain reaction (PCR) technique, results from both methods generally agreed well for both field samples (from eastern Long Island Sound) spiked with cultured P. piscicida and those containing naturally occurring P. piscicida (from Chesapeake Bay tributaries).     

Calculations of the spatial distribution of charge density in the environment of DNA

A technique for modeling the structured environmental charge distribution about isolated polyions of arbitrary geometry is presented and applied to B-DNA. It describes the three-dimensional variation of the continuous space charge and allows estimation of local electrostatic potentials and fields that the electrolytic environment induces at nuclei of the polyion. Calculations involve an iterative solution to the set of equations coupling electrostatic potential and average charge density in space. By dividing the region around a DNA segment into finite volume elements, sets of numerically stable atmospheric charge densities have been obtained over a range of concentrations of added monovalent salt. Results are in good agreement with those of Poisson-Boltzmann calculations on comparable systems and are consistent with findings from Monte Carlo simulations of DNA.     

玉米田斜纹夜蛾空间分布型及抽样技术

对秋甜玉米田的斜纹夜蛾不同发生密度田块调查 ,取得了 7组样本资料 ,运用聚集度指标法、Iwao法和Taylor法等对其空间分布型进行测定检验 ,结果表明斜纹夜蛾幼虫呈聚集分布 ,其聚集原因经Blackith种群聚集均数测定 ,当m <3 .2 60 4时 ,其聚集是由于某些环境如气侯、土壤湿度、植株生长状况等所致 ;当m >3 .2 60 4时 ,其聚集是由于害虫本身的群集行为与环境条件综合影响所致。在此基础上 ,通过几种抽样方式比较以五点式为最佳 ,并提出了最佳理论抽样数和最佳序贯抽样模型 :N =1 D2 (3 .8981 m +0 .75 0 3 ) ,To(n) =0 .5n± 2 .865n。     

A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate.     

DNA-DNA dot hybridization technique used as DNA determination method in the alkaline elution analysis of DNA damage

A DNA-DNA (‘Southern’) dot hybridization technique was adapted for use as a quantitative DNA detection method during alkaline elution analysis of irradiated rat cell material. In comparison to standard microfluorometric methods, similar γ-ray-dose-response relationships were obtained with less than 1% of the cell material when the dot hybridization assay was used. When a highly repetitive, long interspersed DNA element of the rat genome is used as a hybridization probe, as few as 10⁴ cells of rat tissue or rat cell culture cells per sample with approx. 50 ng of DNA were sufficient to detect single-strand breaks and protein cross-links in the DNA of rat hepatocytes and cells of the nasal epithelium after in vitro γ-irradiation. Since highly repetative DNA elements are available from nearly all higher eukaryotes, this alternative approach of detecting DNA in alkaline elution analysis is generally proposed for tissues which yield only low amounts of cell material and/or which are difficult to label by radioactive DNA precursors.