1a. Objectives (from AD-416):
The long-term objectives of this project involve the application of new microscopy technologies for the identification and management of agricultural pests and pathogens. The Beltsville Agricultural Research Center (BARC) Electron and Confocal Microscopy Unit (ECMU) serves the research projects of the ARS that require electron and confocal microscopy data necessary to achieve their specific research objectives. The ECMU will use standard protocols as well as develop new technologies and methodologies as needed to meet the needs of its clientele. Over the next 5 years we will focus on the following objectives: 1. Develop new techniques and methodologies in microscopy that generate high-resolution images of biological specimens more efficiently and effectively. [NP303, C1, PS1] 2. Apply novel microscopy approaches to facilitate the systematic identification and characterization of plant pathogens and pests, alone or with their hosts. [NP303, C1, PS1]
1b. Approach (from AD-416):
The Electron and Confocal Microscopy Unit (ECMU), housed on the BARC campus, performs collaborative research with a diverse group of ARS scientists needing microscopic imaging to validate their research hypotheses. The facility is equipped with state-of-the-art electron microscopes [transmission (TEM) and scanning (SEM)], confocal laser scanning microscope (CLSM), wide-field fluorescence and bright field microscope, and a digital video microscope. TEMs and SEMs can discern the internal and external structures of plants, animals, microbes, and materials at high resolution and at magnifications far exceeding those of light microscopes. Structures can be photographed with great depth of field and in stereo revealing their true three dimensional (3D) structures. The Confocal Laser Scanning Microscope (CLSM), a microscope that uses specific wavelengths of light produced by lasers to excite fluorescent compounds, has the ability to optically (non-destructively) slice through specimens and identify fluorescently labeled tissues, proteins, organisms, cells, etc. The ECMU staff, using software, interactively reconstructs the slices to produce 3D renderings. Techniques that will be used include: critical-point drying apparatus, sputter coating devices, glow discharger , carbon and other metal evaporation systems, freeze-etching equipment , ultra-microtomes, centrifuges, a freeze substitution system, stereo microscopes, TEM prep microwave system, vacuum oven, incubators , 60” and 40 “ large screen monitors, computer equipment for image storage, digitization, printing, and associated software as well as conventional laboratory equipment. Members of the ECMU are responsible for training all personnel on the proper use and maintenance of the microscopes and equipment within the facility. The final result is dramatic, high-resolution, digitally-achievable images of many of the most important pests and pathogens affecting agriculture.
3. Progress Report:
This is new project, 8042-22000-305-00D, initiated on March 25, 2017 carried over from project 8042-22000-278-00D which was terminated on March 24, 2017. Progress has been made in the use of a table top Scanning Electron Microscope to visualize the outer surface of pollen grains from a U.S. germplasm collection of varieties of plants from the cucumber family. Studies have been completed for the discernment of chromoplasts and chloroplasts in tomato transformed with genes that regulate fruit ripening. Mites from the U.S. and 6 other countries were imaged for systematic entomological identification using the LT-SEM to determine if the mites carry the citrus leprosis virus. Images of two new species of smut fungi were taken with the LT-SEM. Molecular characterization of the fungi is underway.