1a. Objectives (from AD-416):
The long-term objectives of this project involve the application of electron and confocal microscopy for the observation of a wide range of sample materials impacting agricultural productivity, the goals of which include the protection of plants, animals and humans from various pathogens and parasites. The Beltsville Agricultural Research Center (BARC) Electron and Confocal Microscopy Unit (ECMU) serves the research projects at BARC 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 and apply new techniques and methodologies in electron and confocal microscopy that facilitate the identification, characterization, and systematics of plant pathogens and other pests, and their interaction with their hosts; and 2) Provide technical support and expertise specific to individual research projects with BARC scientists and their collaborators for achieving previously unobtainable data and improvement of the quality of imaging results.
1b. Approach (from AD-416):
The Electron and Confocal Microscopy Unit (ECMU) is a core facility which provides collaborative assistance for the Beltsville Agricultural Research Center scientists in need of high resolution imaging in their research programs. The facility is equipped with state-of-the-art electron microscopes [transmission (TEM) and scanning (SEM)] and a confocal laser scanning microscope (CLSM). A scanning electron microscope equipped with a cryostage allows samples to be studied in the frozen state for the observation of ultra-delicate external structures of organisms and the study of behavioral interactions on their hosts. Variable pressure SEM requires minimal specimen preparation for rapid observation of specimens. The ECMU also possesses a high-resolution transmission electron microscope that is used to obtain detailed internal ultra-structural information from very thin sections of plastic embedded material. The CLSM permits the study of fluorescent organelles, tissue, proteins, cells with high color contrast and in 3 dimensions. Cellular and subcellular structures are easily observed and photographed using these approaches to provide compositional information as well. Techniques including critical point drying, freeze drying, freeze substitution, freeze etching, rapid freezing, negative staining, thin sectioning, sputter coating, high vacuum evaporation, immunogold labeling, single and multiplex fluorescent labeling are performed on a wide variety of sample types that arise from research projects at BARC. If existing methods are inadequate for appropriately viewing a particular specimen, new techniques, preparative methodologies, fluorochromes, specimen holders and equipment are designed and developed. Training will be provided to staff of collaborating scientists to assist in creating and enhancing images. The final result is dramatic, high-resolution, digitally-achievable images of many of the most important organisms affecting agriculture.
3. Progress Report:
Scientists at the Beltsville Agricultural Research Center and their collaborators are working on numerous and varied research projects and many of them have an occasional or frequent need to visualize samples using microscopy techniques provided by the Electron and Confocal Microscopy Unit (ECMU). There were ten different projects for which the ECMU provided support which included: characterization of viruses attacking bees; studies of animal parasites (Giardia and yeast tachyozoites); investigative studies of insects (mosquitoes and Hemlock Woolly Adelgid); detailed morphological examination of three genera of mites affecting citrus, oranges, lemons, and grapefruits; localization of gene insertions in tomato and soybeans; identification of plant tissues and organelles which store heavy metals for decontamination of soils; and food safety issues including: bacterial contamination of spinach and cabbage and cyptosporidium on the surface of peach, tomato, and apples.
1. Flat mites related to the red palm mite feed on stomata of plants. Flat mites such as false spider mites, red palm mites and peacock mites are devastating pests on bananas, coconuts, date palms, olive, eucalyptus, and ornamental palms. ARS researchers at Beltsville, MD described these mites in detail and through low temperature scanning electron microscopy studies discovered that these mites all specialize in feeding on the stomata of plants. Detection of this feeding method will lead to the discovery of how best to control these major pests by farmers, extension agents, state and university researchers and their identification by APHIS quarantine specialists.
2. Web based flat mite identification tool available. Flat mites are major pests of horticultural and crop plants and are difficult to identify. Collaborative efforts with ARS researchers at Beltsville, MD; University of Maryland, College Park, MD; and the Animal and Plant Health Inspection Service (APHIS) led to the development of an on-line flat mite identification tool. This web based interactive key uses descriptions and hundreds of images obtained with the use of dissecting microscopes, light microscopes and a low temperature scanning electron microscope to correctly identify these microscopic organisms. This is a practical identification tool which can be used by researchers, non-professionals, and APHIS in its duties to protect our country from invasive harmful mite species.
3. Fluorescently tagged fungi used to determine pathogenesis in plants. The observation of pathogenic fungi as they grow along the surface and penetrate plants is difficult to study. A cyan colored fluorescent tag was inserted into a fungus which attacks gladiolus bulbs in order for ARS researchers at Beltsville, MD to follow its growth in 3D using a Confocal Laser Scanning Microscope. This new and novel technique will be used to study how the fungus enters the plant tissue in order to identify methods for preventing infection.
Beard, J.J., Ochoa, R., Bauchan, G.R., Welbourn, W.C., Pooley, C.D., Dowling, A.G. 2012. Flat mite mouthparts and feeding: Raoiella a case study (Tetranychoidea: Tenuipalpidae). Experimental and Applied Acarology. 57(3-4):227-255.