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ARS Home Northeast Area Beltsville, Maryland (BARC) Beltsville Agricultural Research Center Systematic Entomology Laboratory Research





- Jansen, Michael - Andrew
- 2023
- Plant Diseases (303)

Research Project: Microscopy Applications for the Identification and Management of Agricultural Pests and Pathogens (Bridge) **Location:
                            Systematic Entomology Laboratory** 2023 Annual Report **Objectives**
                                

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] **Approach**
                                

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. **Progress Report**
                                

The first objective of this project is to develop new techniques and methodologies in microscopy that generate high resolution images of biological specimens more efficiently and effectively. Progress was made on fifteen different projects where studies were initiated, continued, or finalized by the Electron and Confocal Microscopy Unit in collaboration with USDA-ARS [MNGDBL, SASL, FQL, GIFVL, MPPL, APDL], U.S. National Arboretum [FNPRU], FDA [NCTR Division of Microbiology], and Smithsonian Institute [Arachnology], university [Purdue University, University of Florida], and international [University of Bonn, University of Cologne, Imperial College of London] scientists. Of these projects, 1 manuscript is undergoing peer review, 3 are currently in ARS internal review, while 2 others are in the active writing phase.

Of note, a method was developed to visualize floating live organisms from multiple viewpoints, including lateral and ventral views. This method allows for full immersion of the specimens and provides live, in-situ views with fully microscopic resolution and epifluorescence, if desired. The method relies on a specially designed immersion tank, which is currently being adapted for rapid production with 3D printing. This innovation will facilitate additional live-view studies of both macroscopic and microscopic organisms and has a wide application space.

The second objective of this project is to apply novel microscopy approaches to facilitate the systematic identification and characterization of plant pathogens and pests, alone or with their hosts. Several new methods were developed, as described briefly below.

A new method was developed for rapid, high-throughput screening of soil samples, which was tested on the phytopathogenic bacterium Ralstonia solanacearum, which is a major pest of tomatoes and other solanaceous crops. The method relies on the combined application of fluorescent labeling, the most-probable-number assay, and liquid handling in microtiter plates. The plates can be rapidly read and analyzed to produce accurate pathogen counts using a plate-reader in a semi-automated fashion, thereby enabling scaling to large numbers of plates and samples.

A new program was written to automate the production of specimen data and locality labels, which is currently being optimized for online deployment. The software [Title: L-gen, Platforms: Windows/Linux, Languages: Python3/LATEX] was designed to perform automated data parsing, style formatting, and generation of entomological labels from bulk sample collecting data. It is currently being used by members of Systematic Entomology Laboratory stationed at the Smithsonian Institution in the Entomological Collections of the Natural History Museum. This method eliminated the need for tedious data-copying and formatting from databases and can potentially save specialists and technicians weeks of work on this task. **Accomplishments**


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