Project : USDA ARS

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Research Project: Microscopy Applications for the Identification and Management of Agricultural Pests and Pathogens

Location: Systematic Entomology Laboratory

2020 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 fifty different projects where studies were initiated, continued, and finalized by the staff in the Electron and Confocal Microscopy Unit by collaborating with USDA ARS, university and international scientists. Some of the projects are highlighted. A new species of aphid that feeds on strawberries was discovered using Low-Temperature Scanning Electron Microscopy (LT-SEM). Due to the waxy coating, delicate ornamentation, and high aqueous content of the aphid, imaging of the aphid would not have been possible using standard SEM techniques; thus, freezing with liquid nitrogen and observation in the LT-SEM made these studies possible. This aphid is a new pest on strawberries in Mississippi. Two new species of white mites were discovered and described on cocoa trees in Brazil. Multiple microscopy techniques were used including differential interference contrast (DIC) microscopy, LT-SEM and for the first-time, confocal laser scanning microscopy (CLSM). These microscopy techniques discovered unique morphological structures which proved to be critical for distinguishing these new species of mites on cocoa trees. These mites appear to feed on fungi which may include pathogenic fungi that attack cocoa trees. A very small, four-legged mite was found for the first time in the U.S. in Florida on lychee trees. These mites cause significant damage to the lychee tree. The mites produce leaf galls and stimulate the overproduction of trichomes which appears as a velvet patch that reduces fruit production by as much as 80%. Molecular and LT-SEM analysis determined that the mites are the same species found in India, Hawaii, Brazil, and Taiwan. This discovery caused an alert for quarantine of the plants and area by the Florida Department of Agriculture and USDA-APHIS. Extreme measures have been taken to eradicate the pest by implementing pruning infected branches, removing infected trees and burning them. Confocal laser scanning microscopy was used to identify key characteristics of tobacco budworm and bollworm eggs which led to the development of 3D renderings of the top of the eggs and their conversion to 3D printed plastic models. These models can be used by the USDA-APHIS port inspectors in the identification of invasive insect eggs. Transmission electron microscopy (TEM) studies were conducted to describe a new species of purple pigmented bacteria found in tidal portions of the Potomac and James Rivers in Maryland and Virginia. Some of the species related to this new bacterium have been reported to produce insecticidal factors. Research on the use of several microscopy techniques has been used to redescribe several species and describe 13 new species of mites found in the Brazilian Amazon River basin. Most of these mites feed on fungi and bacteria and glide between trees, having the potential of spreading pathogenic fungi and bacteria to other trees. Confocal laser scanning microscopy along with computerized imaging enhancement techniques have been instrumental in describing the intricate reproductive systems of mites associated with dung beetles. The genitalia of the mites are found on the pincer-like mouth parts of the mite with the male exhibiting a colossal, coiled spring-like structure which unfurls inside the equally complex coiled structure internal to the female’s lower pincher. The images proved that the male reproductive system largely matches the internal spiral structure of the female which is valuable for study of sexual selection among mite species. These beetles and their associated mites contribute to the ecological health of soils. A new coupling agent was tested to improve adhesion of epoxy resins to waxy biological samples for fixing and embedding plant tissue for TEM studies. Binding resins to biological samples allows for the tissue of interest to retain its structure so it is not distorted when sectioned in an ultramicrotome for TEM observations. Many insects and plants produce waxy outer coverings which are difficult to section because the epoxy resin does not stick to the surfaces. This new process will enable the preparation of difficult tissues for analysis in the TEM. A new glow discharge cleaning system that also allows aqueous solutions to spread easily was used for preparing TEM grids for negative staining of iflaviruses on stink bugs. TEM observations of the shape of isolated iflaviruses confirmed molecular studies of the iflaviruses found on U.S. and Brazilian stink bugs. The importance of this discovery is that these viruses can produce large quantities of their RNA without causing any obvious deleterious effects to the insects unlike other iflaviruses on insects. A tropical fowl mite (chigger) typically found on domestic and wild birds in Brazil was found to cause dermatitis in humans. LT-SEM observations of the chigger were the first to reveal the natural state of the chigger. It is likely that the process of freezing the organism for microscopy prevented artifacts typically found in standard SEM preparations. Infestation on humans appears to occur mainly when the abandoned nests are close to a home. The effect of various glucosinolate hydrolysis compounds found in mustard plants was tested to determine the mode of action these compounds have on bacteria. Application of the compounds to E. coli and Salmonella proved that the compounds were effective at stopping bacterial growth. The compound benzyl isothiocyanate (BIC) was the most effective. Additional experiments showed that motility and toxin production were noticeably reduced after treatment with BIC. Fluorescent labeling of live and dead bacteria following treatment and observation in the CLSM confirmed the effectiveness of BIC. TEM was used to visualize the ultrastructural changes at the cellular level, revealing that the cell membranes were disrupted by lysis. These investigations suggest that BIC could be used to attenuate E. coli and Salmonella. 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. Beech tree leaves displaying disease symptoms were frozen in liquid nitrogen, freeze fractured and observed in a LT-SEM. This method exposed the nematodes within the leaves. This discovery is unique as typically nematodes are found in soils, not inside plant leaves. A novel feeding chamber was designed for the observation of predatory mites feeding on free-living nematodes and root-knot nematodes. This allowed for the video recording of the feeding and the subsequent flash freezing of the mites feeding, which allowed imaging in a LT-SEM. The necessity of the availability of free-living nematodes as a food source for the viability of maintaining predatory mites for their use in biocontrol of plant parasitic nematodes is a significant finding.

Accomplishments
1. Description, transmission and distribution of a nematode subspecies causing beech leaf disease in North America. The American beech tree is a major tree species in North American deciduous forests and in 2012, the green and yellow stripes of beech leaf disease (BLD) were first discovered to be a problem near Cleveland, OH. Wanting to understand the disease, scientists from USDA ARS, Beltsville, Maryland, USDA Forest Service (Delaware, Ohio and Morgantown, West Virginia), the state of Ohio (Reynoldsburg), Holden Arboretum (Kirtland, Ohio) and the province of Ontario, Canada (Ottawa and Sault Ste. Marie), used multiple microscopic methods and molecular biology to identify and describe a new subspecies of foliar plant-parasitic nematodes in the beech leaves. The scientists demonstrated nematode transmission to beach buds and leaves, and described its eastward movement and distribution within North America during different seasons. This potentially lethal BLD nematode is of international concern because it is believed to be an invasive species from Asia where it causes relatively minor damage. These results are important to pathologists, arborists, and regulators of domestic and international trade who want to contain this nematode and reduce its destruction.

2. Life cycle of human parasite Cyclospora explained. A growing threat to food safety is posed by the enigmatic parasite Cyclospora cayetanensis that caused 2,299 cases in 33 states with more than 160 hospitalizations in 2018; further multi-state outbreaks of enteric disease in people eating contaminated basil, cilantro, raspberries, and other fresh produce resulted in several produce products being recalled. Proper diagnosis of Cyclospora has been hampered by previous failures to understand how it develops after it is ingested. It previously was thought to develop in the gut lumen and then invade cells lining the small intestine. In a landmark study, USDA ARS, Beltsville, Maryland, scientists working with collaborators at the Food and Drug Administration used light microscopy, transmission electron microscopy and molecular biology techniques to obtain unprecedentedly detailed observations of the morphology and nuclear components of Cyclospora at each stage of its life cycle. Doing so discovered new avenues by which the parasite multiplies during infection in the intestinal and gallbladder epithelia cells. These findings will aid the work of pathologists in diagnosing Cyclospora infection and may help agricultural producers better understand and manage a growing public health burden.

U.S. DEPARTMENT OF AGRICULTURE

Systematic Entomology Laboratory: Beltsville, MD