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:
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 by purchasing a table top scanning electron microscope which was used to characterize the pollen of plant species closely related to cucumber in a germplasm collection. Confocal laser scanning microscopy studies of transgenic tomato fruits demonstrated that genes inserted into tomatoes could cause delayed fruit ripening. Additional progress was made on sixty different projects where staff of the Electron and Confocal Microscopy Unit collaborated with scientists to describe four new species of mites, explain the unique feeding system of the tea mite, elucidate the evolutionary pathway of four-legged plant feeding mites, characterize four new parasitic Sacrocystis in mammals and birds, illustrate the morphological and molecular lineages of alpha-baculoviruses which attack insects, re-discover how aphids walk along surfaces, describe the proteomic biochemistry for symbiotic nitrogen fixation, and visualize how bacteria can get inside harvested tomato fruits. 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. Scanning electron microscopy was used in tandem with molecular studies to characterize new species of Bipolaris and Curvularia that are associated with browntop millet. Light microscopy and low-temperature scanning electron microscopy confirmed the presence of Brevipalpus yothersi, the mite that carries citrus leprosis virus in Brazil. Confocal laser scanning microscopy studies showed that the coat protein sequence of Lolium latent virus could be manipulated to reduce its level of movement from cell to cell, thus stopping its spread. Bright-field microscopy and transmission electron microscopy studies of the root-lesion nematode revealed that the nematodes penetrated the roots and fed on the cell contents producing necrosis, a browning and cell death at the outside layers of the roots; however, tannins and secondary metabolites produced by the plant prevented the nematodes from feeding on the nutrient rich vascular cylinder.
1. Infection of root-lesion nematodes on Easter lilies. Easter lilies are an important floral crop with an annual wholesale value of over $26 million in the United States. Root-lesion nematodes cause disease on Easter lilies. USDA ARS scientists in Beltsville, Maryland, developed a system for growing Easter lilies in a sterile environment which allowed roots to be visualized during growth and which allowed for microscopic study of the nematode’s infection process. Bright-field microscopy and transmission electron microscopy studies of the root-lesion nematode revealed that the nematodes penetrated the roots and fed on the cell contents producing necrosis, a browning and cell death at the outside layers of the roots. However, tannins and secondary metabolites produced by the plant prevented the nematodes from feeding on the nutrient rich vascular cylinder. This system can be used by scientists, breeders and horticulturalists as a fast and efficient method of screening new cultivars or plants to determine resistance to the root-lesion nematode.
2. A newly discovered interaction between Lolium latent virus and a plant protein. Lolium latent virus can cause significant damage and disease losses in crops such as ryegrass. Understanding how viruses interact with their plant hosts is a step towards protecting plants from viral disease. USDA ARS scientists in Beltsville, Maryland, examined the outer proteins that coat Lolium latent virus. The scientists demonstrated that specific molecules in the coat target proteins associated with chloroplasts of the plant cell. The scientists showed that this interaction ultimately facilitates the transfer and spread of the virus. It is expected that these findings will enable virologists and pathologists at companies and research institutions to develop countermeasures to mitigate the disease and to reduce disease loss in plants like ryegrass.
De Araujo, M.S., Bichuette, M.E., Bauchan, G.R., Ochoa, R., Feres, R.F. 2018. A new species of cave dwelling Neocarus (Acari: Opilioacaridae) from Bahia State, Brazil, with remarks on taxonomic characters. Zootaxa. 4402:303-322.
Skvarla, M.J., Miller, G.L., Bauchan, G.R., Lewis, M.L., Foottit, R., Maw, E. 2018. Taxonomy and natural history of a cattail aphid, Rhopalosiphum enigmae Hottes & Frison (Hemiptera: Aphidomorpha: Aphididae), including a new synonymy and notes on ant and parasitoid associates of Rhopalosiphum. Insect Systematics and Diversity. 2(2):1-14.
Jacinavicius, F.C., Bassini-Silva, R., Munoz-Leal, S., Hingst-Zaher, E., Ochoa, R., Bauchan, G.R., Barros-Battesti, D.M. 2018. Quadraseta brasiliensis Goff & Gettinger, 1 1989 (Acari: Trombidiformes: Trombiculidae): neotype designation, redescription of larva and description of deutonymph. Acarology International Congress Proceedings. 58(2):442-456.
Childers, C.C., De Lillo, E., Bauchan, G.R., Rogers, M.E., Ochoa, R., Robinson, C. 2018. The external morphology of the mouthparts, and observations on feeding and behavior of Tuckerella japonica on Camellia sinensis in the continental United States. Experimental and Applied Acarology. 74(1):55-71.
Klimov, P., Oconnor, B.M., Chetverikov, P.E., Bolton, S.J., Amir, R., Abdolazim, L., Andrey, V., Bauchan, G.R., Ochoa, R. 2018. Comprehensive phylogeny of acariform mites (Acariformes) provides insights on the origin of the four-legged mites (Eriophyoidea). Molecular Phylogenetics and Evolution. 119:105-117.
Cooper, B., Campbell, K., Beard, H.S., Garrett, W.M., Mowery, J.D., Bauchan, G.R., Elia, P.E. 2018. A proteomic network for symbiotic nitrogen fixation efficiency in Bradyrhizobium elkanii . Molecular Plant-Microbe Interactions. 31(3):334-343. https://doi.org/10.1094/MPMI-10-17-0243-R.
Zhou, B., Luo, Y., Bauchan, G.R., Feng, H., Stommel, J.R. 2017. Visualizing pathogen internalization pathways in fresh tomatoes using MicroCT and confocal laser scanning microscopy. Food Control. 85:276-282.
Dubey, J.P., Naji, N., Mowery, J.D., Verma, S., Calero-Bernal, R. 2017. Identification of macroscopic sarcocysts of Sarcocystis cameli from camels (Camelus dromedarius) in Iraq. Journal of Parasitic Diseases. 103(2):168-169.
Vieira, P., Mowery, J.D., Kilcrease, J., Eisenback, J.D., Kamo, K.K. 2017. Cytological changes of Easter lily (Lilium longiflorum) upon root lesion nematode (Pratylenchus penetrans) infection. Plant Pathology. 49:1-11.
Verma, S., Lindsay, D., Mowery, J.D., Rosenthal, B.M., Dubey, J.P. 2017. Sarcocystis pantherophis, n. sp. from eastern rat snakes (Pantherophis alleghaniensis) definitive hosts and interferongamma gene knockout mice as experimental intermediate hosts. Journal of Parasitology. 103:547-554.
Harrison, R.L., Rowley, D.L., Mowery, J.D., Bauchan, G.R., Burand, J.P. 2017. The Operophtera brumata nucleopolyhedrovirus (OpbuNPV) represents an early, divergent lineage within genus Alphabaculovirus. Viruses. 9(10):307.
Hermandes, F.A., Bauchan, G.R., Ochoa, R. 2017. New and little known feather mites (Acari). Parasitology Research. 43(7):499-517.
Metz, M., Miller, D.R., Dickey, A.M., Bauchan, G.R., Ochoa, R., Skvarla, M.J., Miller, G.L. 2017. Rediscovering digitules in Aphidomorpha and the question of homology among Sternorrhyncha (Insecta: Hemiptera). ZooKeys. 4276(1):139-144.
Cezar, C., Thompson, P., Verma, S., Mowery, J., Caler-Bernal, R., Sinnett, D., Van Hemert, C., Rosenthal, B.M., Dubey, J.P. 2017. Morphological and molecular characterization of Sarcocystis arctica-like sarcocysts from the Arctic fox (Vulpes lagopus)from Alaska, USA. Veterinary Parasitology. 116:1871-1878. https://doi.org/10.1007/s00436-017-5462-6
Verma, S., Rosypal Von Dohlen, A., Mowery, J.D., Scott, D., Rosenthal, B.M., Dubey, J.P., Lindsay, D. 2017. Sarcocystis jamaicensis, n. sp. from red-tailed hawks (Buteo jamaicensis) definitive host and IFN-Gamma gene knockout mice as experimental intermediate host. Journal of Parasitology. 103:555-564. https://doi.org.10.1645/17-10
Hermandes, F.A., O'Connor, B.M., Bauchan, G.R., Ochoa, R. 2017. A new species of Proctophyllodes Robin, 1877 from the Scarlet Tanager Piranga olivacea (Gmelin) (Passeriformes: Cardinalidae) from North America. International Journal of Acarology. 51:2407-2416.