Progress 03/25/12 to 03/24/17
Outputs
Progress Report 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. 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. This is the final report for project 8042-22000-278-00D which terminated in March 24, 2017. Extensive results were realized over the 5 years of the project. Over 60 different collaborative research projects were completed which facilitated accomplishment of the first objective of this project: the identification, characterization, and systematics of plant pathogens and other pests, and their interaction with their hosts. Some examples are: 1) Discoveries have been made by the use of fluorescent tags and confocal laser scanning microscopy to study plant virus movement between cells in plant leaves. The results helped prove that some virus spread through the vascular tissue while others do not. 2) Low- temperature scanning electron microscopy (LT-SEM) and transmission electron microscopy (TEM) was used to prove that Chrysanthemum white rust is a systemic disease in Chrysanthemum. 3) Fusarium oxysoporum transformed with a fluorescent tag was used to study its initial growth on the surface of resistant Gladiolus plants. 4) Fluorescent microscopy was used to study the growth of blue mold decay in stored apples. The results showed the potential usefulness of germplasm obtained from Kazakhstan for reducing growth of blue mold. 5) Confocal microscopy studies of soybean cyst and root-knot nematodes on plants with resistance genes showed that the genes were responsible for thickened cell walls of steel cells in the root which helped protect the vascular tissue from penetration by the nematodes. 6) LT-SEM was used to discover 10 new species of mites that feed on agriculturally important plants. These data alerted scientists and border control agents of these potentially invasive species. 7) Confocal microscopy, SEM and TEM of various mites helped determine which species of mite carries the citrus leprosis virus. These data help to inform quarantine agents of these invasive species. 8) Microscopy studies of the mite that transmits the rose rosette disease revealed that the mites feed primarily at the base of the sepals of the flower where they are protected by plant trichome hairs. Because of this protection, it may be difficult to reach the mites with chemical sprays. 9) LT-SEM studies showed that the red palm mite feeds only in the stomata of leaves. The results show that systemic or biological controls will be better suited than contact miticides to manage this invasive species. All together, research on this objective led to 30 scholarly scientific publications through 50 collaborations with scientists from ARS and other federal government agencies, universities and international organizations. The second objective of this project was to provide technical support and expertise specific to individual research projects with Beltsville Agricultural Research Center scientists and their collaborators for achieving previously unobtainable data and to improve the quality of imaging results. The staff of the Electron and Confocal Microscopy Unit (ECMU) collaborated with more than 80 scientists. Projects included the characterization of life forms including viruses (citrus, roses, tobacco, bees, and army worm), bacteria (on spinach, lettuce, apple, citrus, and bees), fungi (on Chrysanthemum, Gladiolus, and roses), animal parasites (Giardia, cryptosporidium, and sarcosystis), nematodes (3 genera), insects (aphids, pysllids, mosquitoes, coffee bean borer, thrips, and Hemlock Woolly Adelgid), and mites (18 genera). In addition, the laboratory provided microscopy assistance to visualize proteins experimentally expressed in tomato, tobacco, spinach, and soybeans; to characterize healthy and infected plant and chicken tissues; and to study biodegradable plastics made from chicken feathers. Projects related to food safety included the study of bacterial biofilms, bacterial contamination of tomatoes, spinach, and lettuce, and cyptosporidium contamination of spinach, lettuce, and apples. Over the term of the project, 42 scholarly scientific studies were published. Images obtained through these research projects appeared on the covers of 15 scientific journals, and have been displayed on USDA and ARS web pages, in text books, in museum displays, and on multiple web pages. This project has supported collaborations with more than 90 scientists from around the world. Members of the laboratory increased knowledge by providing tours of our facility to more than 50 different groups including visitors from the White House Science Advisory Council, USDA and ARS, National Parks Service, Smithsonian Museums, and the National Science Foundation. Routine visitors included university professors, postdoctoral scientists, graduate, undergraduate, and high school students. International visitors came from from Australia, Brazil, Canada, Chile, China, Colombia, Costa Rica, Egypt, Germany, Great Britain, Israel, Jamaica, Mexico, The Netherlands, New Zealand, Poland, Peru, Russia, South Africa, Spain, and Venezuela. A highlight for the project was a 30 minute live video tour of the microscopy facility by PBS News Hour. The tour was shown on the PBS Facebook Science page and was viewed 26,000 times. Accomplishments 01 Bee mite identification on the web. Bees play a crucial role in U.S. agriculture as pollinators of many important crops. There are mites that live on bees and some are parasitic to bees, spreading viruses that contribute to colony collapse, while other mites have little detrimental effect on bees. ARS scientists in Beltsville, Maryland, along with researchers at the University of Michigan and USDA-APHIS designed an interactive, web-based tool for the identification of mites found on bees. The searchable image gallery contains over 850 mite images with an emphasis on mites associated with important pollinators, including honey bees, mason bees, and bumble bees in temperate regions, and stingless bees and large carpenter bees in the tropics. This interactive site is useful to bee keepers, scientists, extension agents, and quarantine officers worldwide who want to distinguish harmless mites from those that might harm bees or their colonies.
Impacts
(N/A)
Publications
- Kendra, P.E., Owens, D., Montgomery, W.S., Narvaez, T.I., Bauchan, G.R., Schnell, E.Q., Tabanca, N., Carrillo, D. 2017. a-Copaene is an attractant, synergistic with quercivorol, for improved detection of Euwallacea nr. fornicatus (Coleoptera: Curculionidae: Scolytinae). PLoS One. 12(6) :e0179416.
- Harrison, R.L., Rowley, D.L., Mowery, J.D., Bauchan, G.R., Theilman, D.A., Rohrmann, G.F. 2017. The complete genome sequence of a second distinct betabaculovirus from the true armyworm, Mythimna unipuncta. PLoS One. 12(1) :e0170510.
- 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.
- Vega, F.E., Bauchan, G.R., Infante, F., Davis, S. 2017. Mouthpart structure and elemental composition of the mandibles in the coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae). Annals of the Entomological Society of America. 110:381-389.
- Chetverikov, P.E., Amrine, J., Bauchan, G.R., Ochoa, R., Sujhareva, S.I., Vishnyakov, A.E. 2017. Supplementary description of Novophytoptus stipae Keifer 1962 (Acariformes, Eriophyoidea) with LT-SEM observation on mites from putatively conspecific populations: cryptic speciation or polyphagy. Systematic and Applied Acarology. 22(2):253-270.
- Castro, E.B., Ramos, F.M., Feres, R.F., Ochoa, R., Bauchan, G.R. 2017. Redescription of Tenuipalpus heveae Baker (Acari: Tenuipalpidae) and description of a new species collected on rubber tree from Amazonia, Brazil. Acarologia. 57(2):421-458.
- 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.
- Calero-Bernal, R., Cerqueira-Cezar, C., Verma, S., Mowery, J.D., Carmena, D., Beckman, K., Dubey, J.P. 2016. Sarcocystis arctica (Apicomplexa: Sarcocystidae): ultrastructural description and its new host record, the Alaskan wolf (Canis lupus. Parasitology Research. 115:2893-2897.
- Castro, E.B., Feres, R.F., Ochoa, R., Bauchan, G.R. 2016. Tenuipalpus sensu stricto (Acari: Tenuipalpidae) from Brazil, with ontogeny and a key to the known species. Zootaxa. 4088(3):355-378.
- Broadhurst, L., Schmidt, W.F., Kim, M.S., Nguyen, J.K., Qin, J., Chao, K., Bauchan, G.R., Shelton, D.R. 2016. Continuous gradient temperature Raman spectroscopy of n-6 DPA and DHA from -100 C to 20�C. Chemistry and Physics of Lipids. 200:1-10.
- Han, J., Chung, J., Kim, J., Seo, E., Kilcrease, J.P., Bauchan, G.R., Lim, S., Hammond, J., Lim, H. 2016. Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus. Virus Genes. 52:592-596.
- Borges, L.M., Li, A.Y., Olafson, P.U., Renthal, R., Bauchan, G.R., Lohmeyer, K.H., Perez De Leon, A.A. 2016. Neuronal projections from the Haller's organ and palp sensilla to the synganglion of Amblyomma americanum. Revista Brasileira de Parasitologia Veterinaria. 25(2):217-224.
- Gou, A., Xu, Y., Mowery, A.K., Nagy, A., Bauchan, G.R., Nou, X. 2016. Ralstonia insidiosa induces cell aggregation by Listeria monocytogenes. Food Control. 67:303-309.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report 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. 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. The first objective of this project is to 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. Progress was made by purchasing and using a wide field fluorescence microscope to observe multiple resistance mechanisms against blue mold decay produced by wild apple wound responses. Low-temperature scanning electron microscopy using a standard and tilted specimen holder was used to differentiate several new species of mites found in the Brazilian Amazon River Basin. Three different confocal laser scanning microscopy (CLSM) fluorochromes were used to visualize a triple acting lytic enzyme (fluorescent green) treatment of drug resistant Staphylococcus aureus (fluorescent red) and nucleus and cell membranes (fluorescent blue). Transmission electron microscopy (TEM) methods were developed to extract paraffin embedded muscle tissue so that it could be embedded in resin for ultra-thin sectioning for the observation of neurological sarcocystosis in cattle. Critical point drying was used to help observe a new Trypanosomatidae parasite of bees by SEM. The second objective of this project is to provide technical support and expertise specific to individual research projects with Beltsville Agricultural Research Center scientists and their collaborators for achieving previously unobtainable data and to improve the quality of imaging results. Progress was made on seventy-two different projects where staff of the Electron and Confocal Microscopy Unit (ECMU) collaborated with scientists. This year�s projects included: discovery of multiple resistance mechanisms against blue mold decay produced by wild apple wound responses; discovery of an ethanol extract for Serratia marcescans compatible with Trichoderma isolates for control of damping off of cucumber seeds; ultra-structural morphological examination and clarification of the taxonomy of citrus mites that vector citrus leprosis viruses; discovery of several new species of mites found in the Brazilian Amazon River Basin; a triple acting lytic enzyme treatment of drug resistant Staphylococcus aureus; discovery of neurological sarcocystosis in cattle through pathological, immunohistochemistry, and ultrastructural research; discovery of a new Trypanosomatidae parasite of bees; discovery that the bacteria Ralstonia insidiosa can serve as a bridge species for several food borne pathogens; and discovery that Escherichia coli can proliferate on microgreens following inoculation of the original seed. Accomplishments 01 Wound responses in wild apple provide postharvest resistance to mold. Blue mold, caused primarily by Penicillium expansum, is the most destructive disease of stored apples in the U.S. and worldwide. It was recently shown that resistance to blue mold exists in wild apples (Malus sieversii) from Kazakhstan and in other wild apple species in a USDA collection in Geneva, New York. ARS scientists in Kearneysville, West Virginia and Beltsville, Maryland, determined the durability of resistance and the possibility of multiple mechanisms of resistance in some of the wild apples. Inoculation studies of the apples showed that resistant apples had quicker wound responses. Cytological studies showed that reactive oxygen molecules (peroxides, super oxides, free oxygen), commonly associated with apple resistance, were often detected at very high levels immediately after wounding even in susceptible accessions. The results indicate that the resistance mechanism(s) in wild apples is durable and complex and may be used in breeding programs for variety improvement.
Impacts
(N/A)
Publications
- Roy, A., Hartung, J.S., Schneider, W.L., Shao, J.Y., Leon, G.M., Melzer, M. J., Beard, J.J., Otero-Colina, G., Bauchan, G.R., Ochoa, R., Brlansky, R.H. 2015. Role bending: complex relationships between viruses, hosts and vectors related to citrus leprosis, an emerging disease. Phytopathology. DOI: 10.1094/PHYTO-12-14-0375-FI.
- Schwarz, R.S., Bauchan, G.R., Murphy, C.A., Ravoet, J., De Graaf, D.C., Evans, J.D. 2015. Genetic and ultrastructure characterization of a known and a new species of trypanosomatidae from the honey bee Apis mellifera: Crithidia mellificae Langridge and McGhee, 1967 and Leptomonas passim sp. n. Protist. 1:1-17.
- Xiao, Z., Bauchan, G.R., Nichols-Russell, L.K., Luo, Y., Wang, Q., Nou, X. 2015. Proliferation of Escherichia coli O157:H7 in soil and hydroponic microgreen production systems. Journal of Food Protection. doi: 10.4315/ 0362-028X.JFP-15-063.
- Bolton, S.J., Bauchan, G.R., Ochoa, R., Klompen, H. 2015. The Structure, Function and Evolution of a Novel Form of Fluid-feeding Apparatus for Microbivory. International Journal of Acarology. 44:313-325.
- Castro, E.B., Kane, E.C., Feres, R.F., Ochoa, R., Bauchan, G.R. 2016. Definition of Tenuipalpus sensu stricto (Acari, Tenuipalpidae), with redescription of Tenuipalpus caudatus (Dug�s) and description of a new species from Costa Rica. International Journal of Acarology. 42:106-126.
- Welbourn, W.C., Beard, J.J., Bauchan, G.R., Ochoa, R. 2016. Description of two species of Tenuipalpus (Acari: Trombidiformes) from succulent plants. International Journal of Acarology. 4088:(3):355-378.
- Favret, C., Tzaud, M., Erbe, E.F., Bauchan, G.R., Ochoa, R. 2015. Collophore may help direct springtail jump. International Congress of Acarology. 108:1-6.
- Rezende, J.M., Lofego, A.C., Ochoa, R. 2015. Two new species of Daidalotarsonemus (Acari: Prostigmata: Tarsonemidae) from Brazil. Acarologia. 55(4):435-448.
- Dubey, J.P., Calero-Bernal, R., Verma, S.K., Mowery, J. 2016. Pathology, immunohistochemistry, and ultrastructural findings associated with neurological sarcocystosis in cattle. Veterinary Parasitology. 223:147�152.
- Trupkiewicz, J.G., Calero-Bernal, R., Verma, S., Mowery, J.D., Davison, S., Habecker, P., Georoff, T.A., Ialeggio, D.M., Dubey, J.P. 2015. Acute, fatal Sarcocystis calchasi-associated hepatitis in Roller pigeons (Columbia livia f. dom.) at Philadelphia Zoo. Veterinary Parasitology. 216:52-58.
- Bakst, M.R., Bauchan, G.R. 2016. Lectin staining of epithelia lining the uterovaginal junction and sperm-storage tubules in chicken hens. Poultry Science. 95:948-955.
- Hernanes, F.A., Mironov, S.V., Bauchan, G.R., Ochoa, R. 2016. A new asymmetrical feather mite of the genus MIchaelia Trouessart, 1884 (Astigmata: Freyanidae) from the neotropical cormorant, Phalacrocorax brasilianus (Pelecaniformes). Acarologia. 56(1):45-61.
- Janisiewicz, W.J., Evans, B.E., Bauchan, G.R., Chao, C.T., Jurick II, W.M. 2016. Wound responses of wild apples suggest multiple resistance mechanism against blue mold decay. Postharvest Biology and Technology. 117:132-140.
- Kovalskaya, N.Y., Foster Frey, J.A., Donovan, D.M., Bauchan, G.R., Hammond, R. 2015. Expression of a bioactive bacteriophage endolysin in Nicotiana benthamiana plants. Journal of Microbiology and Biotechnology. 26:160-170.
- Nagy, A., Mowery, J.D., Bauchan, G.R., Wang, L., Nichols-Russell, L., Nou, X. 2015. Role of major surface structures of Escherichia coli O157:H7 in initial attachment to biotic and abiotic surfaces. Applied and Environmental Microbiology. 81:4720-4727.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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. There were fifty-five different projects supported by The Electron and Confocal Microscopy Unit (ECMU). The ECMU is a core facility where scientists at the Beltsville Agricultural Research Center and their collaborators apply microscopy techniques to their numerous and varied research projects. This year�s projects included: ultra-structural studies of the systemic infection of the fungus causing Chrysanthemum white rust in Chrysanthemum; ultra-structural morphological examination and clarification of the taxonomy of citrus mites that vector citrus leprosis viruses; discovery of new species of mites found in the Brazilian Amazon River Basin; investigative studies of the breathing organs and wing spurs of the Coffee Berry Borer; the use of fluorescent markers to discover important proteins in the Cryptosporidium parasite; expression of a synthetic antimicrobial peptide in Gladiolus plants; and the study of the surfaces of plants, the attachment of bacteria to plant surfaces, and the production of bacterial biofilms for surface attachment, which were all part of a larger food safety research effort. Accomplishments 01 Clarification of the identification of mites that spread the leprosies citrus virus. Brevipalpus mites feed on citrus plants and while feeding infect the plants with citrus leprosies viruses. The virus can kill a citrus tree within a few short years. ARS Researchers at Beltsville, Maryland, in collaboration with researchers in Animal Plant Health Inspection Service (APHIS) in Edinburg, Texas and the Queensland Museum in Australia clarified the identification of the mites that feed on citrus. Extensive observations were made and measurements of mites collected using differential contrast light microscopy and low temperature scanning electron microscopy. These techniques allowed the researchers to define characteristic structures and patterns for the correct identification of the citrus mites. The data allowed the scientists to determine which mites were vectors for the citrus leporsis viruses. This information was disseminated by a published monograph describing the various species and the pictures were shown on- line. The project web page had more than 123,800 visits and generated inquiries from more than 180 countries. This research will be used by researchers, citrus growers, and border inspection agents to safeguard our country from invasive, destructive diseases.
Impacts
(N/A)
Publications
- Beard, J.J., Ochoa, R., Braswell, W., Bauchan, G.R. 2015. Brevipalpus Phoenicis (Geijskes) species complex � a closer look. Zootaxa. 3944(1):1- 67.
- Bolton, S.J., Bauchan, G.R., Ochoa, R., Pooley, C.D., Klompen, H. 2015. The role of the integument with respect to different modes of locomotion in the Nematalycidae (Endeostigmata). Experimental and Applied Acarology. 65:149-161.
- Bonde, M.R., Murphy, C.A., Bauchan, G.R., Luster, D.G., Palmer, C.L., Nester, S.E., Revell, J., Berner, D.K. 2014. Evidence for systemic infection by Puccinia horiana, causal agent of Chrysanthemum White Rust, in Chrysanthemum. Phytopathology.
- Castro, E., Ochoa, R., Feres, R.J., Beard, J.J., Bauchan, G.R. 2015. Reinstatement of the genus Colopalpus Pritchard and Baker (1958) and re- description of Colopalpus matthyssei Pritchard and Baker (1958), the type species of this genus (Acari, Tenuipalpidae). International Journal of Acarology. 41:310-328.
- Bakst, M.R., Bauchan, G.R. 2015. Apical blebs on sperm-storage tubule epithelial cell microvilli: their release and interaction with resident sperm in the turkey hen oviduct. Theriogenology. 83:1438-1444.
- Fisher, R., Fisher, D.M., Nelson, W.A., O'Neill, J.C., Skvaria, M.J., Ochoa, R., Bauchan, G.R., Radwell, A.J., Dowling, A.G. 2015. Torrenticola trimaculata sp. nov. (Parasitengona: Torrenticolidae), a three-spotted water mite from eastern North America: taxonomic history, species delimitation, and survey of external morphology. Acarologia. 55(1):71-116.
- Kamo, K.K., Lakshman, D.K., Bauchan, G.R., Rajasekaran, K., Cary, J.W., Jaynes, J. 2015. Expression of a synthetic antimicrobial peptide, D4E1, in Gladiolus plants for resistance to Fusarium oxysporum f. sp. gladioli. Plant Cell Tissue And Organ Culture. 121:459-467.
- Kendra, P.E., Montgomery, W.S., Niogret, J., Pruett, G.E., Mayfield Iii, A. E., Mackenzie, M., Deyrup, M.A., Bauchan, G.R., Ploetz, R.C., Epsky, N.D. 2014. North American Lauraceae: Terpenoid emissions, relative attraction and boring preferences of redbay ambrosia beetle, Xyleborus glabratus (Coleoptera: Curculionidae: Scolytinae). PLoS One. 9(7):e102086.
- Rezende, J.M., Lofego, A.C., Ochoa, R., Bauchan, G.R. 2015. New species of Daidalotarsonemus and Excelsotarsonemus (Acari: Tarsonemidae) from the Brazilian rainforest including new morphological characters. Zoological Journal of the Linnean Society. 475(1):1-36.
- Vega, F.E., Bauchan, G.R., Infante, F., Valdez-Carrasco, J.M., Beaver, R. 2015. Visualizing the mesothoracic spiracles in a bark beetle: The coffee berry borer, Hypothenemus hampei. Annals of the Entomological Society of America. 108:446-448.
- Vega, F.E., Simpkins, A., Bauchan, G.R., Valdez-Carrasco, J.M., Castillo, A., Infante, F. 2015. A mysterious wing spine in male coffee berry borers. Florida Entomologist. 98:352-353.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. There were thirty different projects for which The Electron and Confocal Microscopy Unit (ECMU) provided support. The ECMU is a core facility for scientists at the Beltsville Agricultural Research Center and their Collaborators who are working on numerous and varied research projects, and have a frequent need to visualize samples using microscopy techniques. Projects in FY14 included: detailed morphological examination of a new genus and species of mite found in soils in Ohio; three genera of mites affecting citrus, coffee, and Australian oaks; studies of animal parasites (Eimeria and Bee viruses); investigative studies of the eye of the Coffee Berry Borer; identification of plant tissues and organelles which store heavy metals for decontamination of soils; the use of fluorescent markers to discover how viruses move from cell to cell within a plant; drought responses to elevated levels of carbon dioxide in corn; discovery of calcium crystals in plants which potentially provide insect resistance; and food safety issues including enhanced inactivation of bacteria in fresh produce and spinach and variation in bacterial attachment to the surfaces of plants. Accomplishments 01 Discovery of a new genus and species of soil mite. Soil mites are important for the health of soils as they provide aeration to the soils, help with the decomposition of organisms, and are a food source for other mites and insects. ARS researchers at Beltsville, MD in collaboration with researchers at The Ohio State University discovered a new genus and species of a strange and unique worm-like soil mite termed the Buckeye Dragon Mite. The research team used light microscopy and low temperature scanning electron microscopy to provide comprehensive morphological and systematic studies of this very unusual looking new mite. The publication of this finding is the most highly read scientific article published in the Journal of Natural History, with over 9,000 downloads. Since the publication of this new genus and species of mite in March 2014 over 80 million visits of people from all over the world have viewed the scanning electron microscopy images produced by scientists at Beltsville, MD. Findings based on the study of this mite will help researchers to understand mobility and feeding mechanisms of more evolved mites including plant feeding mites.
Impacts
(N/A)
Publications
- Beard, J.J., Seeman, O.D., Bauchan, G.R. 2014. Tenuipalpidae (Acari: Trombidiformes) from Casuarinaceae. Zootaxa. 3778:001-157.
- Barnaby, J.Y., Kim, M.S., Bauchan, G.R., Bunce, J.A., Reddy, V., Sicher Jr, R.C. 2013. Drought responses of foliar metabolites in three maize hybrids differing in water stress tolerance. PLoS One. 8(10):1-10.
- Bolton, S.J., Klompen, H., Bauchan, G.R., Ochoa, R. 2014. A new genus and species of Nematalycidae (Acari: Endeostigmata). Journal of Natural History. 48(23-24):1359-1373.
- Boncristiani, H., Evans, J.D., Chen, Y., Pettis, J.S., Murphy, C.A., Lopez, D.L., Simone-Finstrom, M., Strand, M., Tarpy, D., Rueppell, O. 2013. In- vitro infection of pupae with Israeli Acute Paralysis Virus suggests variation for susceptibility and disturbance of transcriptional homeostasis in honey bees (Apis mellifera). PLoS One. DOI: 10.1371/journal. pone.0073429.
- Broadhurst, C.L., Bauchan, G.R., Murphy, C.A., Tang, Y., Pooley, C.D., Davis, A.P., Chaney, R.L. 2013. Accumulation of zinc and cadmium and localization of zinc in Picris divaricata. Environmental and Experimental Botany. 87:1-9.
- Burchi, G., Bauchan, G.R., Murphy, C.A., Roh, M.S. 2014. Characterization of calcium crystals in Abelia using x-ray diffraction and electron microscopes. Journal of Horticultural Science and Biotechnology. 89:61-68.
- Fetterer, R.H., Schwarz, R., Miska, K.B., Jenkins, M.C., Barfield, R.C., Murphy, C. 2013. Characterization and localization of an Eimeria-specific protein in Eimeria maxima. Journal of Parasitology. 112(10):3401-3408.
- Citation: Lee, SH, Lillehoj, HS, Tuo, W., Murphy, CA, Hong, YH, Lillehoj, EP. 2013. Parasiticidal activity of a novel synthetic peptide from the core a-helical region of NK-lysin. Veterinary Parasitology 197:113-121.
- Vega, F.E., Simpkins, A., Bauchan, G.R., Infante, F., Kramer, M.H., Land, M.F. 2014. On the eyes of the coffee berry borer as rudimentary organs. PLoS One. 9(1):e85860.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. The Electron and Confocal Microscopy Unit (ECMU) is a core facility where scientists at the Beltsville Agricultural Research Center and their collaborators are working on numerous and varied research projects that have an occasional or frequent need to visualize samples using microscopy techniques. There were thirty different projects for which the ECMU provided support which included: detailed morphological examination of a new species of mite on skunk vine, an invasive plant in Florida; three genera of mites affecting citrus, tea, and palms; studies of animal parasites (Giardia and yeast tachyozoites); investigative studies of the Hemlock Woolly Adelgid; utilization of genetically transformed fluorescent fungi to observe how they invade roots of gladiolus; the use of fluorescent markers to discover how viruses move from cell to cell; identification of plant tissues and organelles which store heavy metals for decontamination of soils; and food safety issues including the development of biofilms; bacterial contamination of spinach and cabbage and cyptosporidium on the surface of peach, cucumber, tomato and apples. Accomplishments 01 Flat mite identification on the web. Flat mites such as false spider mites, red palm mites, citrus mites and peacock mites are devastating pests on citrus, tea, bananas, coconuts, date palms, olive, eucalyptus, and ornamental palms. ARS researchers at Beltsville, MD in collaboration with the Animal Plant Health Inspection Service (APHIS) have developed an on-line lucid identification key with descriptors and numerous images using light microscopy and low temperature scanning electron microscopy. Since its launch one year ago there have been over 123,800 visits to the web site with inquiries from 180 countries. This has been a tremendous identification tool for use by farmers, extension agents, state and university researchers, government agencies and APHIS quarantine specialists.
Impacts
(N/A)
Publications
- Lee, A.K., Hong, J., Bauchan, G.R., Park, S.H., Joung, Y.H. 2012. Confirmation of hybrid origin of Cyrtanthus based on the sequence analysis of internal transcribed spacer. Scientia Horticulturae. 144:153-160.
- Oten, K., Bauchan, G.R., Frampton, F.P., Hain, F.P. 2012. Biophysical characteristics of Adelges tsugae feeding sites on six hemlock (Tsuga) species and a hybrid: implications for resistance. Journal of Botany. 90:1170-1178.
- Beard, J.J., Ochoa, R., Childers, C.C., Bauchan, G.R., Shepard, M. 2013. Travelling with tea: a Tuckerella�s tale. Experimental and Applied Acarology. 59(1-2):177-202.
- Fayer, R., Santin, M., Macarisin, D., Bauchan, G.R. 2013. Adhesive-tape recovery combined with molecular and microscopic testing for the detection of Cryptosporidium oocysts on experimentally contaminated fresh produce. Parasitology Research. 112(4):1567-1574.
- Yossa, N., Patel, J.R., Millner, P.D., Murphy, C.F., Bauchan, G.R., Lo, M. 2012. Antibacterial activity of cinnamaldehyde and Sporan against Escherichia coli O157:H7 and Salmonella. Journal of Food Processing and Preservation. DOI: 10.1111/jfpp.12026.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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, a ornamental palms. ARS researchers at Beltsville, MD described these mit in detail and through low temperature scanning electron microscopy studi 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, stat and university researchers and their identification by APHIS quarantine specialists. 02 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 Inspectio Service (APHIS) led to the development of an on-line flat mite identification tool. This web based interactive key uses descriptions a hundreds of images obtained with the use of dissecting microscopes, ligh 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 mit species. 03 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 AR researchers at Beltsville, MD to follow its growth in 3D using a Confoca Laser Scanning Microscope. This new and novel technique will be used to study how the fungus enters the plant tissue in order to identify method for preventing infection.
Impacts
(N/A)
Publications
- 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.
- Macarisin, D., Patel, J.R., Bauchan, G.R., Giron, J., Sharma, V.K. 2012. Role of curli and cellulose expression by Escherichia coli O157:H7 on the cell�s ability to attach to spinach. Foodborne Pathogens and Disease. 9(2) :160-167.
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