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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Systematic Entomology Laboratory » Research » Research Project #432685

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

Location: Systematic Entomology Laboratory

2021 Annual Report

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]

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-two different projects where studies were initiated, continued, and finalized by the Electron and Confocal Microscopy Unit (ECMU) staff by collaborating with USDA-ARS, university, and international scientists. Some of the projects are highlighted. Acquisition and installation of a new, cutting-edge Cryo-Scanning Electron Microscope (Cryo-SEM) were completed. The new Cryo-SEM provides an all-in-one solution that enables biological samples to be frozen in liquid nitrogen and analyzed in their native state without the chemical artifacts characteristic of conventional SEM while also allowing variable pressure imaging of sensitive hydrated samples at room temperature. The variable pressure mode combined with novel beam deceleration techniques allows the vacuum pressure in the microscopy chamber to be lowered so that the otherwise high-vacuum condition will not destroy sensitive hydrated samples. The new Cryco-SEM features 6 state-of-the-art electron detectors enabling a wide range of imaging modalities, including Energy-Dispersive X-ray Spectroscopy for elemental analyses and mapping. The new Cryo-SEM incorporates numerous significant improvements and efficiencies over the previous model and will enable an even greater range of specimens to be analyzed, leading to further novel microscopy approaches and facilitating the next generation of scientific discoveries. A Cryo-SEM comparison was conducted on saltgrass leaves grown in highly saline areas compared to leaves grown in freshwater. Leaf tissues of both samples were frozen in liquid nitrogen, freeze-fractured, and observed in the Cryo-SEM. The Energy-Dispersive X-ray Spectroscopy detector was used to analyze the composition of elements in the interior of the frozen/fractured leaf, revealing a higher proportion of sodium and calcium uptake in particular cells in the saltgrass leaves grown in saltwater. The combined use of Cryo-SEM, freeze-fracture and elemental analysis of internal structures is a novel technique that has yielded promising results, which may open the door to further research and discoveries. A new technique was developed for imaging soft-bodied insects, pests, and pathogens. Soft-bodied insects such as nematodes are typically stored in an aqueous solution to prevent desiccation. This presents a challenge for imaging because wet specimens must be dried before observation in an SEM. A new technique was developed, allowing multiple wet specimens to be mounted onto a drop of alcohol on a microscopic screen on an SEM stub and properly positioned; then, the liquid droplet can be wicked away from below the screen. This new technique has significantly improved the ability to image nematodes, resulting in stunning, high-resolution images and enabling the morphological description of three new nematodes species. Work is now underway to apply this technique to soft-bodied scale insects as well. A new technique was developed for characterizing bacteria growing on the surface of fresh-cut carrots by using liquid agar to encapsulated bacteria. The addition of agar onto the surface of the carrot prevented the bacteria from washing off the carrot during the numerous chemical reagent and resin transfers required to embed tissue for transmission electron microscopy (TEM). This new procedure has allowed the production of cross-sections showing the ultrastructural morphology of bacteria growing on the surface of the carrot. This technique is being utilized to study the antimicrobial properties of cut carrot slices compared to other root vegetables. It has revealed details of a rare mesosome structure within Listeria monocytogenes that may explain the mechanism underlying the antimicrobial property of carrots. This research has the potential to increase the understanding of bacteria growth and antimicrobial resistance. 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. TEM and confocal microscopy were used to evaluate the susceptibility of three common foodborne pathogens to sanitizers in the simulated leafy-green wash water. Chlorine and peracetic acid (PAA) are chemical disinfectants (sanitizers) commonly used in the fresh produce industry to reduce contamination from harmful microorganisms that may cause foodborne infections. Both sanitizers effectively killed foodborne pathogens; however, treatment with PAA might have induced some of the treated pathogens to enter a viable but non-culturable (VBNC) state. Additional studies to evaluate the risks of VBNC are needed to improve the sanitation practices of the fresh produce industry. Egg parasitoids are tiny wasps that are particularly effective as biological control agents due to their ability to reduce insects detrimental to agriculture. A study was conducted to examine the behavior of the wasps on egg masses of the common squash bug and the horned squash bug. Parasitoid wasps were observed to spend significantly more time drilling into common squash bug eggs than horned squash bug eggs. Measurements were taken using TEM, which determined that squash bug eggs had significantly thicker shells than those of horned squash bugs. This difference may account for the lower rates of parasitism and for the increased time spent drilling into squash bug eggs compared to horned squash bug eggs. This study provides the basis for future studies that will improve biological control programs in squash fields. Morphological characterization was performed by Cryo-SEM and light microscopy, which revealed a new species of lesion nematode infecting soybeans from North Dakota. Lesion nematodes are among the most economically important plant-parasitic nematodes that damage the roots of crops. Microscopy staff also created new, representative line drawings of the nematodes with diagnostic characteristics from multiple specimens in one concise drawing. The discovery that several species of Chromobacterium produce insecticidal compounds with activity against many pest insects has increased interest in this genus of bacteria. We used TEM to study the morphology of a previously unknown species of Chromobacterium discovered in a marsh near the mouth of the Nanticoke River in Maryland. The bacterium, named Chromobacterium paludis, was determined to be a new species based on comparisons of its genome sequence with those of known Choromobacterium. C. paludism was found to be most closely related to C. phragmitis and C. amazonense. Although C. paludis shows little insecticidal activity itself, detailed genomic comparisons with the closely related insecticidal C. phragmitis may reveal the genes responsible for the insect-killing compounds produced by the latter. These findings will interest scientists searching for ways to boost the production of the insecticidal compounds produced by Chromobacterium. A new species of sheath nematode that feeds on a large variety of plants was discovered using Cryo-SEM. Due to an uncommon outer loosened cortical layer that forms a cuticular sheath, imaging of the nematode would not have been possible using standard SEM techniques; rather, freezing with liquid nitrogen and observation in the Cryo-SEM made these studies possible. This sheath nematode is a pest newly discovered in Upper Marlboro, Maryland, isolated from the roots and around soil from tall fescue. Microscopy staff also created new line drawings of the nematodes showing representative diagnostic characteristics from multiple specimens in one concise drawing.

1. An emerging plant-parasitic cyst nematode infecting turf grass. Cyst nematodes are microscopic worms that cause billions of dollars of crop losses each year in the United States. Some cyst nematodes infect turf grass and ruin golf courses and other recreational areas. Scientists from USDA-ARS, Beltsville, Maryland, and Oregon State University used advanced cryo-scanning electron microscopy to visualize cyst nematodes isolated from turf grass in Bandon, Coos County, Oregon. They found a new cyst nematode species. This discovery is valuable for scientists and turf management experts developing new methods to more quickly survey turf areas for this detrimental nematode and implement management practices to reduce turf damage.

Review Publications
Medina-Salguero, A., Comejo-Franco, J., Grinstead, S.C., Mowery, J.D., Mollov, D.S., Quito-Avila, D. 2021. Characterization of a mild isolate of papaya ringspot virus type-P (PRSV-P) and assessment of its cross-protection potential. Plant Disease.
Handoo, Z.A., Yan, G., Kantor, M., Huang, D., Chowdhury, I.A., Plaisance, A., Bauchan, G.R., Mowery, J.D. 2021. Morphological and molecular characterization of Pratylenchus dakotiensis n. sp. (Nematoda: Pratylenchidae), a new root-lesion nematode species on soybean in North Dakota, USA. Plants. 10(168):1-13. https://doi:10.3390/plants10010168.
Kantor, M.A., Handoo, Z.A., Skantar, A.M., Hult, M.N., Ingham, R., Wade, N., Ye, W., Bauchan, G.R., Mowery, J.D. 2020. Morphological and molecular characterisation of Punctodera mulveyi n. sp. (Nematoda: Punctoderidae) from a golf course green in Oregon, USA, with a key to species of the Punctodera. Nematology.
Cornelius, M.L., Vinyard, B.T., Mowery, J.D., Hu, J.S. 2020. Ovipositional behavior of the egg parasitoid Gryon pennsylvanicum (Hymenoptera: scelionidae) on two squash bug species Anasa tristis and Anasa armigera (Hemiptera: coreidae). Environmental Entomology.
Gu, G., Bolten, S., Mowery, J., Mowery, J.D., Luo, Y., Nou, X. 2020. Susceptibility of foodborne pathogens to sanitizers in produce rinse water and potential induction of viable but non-culturable state. Food Control. 112:107138.
Bakshi, M., Hebert, D.A., Gulbronson, C., Bauchan, G.R., Tuo, W., Zarlenga, D.S. 2021. Ostertagia ostertagi mediates early host immune responses via macrophage and Toll like receptor pathways. Infection and Immunity. 1-42.
Liu, M., Yi, T., Gulbronson, C., Bauchan, G.R., Ochoa, R. 2020. Ontogenetic and morphological studies on Tetranychus canadensis (Acari:Tetranychidae). Zootaxa. 4857(1):215-250.
Vieira, P., Vicente, C.S., Branco, J., Bauchan, G.R., Mota, M., Nemchinov, L.G. 2021. The root lesion nematode effector Ppen10370 is essential for parasitism of Pratylenchus penetrans. Molecular Plant-Microbe Interactions.
Blackburn, M.B., Farrar, R.R., Sparks, M., Kuhar, D.J., Mowery, J.D., Mitchell, A.D., Gundersen, D.E. 2020. Chromobacterium paludis sp. nov., a novel bacterium isolated from a Chesapeake Bay marsh. International Journal of Systematic and Evolutionary Microbiology.