INSECT TRANSMISSION OF PLANT AND HUMAN PATHOGENS TO PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1004326
• Project Start Date: Oct 23, 2014
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078

Performing Department
Entomology And Plant Pathology

Non Technical Summary
Many plant and animal disease organisms including bacteria, viruses, and other microbes, are transmitted by arthropod vectors (Gray and Banerjee 1999). Vectors provide transportation to new host and habitats and often have intimate and specific relationships with the pathogens they transmit (Nault 1990). This project primarily addresses insect transmission of plant pathogens, but the mechanisms can be very similar to those of vector-animal pathogen systems. This project addresses the mechanisms of pathogen attachment and release from insect tissues. This includes bacterial attachment to fly cuticle and bacterial and viral vector competence of hemipteran vectors. My overall vision for this project is to interrupt transmission of insect-borne pathogens to plants.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	0780	1130	50%
712	1430	1130	50%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
0780 - Grasslands, other; 1430 - Greens and leafy vegetables;

Field Of Science
1130 - Entomology and acarology;

Keywords

hemiptera

diptera

phloem-inhabiting bacteria

plant virus

leafhoppers

filth flies

transmission

acquisition

inoculation

food safety

Goals / Objectives
1. Identify and characterize probing activities associated with pathogen acquisition and inoculation with the goal of identifying plant germplasm which prevents or inhibits pathogen transmission2. Identify known and unknown vectors of grass and forb infecting plant viruses and bacteria in the central states3. Identify factors which determine filth fly transmission of human enteric bacteria to leafy greens and fresh vegetables/fruits4. Development of novel methods of intervention to prevent or reduce food contamination by flies

Project Methods
- Objective 1. Identify and characterize those probing activities associated with pathogen acquisition and inoculation with the goal of identifying plant germplasm which prevents or inhibits pathogen transmission This ongoing project focuses on the feeding activities of leafhoppers and other hemipterans using Electrical Penetration Graph )EPG) technology. EPG monitoring and analysis capability of hemipteran probing behavior is already in place, housed within the Department of Entomology and Plant Pathology, Oklahoma State University, and will be used to study Macrosteles quadrilineatus (aster yellows phytoplasma vector), Exitianus exitiosus ( grass virus and spiroplasma vector), and Endria inimica (American wheat striate rhabdovirus vector). Efforts to obtain funding to compare canola, wheat, and sorghum germplasm for resistance are underway. Similar comparisons and ultimately development of resistance indices will be compiled for other hemipteran vectors of plant disease pathogens, particularly those that pose an imminent threat to the US agricultural economy as invasive species or select agents. Objective 2. Initiate surveillance for known and unknown vectors of grass and forb infecting plant viruses and bacteria in the central states. This project is a continuance of the successful Virus Ecology survey carried out by Ulrich Melcher (BMB), in which he and others identified cryptic plant viruses in plants within the Tallgrass Prairie in Northeastern Oklahoma. Out of that project came the obvious question as to how these viruses were being moved around. To help establish the role of insect vectors in cryptic and invasive plant pathogens, leafhoppers will be collected from regions within and surrounding Oklahoma and tested for presence of virus and bacteria. At the conclusion of this project, we should have a regional map of the central states pinpointing the plant and leafhopper reservoir populations of infectious agents.2.1 Develop a multiplex PCR platform assay for multiple viruses and bacteria in plants and in leafhoppers. Four to five plant RNA viruses and Ca. Phytoplasma asteris will be targets for this multiplex PCR assay to be developed in conjunction with Dr. Francisco Ochoa Corona (NIMFFAB) and Dr. Anna Whitfield, Kansas State University.2.2 Collect and identify grass-inhabiting leafhoppers in mixed grasses, cultivated grasses, and native prairies throughout Oklahoma. This list will be compared to a previous study made over 40 years ago to determine if new species are detected. This is important as part of biosecurity surveillance of vectors and pathogens they may carry into the country.2.3 Determine host and geographical range of the invasive leafhopper, Balclutha rubrostriata. This species is moving north rapidly after introduction to Texas in 2010. Found on the invasive grass, Bothriochloa ischaemum, this leafhopper has the potential to spread throughout the Midwest. Summer surveillance of B. rubrostriata in OK and surrounding states as well as vector competence parameters will be carried out and determined.2.4 Next generation sequencing of infected plants and vectors. This is the obvious next step to understand pathogen movement in the environment. Costs for NGS are dropping rapidly, and so whole genome analysis of vectors will give a deeper analysis of pathogens carried and perhaps more insight as to why some pathogens are not transmitted. Several leafhopper species will be sequenced including universal vectors Macrosteles quadrilineatus and Circulifer tenellus and specialists such as Exitianus exitiosus and Dalbulus maidis. The resulting genomes will serve as a starting place for novel pathogen detection strategies, such as electronic diagnostic nucleic acid probes (EDNA, Stobbe et al. 2013) and helicase dependent amplification.Insect vectors of human pathogens to plantsIdentification of factors important to fly attraction to lettuce and other fresh produce is currently underway using modified fly transmission tests. A large, Plexiglas flight chamber was constructed to study fly orientation and movement to lettuce and spinach using standard protocols. A number of filth fly species, enteric bacterial pathogens, and target plant crop species will be examined. Impact of fly defecation and regurgitation spots on E. coli O157:H7 colonization of the leaf phyllosphere will be determined by fluorescent and confocal microscopy. This work will be carried out in laboratories in Noble Research Center and OSU Imaging Center.Objective 3. Investigate mechanisms of specific attachment and release of bacterial pathogens to and from filth flies. 3.1 Rapid dissemination of enteric bacteria to different surfaces. The "Five Second Rule" for food safety will be tested by allowing flies very short contact with various surfaces and then assaying those surfaces for pathogens.3.2 Blocking E. coli and Salmonella attachment to fly surfaces. Chemical and biological blocking agents will be tested for ability to reduce binding of enteric bacteria to tarsi and labellae of house flies and blow flies. If successful, a blocking strategy may be useful in reducing pathogen load on adult flies in animal confinement operations.Objective 4. Identify plant products that are at risk for fly-borne contamination of human enteric bacteria and develop novel methods of fly intervention to prevent or reduce food contamination by flies.4.1 Compare transmission efficiency of E. coli and Salmonella by blow flies to different fruit species. Preliminary data suggested a marked difference in pathogen release to blueberries compared to tomatoes and grapes. Properties of fruit will be investigated to determine mechanisms of differential transmission. Blow flies and house flies will be tested for vector competence to transmit pathogens from contaminated manure to common fruits using short acquisition and inoculation periods. Intervention methods such as netting, protective sprays and trap crops will be tested to determine if these might be viable pathogen reduction strategies.

Progress 10/23/14 to 09/30/19

Outputs
Target Audience:The data generated by this project would be of interest to the scientific community, such as researchers active in plant pathological and entomological projects. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In 2018-2019, two graduate students completed their research and requirements, and three undergraduate students worked in the lab gaining valuable research experience. Impact of these opportunities: one undergraduate student graduated and has moved on to graduate school, another undergraduate student graduated and left for a job at another university in the genetics core and now analyzes genomic and transcriptomic data, one graduate student used his training to obtain a post-doctoral position at the FBI research facility at Quantico, VA, and the second graduate student is graduating and moving to another lab to conduct bioinformatics. How have the results been disseminated to communities of interest?In 2018-2019, results were presented at the Entomological Society of America Branch meeting in Tulsa, OK, at the American Phytopathological Society national meeting in Cleveland, OH, at the Arthropod Genomics Symposium in Manhattan, KS, and at the Entomological Society of America national meeting in St. Louis, MO. Four manuscripts were published. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Under Objective 1, (Identify and characterize those probing activities associated with pathogen acquisition and inoculation with the goal of identifying plant germplasm which prevents or inhibits pathogen transmission), the primary accomplishment was the completion of the electropenetrograph (EPG) recordings of CRISPR Cas9 mutant planthoppers, native planthoppers, and the endosymbiont-free aphids that were generated for the DARPA project. In that project, Team Maize Hopper was charged with generating modified vectors to deliver a positive trait genetic message maize.My role in the project was to determine if genetic or biological modification negatively impacted the ability of the modified vectors to deliver the virus package to maize.EPG recordings were completed at North Carolina State University where the modified vectors are housed.A total of 18 CRISPR Cas9 mutant Perigrinus maidis, 13 native P. maidis, 12 Rifampicin-fed Rhopalosiphon padi aphids, and 10 water-fed aphids were recorded while feeding on maize.Both types of P. maidis planthoppers probed maize, typically producing 10-20 probes in a 6 hour recording period.All recorded insects reached both phloem and xylem with no discernable qualitative differences in probing activities.It was determined that both the native and the CRISPR Cas9 mutant planthoppers performed the probing activities required for virus delivery to maize during the feeding process.Endosymbiont-free (EF) and intact aphids were measured for 1 hour each to determine the number of potential drops (virus inoculation behavior) performed on maize.EF aphids produced 6 potential drops/hour whereas intact aphids produced 8 potential drops/hour, indicating no difference in virus-inoculation behaviors between these two phenotypes. Under Objective 2 (Initiate surveillance for known and unknown vectors of grass and forb infecting plant viruses and bacteria in the central states), the generation of genomes and transcriptomes of six deltocephaline leafhoppers was accomplished.Funded by NSF, we are working to identify vector competence genes by comparing vector and non-vector leafhopper genomes and transcriptomes.Assembly and annotation of six draft genomes for Exitianus exitiosus, Macrosteles quadrilineatus, Graminella nigrifrons, Balclutha neglecta, Balclutha rubrostriata, and Dalbulus maidis were completed.Raw reads were uploaded into the NCBI database.925 unique genes that shared similarity among vectors but not the nonvectors were identified.Candidate genes from this pool have been identified for further study for functionality. Also, under this objective, diagnostic tools to identify major wheat-infecting pathogens that are transmitted by arthropods were developed. A multiplex-PCR protocol was constructed to identify wheat streak mosaic virus, three variants of Barley yellow dwarf virus, and maize streak virus.Electronic probe diagnostics to identify Wheat streak mosaic virus in wheat samples was developed and validated in infected wheat. A manuscript reporting this new protocol was submitted in November, 2019 to J. Virological Methods. Under Objective 3 (Identify factors which determine filth fly transmission of human enteric bacteria to leafy greens and fresh vegetables), a 16S rRNA genomic analysis of flies exposed to rat carcasses over time was accomplished.Samples taken from Sarcophaga bullata exposed to rotten rat carcasses at 0, 2, 4, 6, and 8 days revealed few differences in bacterial load between the treatments.What was revealed were populations of antibiotic resistance genes present in the bacteria carried by these flies.There was not a demonstrated relationship between presence of bacterial genera and the aging of the rat carcasses. Under Objective 4 (Identify plant products that are at risk for fly-borne contamination of human enteric bacteria and develop novel methods of fly intervention to prevent or reduce food contamination by flies), the final manuscript from a USDA NIFSI-funded project was published.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Pickens, V. and A. Wayadande Surfing the Waves: Electropenetrographs of mosquito feeding on different hosts. Entomological Society of America Southwestern Branch meeting, Tulsa OK, April 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Wayadande Flies, Food Safety, and One Health. American Phytopathological Society national meeting, August 2019, Cleveland, OH
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Ayala, C., T. Blagden, E. Bird, B. Couger, and A. Wayadande. Comparative transcriptomics of six leafhoppers (Hemiptera: Cicadellidae). Arthropod Genomics Symposium, June 2019, Manhattan, KS.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Wayadande, A. So you want a career in academia? Navigating those rough waters without a rudder. Entomological Society of America Southwestern Branch meeting, Tulsa OK, April 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Wayadande, A. Are we really to busy to take sabbatical leave? Entomology and Plant Pathology seminar, September, 2019, Oklahoma State University, Stillwater, OK.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Rydzak, P. Detection of arthropod-transmitted viruses in cereal crops and RNAi induced resistance to Wheat streak mosaic virus. Ph.D defense seminar, April, 2019, Oklahoma State University, Stillwater, OK.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Backus, E., F. Cervantes, R. Guedes, A. Li and A. Wayadande. 2019. ACDC electropenetrography for in-depth studies of feeding and oviposition behaviors. Ann. Ent. Soc. Am. 112: 236-248
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wayadande, A., E. Backus, B. Noden, and T. Ebert. 2019. Waveforms From Stylet Probing of the Mosquito Aedes aegypti (Diptera: Culicidae) Measured by ACDC Electropenetrography. J. Medical Entomology. https://academic.oup.com/jme/advance-article/doi/10.1093/jme/tjz188/5611060?guestAccessKey=1e73fc48-423d-4580-b0ba-fe044f80225d
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Rydzak, Patrick. 2019. DETECTION OF ARTHROPOD TRANSMITTED VIRUSES OF CEREAL CROPS AND RNAi INDUCED RESISTANCE TO WHEAT STREAK MOSAIC VIRUS. PhD Dissertation. Oklahoma State University, Stillwater, OK 74078

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Academia, USDA, NSF. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students are associated with this project; one is a doctoral student who has been training in molecular virology and vector biology. The other student is a master's student who has become proficient in sample preparation for sequencing, rNAi, and bioinformatics. There was also an undergraduate student who learned bioinformatics and molecular techniques. All students have had the opportunity to attend professional meetings to present their research. How have the results been disseminated to communities of interest?All students have attended professional meetings (American Phytopathological Society, Entomological Society of America, Arthropod Genomics Conference) and presented their research results. Two manuscripts have been submitted to scientific journals. What do you plan to do during the next reporting period to accomplish the goals?I plan to continue with all three projects until the funding period ends.

Impacts
What was accomplished under these goals? All work completed involves understanding mechanisms of pathogen transmission by insects. To that end, there were three major projects: 1) leafhopper cell line development to support virus replication technologies; 2) whole genome sequencing of leafhopper vectors and non-vectors to uncover vector competence genes; 3) electropenetrography of insect vectors to understand inoculation activities. The leafhopper cell line, GN1, was successfully initiated in early 2018. Embryonic tissue from the leafhopper, Graminella nigrifrons was homogenized and plated in cell culture media from which immortalized cells were developed. The GN1 line is now being used to test rhabdovirus gene expression. Five leafhopper species have been successfully sequenced using the illumine platform. Assembly and annotation were completed with BUSCO scores ranging from 88-93% indicating fairly complete assemblies and coverage. Electropenetrography of hoppers, Graminella nigrifrons and Perigrinus maidis was accomplished during 2018. Inoculation-associated waveforms were identified in all specimens recorded, confirming that inoculation of persistent plant viruses will likely occur within 1-2 hours after exposure to susceptible plants. The mosquito, Aedes aegypti was recorded while feeding on humans. Five waveform types were identified and the sequence of probing activities is stereotypic for this species.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: AyalaChristian Ayala-Ortiza, Brian Cougerb, Trenna Blagdena, Edward Birda, Chris Dietrichc, Dmitry Dmitrievc, and Astri Wayadandea. Looking For Vector Competence Genes: Transcriptome Comparison Of Vector And Non-vector Deltocephaline Leafhoppers. Arthropod Genomics Symposium, Champaign, IL
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Wayadande. Filth flies as disseminators of human pathogens. 2018. Entomological Society of America, Vancouver, BC.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: P. Rydzak. EDNA-Wheat, Using NGS as a Tool to Detect Viruses in Wheat Metagenomic Data. 2018. American Phytopathological Society, Boston, MA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Pickens ,V., E. Bird, W. W. Hoback & A. Wayadande. 2018. Microbial interactions of necrophagous flies and their impact on bacterial transmission over time. Entomological Society of America, Vancouver, BC.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Nothing to report that is a subset of the original target audience Changes/Problems:New Research Goals for 2017 include 1. development of three leafhopper insect cell lines that are to be used in a rhabdovirus in vivo cell culture system, 2. Genomic sequencing of vector and non-vector leafhoppers and 3. Use of electropenetrography to assess mosquito feeding behavior. All of these changes are due to procurement of extramural funds. What opportunities for training and professional development has the project provided?In 2017, four graduate students and three undergraduate students have been trained under this project. Electropenetrography training has been instrumental in the success of one graduate student, bioinformatics training to understand leafhopper phylogeny and vector competence has been available for one graduate student and one undergraduate student, one graduate student learned leafhopper identification and studied grassland ecology and one undergraduate student learned live fly - pathogen interaction methods and 16S sequencing. Finally, one graduate student has become proficient in development of plant virus diagnostic assays. How have the results been disseminated to communities of interest?Yes, four professional presentations have been made at scientific meetings and two presentations have been made at local Oklahoma State University symposia. One master's thesis has been published and three peer-reviewed papers have been published. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will be very different because the major goals of the project have been fulfilled or have been left unfunded by outside agencies. Goals for the next year include final publication of at least two vector manuscripts, and a switch to vector genomics and development of insect cell lines. These latter two projects are now goals because of extramural funds procured during 2016- 2017.

Impacts
What was accomplished under these goals? 1 Identify and characterize those probing activities associated with pathogen acquisition and inoculation with the goal of identifying plant germplasm which prevents or inhibits pathogen transmission. Host plant resistance studies are often carried out using colony insects during early stages of screening. Colony conditions, particularly for aphids, yield smaller, less fit individuals. To assess how rearing conditions might impact aphid feeding behavior, and thus impact assessment of germplasm resistant to aphid-transmitted viruses, aphids that were reared under stressed conditions were compared to those reared under relaxed conditions. Stressed and relaxed bird cherry oat aphids(Rhopalosiphum padi) were recorded using electropenetrography. Analysis of the data revealed that stressed colony aphids probed less but produced approximately the same number of potential drops as relaxed aphids. Relaxed aphids also performed significantly more salivation events while interacting with the phloem. These data suggest that relaxed aphids perform more activities associated with inoculation of the circulative virus Barley yellow dwarf and for accurate assessment of resistance to this virus, the larger, more relaxed aphid rearing regime should be used in host plant resistance studies. 2. Initiate surveillance for known and unknown vectors of grass and forb infecting plant viruses and bacteria in the central states. This objective was accomplished in 2017 with the completion of a statewide 3 year survey of vectors. Over 20 leafhopper vectors of plant viruses and bacteria were found in native prairies, cultivated pastures, and cereal crops, although individual populations were highly variable from year to year. Some of the most important vectors (Macrosteles quadrilineatus, Graminella nigrifrons) were found episodically, whereas others were found in low numbers throughout the year. These data show that fluctuations in vector populations may be tied to fluctuations in annual disease incidence. 3. Identify factors which determine filth fly transmission of human enteric bacteria to leafy greens and fresh vegetables. Differential transmission of two human pathogens (E. coli O157:H7 and Salmonella) was documented for blow flies compared to house flies. Blow flies are much more efficient vectors of these two pathogens than are house flies. This has implications for food safety because house flies are more commonly associated with foodborne diseases than any other fly. 4. Identify plant products that are at risk for fly-borne contamination of human enteric bacteria and develop novel methods of fly intervention to preven or reduce food contamination by flies. Nothing to report for this objective.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Pace, R. J. Talley, T.Crippen, and A. Wayadande. 2017. Filth fly transmission of E.coli O157:H7 and Salmonella enterica to lettuce, Lactuca sativa. 2017. Annals Ent. Soc. Am. 110: 83-89.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Dubie, T. , J. Talley, J. Payne, A. Wayadande, and J. Dillwith. 2017. Filth fly activity associated with composted and noncomposted beef cadavers and laboratory studies on volatile organic compounds . J. Med. Entomol. 54: 1299-1304.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Andreason, S. J. Brown, Arif, M. F. Ochoa-Corona, J. Fletcher, and A. Wayadande. 2017. Single-target and multiplex discrimination of whiteflies (Hemiptera: Aleyrodidae) Bemisia tabaci and Trialeurodes vaporariorum with modified priming oligonucleotide thermodynamics. J. Econ. Entomol. 110: 1821-1830
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Wayadande, A., B. Bruton, and J. Fletcher. 2017. Cucurbit yellow vine disease. In W. Keinanth, W. Wintermantel, and P. Zitter [eds.] Compendium of Curcurbit Diseases and Pests, Second Edition. 908 pp.
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Webb, S. and A. Wayadande. 2017. Cucumber beetles. in W. Keinanth, W. Wintermantel, and P. Zitter [eds.] Compendium of Cucurbit Diseases and Pests, Second Edition. 908 pg.
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Wayadande, A., J. Fletcher, and B. Bruton. 2017. In J. Brown, Ed. Cucurbit Yellow Vine Disease: A Model for True Bug-Pathogen Relationships. In J. K. Brown. [ed]. Vector-Mediated Transmission of Plant Pathogens. American Phytopathological Society Press. (Refereed).
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Hung, K., McElfresh, Y. Zou, A. Wayadande, and A. Gerry. Identification of insect honeydew volatiles and attraction of house flies (Musca domestica) to insect honeydew volatiles in a semi-field environment. Submitted to J. Chemical Ecol.
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Borrone, J. A. Stobbe, U. DeSilva, J. Talley, P. Hoyt, and A. Wayadande. Characterization of the excreted microbiome of the synathropic fly, Phormia regina (Meigen) (Diptera: Calliphoridae): The Excretome. Submitted to J. Med. Entomol.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Wayadande, A. and B. Noden. Electropenetrography of Bloodsucking Arthropods. Invited seminar, Dept. of Entomology, Louisiana State University, Baton Rouge, LA Host: Dr. Jeff Davis. April 12, 2017
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Rydzak, P. F. Ochoa-Corona, A. Whitfield, and A. Wayadande. Detection and differentiation of arthropod-transmitted viruses of cereal crops using multiplex PCR and high resolution melting. American Phytopathological Society, San Antonio TX, August 4-7, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Gahm, N. A. Whitfield, W. Hoback and A. Wayadande. Salivary sheath recovery from leafhoppers feeding on seed heads of Bothriochloa ischaemum. Entomological Society of America national meeting, Denver, CO, November 5-9, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Wayadande A. and B. Noden. Electropenetrography of the mosquito, Aedes aegypti: R vs emf components.Entomological Society of America national meeting, Denver, CO, November 5-9, 2017.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Professional science societies: these include my colleagues at the Entomological Society of America, American Phytopathological Society, Society for Vector Ecologists, and International Association of Food Protection. Government agencies such as: Centers for Disease Control and Animal and Plant Health Inspection Service. Scientific community at large. . Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In 2016, three graduate students joined, continued or finished their projects in my laboratory. These students learn scientific methodology, scientific writing, and expectations of the professional community. One student graduated and went on to a post-doctoral research position at University of California Riverside, an opportunity made possible by the training and experience gained in my laboratory. Another student was able to attend the International Congress of Entomology to meet other professional entomologists. How have the results been disseminated to communities of interest?Papers have been presented at professional meetings and manuscripts have been submitted and accepted: What do you plan to do during the next reporting period to accomplish the goals?I will continue working on insect transmitted plant and animal pathogens, writing grant proposals to fund the planned work. In addition, I have a new NSF grant starting in January that will fund genomic comparisons of vector and non vector leafhoppers. This will be a tremendous contribution to our understanding of genes and gene products that mediate pathogen transmission.

Impacts
What was accomplished under these goals? Major accomplishments include graduation of one doctoral student, identification of key leafhopper-associated transcripts upregulated in response to Spiroplasma infection, validation of EDNA (electronic diagnostic nucleic acid) probes for detection of Spiroplasma in leafhopper metagenomes, identification of leafhopper vectors in mixed grass ecosystems and in the invasive grass, Bothriochloa ischaemum, development of protocols to study mosquito probing behavior using electropenetrography.

Publications

• Type: Book Chapters Status: Published Year Published: 2016 Citation: Cucurbit yellow vine disease: A model for true bug  bacteria transmission. 2016 In Vector-Mediated Transmission of Plant Pathogens", J.K. Brown Ed. 510 pp. APS Press, Madison, WI
• Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Andreason, Sharon. Doctoral Dissertation 2016 Oklahoma State University. Discrimination and Trancriptional Response Analysis Of Hemipteran Phytopathogen Vectors.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Gahm, N., W. Hoback, and A. Wayadande. Differences in Oklahoma Leafhopper Abundance and Diversity in Mixed Grasses Compared to King Ranch Bluestem. Poster presentation at International Congress of Entomology, Orlando, FL September, 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Wayadande, A. and B. Noden. Electropenetrography of Mosquitoes Feeding on Humans. Electopenetrography Symposium invited presentation, International Congress of Entomology, Orlando FL, September 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Wayadande, A. and E. Backus. Comparison of EPG Waveforms of Mosquitoes and Sand Flies Feeding on Humans. Electropenetrography Conference and Workshop, Lake Alfred, FL, October 2016.

Progress 10/23/14 to 09/30/15

Outputs
Target Audience:Professional community, including university academics, research collaborators Food safety organizations. Changes/Problems:RSL appears to be a very weak invasive. We were unable to keep the colony going and as a consequence, we have no live insects to conduct transmission tests this winter. Alternatively, we will work with congener, Balclutha abdominalis, to test experimental transmissibility of Spiroplasma kunkelii. Other objectives remain unchanged. What opportunities for training and professional development has the project provided?The two students (1 Undergraduate, 1 Graduate) have been trained to sight ID grass inhabiting leafhoppers. The graduate student has learned leafhopper rearing techniques and how to conduct pathogen transmission assays. How have the results been disseminated to communities of interest?Results have been disseminated only to the immediate group who are working on the RSL project. Food safety and flies talks have been presented at the Entomological Society of America and Crop Sciences Society of America. What do you plan to do during the next reporting period to accomplish the goals? Continue with ongoing transmission tests Continue with development of the multiplex PCR assay to detect grass-infecting pathogens Confirm next summer that RSL does not overwinter north of Texas Publish results from the E-probes detection of S. kunkelii work

Impacts
What was accomplished under these goals? There are two major areas of research: Agricultural Biosecurity and Food safety Agricultural Biosecurity: The invasive leafhopper, RSL, project started in April, 2015.Surveys of the invasive grass, Bothriochloa ischaemum and surrounding mixed grasses were completed in Oklahoma and adjacent states to identify populations of the invasive leafhopper, the red streaked leafhopper, Balclutha rubrostriata (RSL).Diagnostic tool development was also initiated.We accomplished the following: Weekly surveys throughout Oklahoma revealed no adult RSL until early November. RSL was detected in central Texas in late October and never detected in central Kansas. This suggests that RSL does NOT overwinter in Oklahoma and other states north of Texas. Native and cultivated grasses were identified and collected throughout Oklahoma and Kansas for later assay for grass-infecting viruses and mollicutes. Primer design for the multiplex PCR platform was initiated. We are collecting RNA and DNA positive controls for assay development. Advanced diagnostics for presence of pathogens in large next generation sequencing datasets was accomplished using electronic probes to detect the presence of Spiroplasma kunkelii in gray lawn leafhopper transcriptomes. Food Safety: Nothing to report

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Andreason, S. M. Gard, and A. Wayadande, 2015. Detection of the invasive leafhopper, Balclutha rubrostriata, on an invasive grass species. Southwestern Entomologist 40:479-485.
• Type: Book Chapters Status: Accepted Year Published: 2015 Citation: Wayadande, A. and J. Fletcher. Squash bug vectors of Serratia marcescens. In Vector Pathosystems, J. Brown, Ed. APS Press.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Andreason, S., R. Davis, W. Schneider, and A. Wayadande, 2015. Electronic probes for the detection of Spiroplasma kunkelii in transcriptomes of the gray lawn leafhopper, Exitianus exitiosus. National Meeting of the Entomological Society of America, Minneapolis, MN, November, 2015.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Wayadande, A. 2015. Filth fly carriers of human pathogens in manure. Invited symposium speaker, National Meeting of the Entomological Society of America, Minneapolis, MN, November, 2015.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Gahm, N. W. Hoback, and A. Wayadande, 2015 Temporal changes in leafhopper abundance in mixed grass ecosystems. Southwestern Branch of the Entomological Society of America, February, 2015, Tulsa, OK.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Wayadande A. and J. Talley. 2015. Fly vectors of human pathogens to plants. Invited symposium speaker, Society of Vector Entomologists, Albuquerque, NM, October, 2015.