r&r other project information field 8 – project narrative...
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R&R Other Project Information Field 8 – Project Narrative
TABLE OF CONTENTS
Section Title Page #s
Executive Summary
2 - 11
1. Project Title 2 - 2 2. Project Type 2 - 2 3. Focus Area 3 - 3 4. Program Staff 3 - 7 5. Critical Stakeholder Need & Project Goals 7 - 7 6. Outreach Plan 7 - 7 7. Economic, Social & Environmental
Benefits 7 - 8
8. Stakeholder Engagement 8 - 8 9. Logic Model 8 - 11 Introduction 12 - 16
1. Long Term Goals, Critical Needs of Citrus Industry, and Outreach Objectives
12 - 13
2. Stakeholder Involvement 13 - 13 3. Body of Knowledge/Past Activities 14 - 14 4. Ongoing/Recently Completed Significant
Activities 14 - 16
5. Preliminary Data/Information 16 - 16 Rationale & Significance
17 - 17
Approach 18 - 36
1. Proposed Activities, Key Personnel, and Sequence of Activities
18 - 21
2. Methods & Feasibility 21 - 28 3. Expected Outcomes 28 - 28 4. Results Analysis 29 - 31 5. Use of Results 31 - 31 6. Outreach Plan 31 - 33 7. Potential Pitfalls 34 - 36 8. Limitations " - " 9. Hazards " - "
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Executive Summary
Throughout the proposal, we will use the following acronyms and key definitions to refer to various elements of importance for the project. Following are a list of acronyms and their descriptions:
Acronym Full Name Description
HLB Huanglongbing Citrus greening disease CLas Candidatus Liberibacter asiaticus Bacterial pathogen associated with HLB
disease ACP Asian citrus psyllid Insect vector of CLas pathogen (aka D. citri) CHMA Citrus Health and Management
Area Outreach model currently utilized in Florida to deploy area-wide pesticide control strategies for the Asian citrus psyllid; recommended by the National Research Council.
D. citri Diaphorina citri Insect vector of CLas pathogen (aka ACP) wtPsyllid Wild type Psyllid Natural population of the invasive ACP pest
currently found in Florida, Texas, California, Arizona, Louisiana, Mississippi and Mexico
nuPsyllid New Psyllid Laboratory isolated or created ACP lacking the ability to vector the CLas pathogen
Driver Genetic system used to spread a trait into a population - the vehicle to replace wtPsyllid with nuPsyllid
Effector Molecular basis or interaction mechanism that blocks the ability of nuPsyllid to vector CLas
Refugia Areas supporting ACP populations that are not subjected to insecticide treatments; i.e., abandoned orchards, urban citrus, and experiment station orchards
1. Project Title
The Project’s title is “Rear and Release Psyllids as Biological Control Agents - An Economical and Feasible Mid-Term Solution for Huanglongbing (HLB) Disease of Citrus.”
2. Project Type
The project will be a Coordinated Agricultural Project (CAP) with a five year timeline. The applicant is the Citrus Research and Development Foundation (CRDF). The CRDF, a not-for- profit Corporation, was established in 2009 as a Direct Support Organization (DSOs) of the University of Florida (UF). Its operations are specifically governed by several other Florida Statutes and UF regulations. Specifically, UF provides space and other resources and services for CRDF, approves the appointment of Board of Directors, employs the CRDF Chief Operating Officer, has oversight of CRDFs expenditures, business practices and requires an annual financial audit to be submitted to the UF Board of Trustees.
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3. Focus Area Our project directly and wholly addresses “Focus Area #2 – Efforts to identify and address threats from pests and diseases” under “PART I—FUNDING OPPORTUNITY DESCRIPTION” of the RFA and the “FY 2012 Invited Coordinated Agriculture Project for Huanglongbing Disease of Citrus” request under the same Part. 100% of efforts and funds will be dedicated to this Focus Area.
4. Program Staff
The program staff and key personnel are listed below using the following acronyms to identify their role.
• PD: Program Director • Co-PD: Co-Program Director • Co-PI: Co-Principal Investigator • KP: Key Personnel
Name & Title Role Affiliation & Address Email
Administrative Team Tom Turpen Program Manager
PD Citrus Research & Development Foundation 700 Experiment Station Road Lake Alfred, FL 33850
Harold Browning Chief Operating Officer
Co-PD Citrus Research & Development Foundation 700 Experiment Station Road Lake Alfred, FL 33850
Jackie Burns Center Director & Professor
Co-PD University of Florida IFAS Citrus Research & Education 700 Experiment Station Road Lake Alfred, FL 33850
Mary Lou Polek Vice President Science & Technology
Co-PD Citrus Research Board 217 N. Encina; PO Box 230 Visalia, CA 93279
The technical teams will focus on the following areas: 1) Driver Systems: Determine method to develop a nuPsyllid population. 2) Effector Mechanisms: Define the molecular basis to block HLB transmission. 3) Rear, Release, Monitor: Develop and deploy nuPsyllid rear, release and monitoring strategy. 4) Socio-Economics/Modeling: Optimization of the release mechanism strategy including
applied biological, social, and economic implications. Includes procedures related to economic impact, grower acceptance, and clear and open communication to the general public to allay possible concerns.
5) Outreach: Communication to stakeholders of nuPsyllid information and benefits; education of public and citrus industry through existing university extension.
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Name & Title Role Affiliation & Address Email
Team to address Driver System - Chromosomal Driver (Team Lead: Bruce Hay) Bruce Hay Professor
Co-PI California Institute of Technology MC156-29, 1200 East California Pasadena, CA 91125
Alfred Handler Research Geneticist
KP USDA - ARS 1700 SW 23rd Drive Gainesville, FL 32608
Omar Akbari Postdoctoral Scholar
KP California Institute of Technology 1200 East California Blvd Pasadena, CA 91125
Philippos Papathanos Postdoctoral Fellow
KP California Institute of Technology MC156-29, 1200 East California Pasadena, CA 91125
Paul Shirk Research Physiologist
KP USDA - ARS 1700 SW 23rd Drive Gainesville, FL 32608
Team to address Driver System – Viral Driver (Team Lead: Bryce Falk) Bryce Falk Professor
Co-PI University of California, Davis One Shields Ave. Davis, CA 95616
Kris Godfrey Project Scientist
KP University of California, Davis 555 Hopkins Road Davis, CA 95616
Da Ha Postdoctoral Scientist
KP University of California, Davis One Shields Avenue Davis, CA 95616
Team to address Driver System – Bacterial Driver (Team Lead: Kirsten Pelz-Stelinski) Kirsten Pelz- Stelinski Assistant Research Scientist
Co-PD University of Florida IFAS Citrus Research & Education 700 Experiment Station Road Lake Alfred, FL 33850
Lukasz Stelinski Assistant Professor
KP University of Florida IFAS Citrus Research & Education 700 Experiment Station Road Lake Alfred, FL 33850
Nian Wang Assistant Professor
KP
University of Florida IFAS Citrus Research & Education 700 Experiment Station Road Lake Alfred, FL 33850
Team to address Effector Mechanism – Library Approach (Team Lead: Bob Shatters) Bob Shatters Research Molecular Biologist
Co-PD USDA Horticultural Research Lab 2001 South Rock Road Fort Pierce, FL 34945
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Name & Title Role Affiliation & Address Email El-Desouky Ammar Research Entomologist
KP USDA: Subtropical Insects Research 2001 South Rock Road Fort Pierce, FL 34945
[email protected] da.gov
Marc Giulianotti Director of Chemistry Operations
KP Torrey Pines Institute for Molecular Studies 11350 SW Village Parkway Port St. Lucie, FL 34987
David Hall Supervisory Entomologist/ Research Leader
KP USDA: Subtropical Insects Research 2001 South Rock Road Fort Pierce, FL 34945
John Hartung Research Plant Pathologist
KP USDA: Molecular Plant Pathology 10300 Baltimore Avenue Beltsville, MD 20705
Wayne Hunter Research Entomologist
KP USDA: Subtropical Insects Research 2001 South Rock Road Fort Pierce, FL 34945
Team to address Effector Mechanism – Genomic Approach (Team Lead: Judy Brown) Judy Brown Professor
Co-PI University of Arizona 1140 E. South Campus Drive Forbes Blvd #303 Tucson, AZ 85721
David Gang Associate Professor & Fellow
KP Washington State University PO Box 646340 Pullman, WA 99164
Team to address Rear, Release & Monitor (Team Lead: Joseph Patt) Joe Patt Research Entomologist
Co-PD USDA Horticultural Research Lab 2001 South Rock Road Fort Pierce, FL 34945
David Bartels Entomologist
KP USDA APHIS PPQ CPHST Moore Air Base, Bldg. 6414 22675 N. Moorefield Road Edinburg, TX 78541-9398
[email protected] da.gov
Daniel Flores Entomologist
KP USDA APHIS PPQ CPHST Moore Air Base, Bldg. 6414 22675 N. Moorefield Road Edinburg, TX 78541-9398
[email protected]. gov
Celestina Galindo Program Supervisor IV
KP California Department of Food & Agriculture 13915 Saticoy Street Van Nuys, CA 91402
Kris Godfrey KP Also listed under “Driver System – Viral Driver”
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Name & Title Role Affiliation & Address Email Tim Gottwald Research Leader
KP USDA Horticultural Research Lab 2001 South Rock Road Fort Pierce, FL 34945
David Hall KP Also listed under “Effector System – Library Approach”
David Morgan Program Supervisor IV
KP California Department of Food & Agriculture 4500 Glenwood Dr., Building E Riverside, CA 92501
Michael Rogers Associate Professor
KP University of Florida IFAS Citrus Research & Education 700 Experiment Station Road Lake Alfred, FL 33850
Eric Rohrig Biological Scientist
KP FDACS, Division of Plant Industry Methods Development & Biological Control 1911 SW 34th Street Gainesville, FL 32608
eric.rohrig@freshfromflor ida.com
Mamoudou Sétamou Associate Professor
KP Texas A&M University 312 N. International Blvd Weslaco, TX 78596
mamoudou.setamou@tam uk.edu
Richard Stouthamer Professor
KP University of California, Riverside Department of Entomology 3401 Watkins Drive Riverside, CA 92521
Team to address Socio-Economics/Modeling (team lead: Neil McRoberts) Neil McRoberts Assistant Professor
Co-PI University of California, Davis One Shields Avenue Davis, CA 95616
Leonard Coop Assistant Professor Chief Developer of OSU IPPC website
KP Oregon State University 2040 Cordley Corvallis, OR 97331
[email protected] e.edu
Mark Lubell Professor Director - Center for Environmental Science and Policy
KP University of California, Davis One Shields Avenue Davis, CA 95616
Paul Mitchell Associate Professor, Agriculture and Applied Economics
KP University of Wisconsin 427 Lorch St. Madison, WI 53706-1503
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Name & Title Role Affiliation & Address Email Team to address Public Outreach (team lead: Elizabeth Grafton-Cardwell) Elizabeth Grafton- Cardwell IPM Specialist and Research Entomologist
KP University of California Riverside Department of Entomology Riverside, CA 92521
Michael Rogers KP Also listed under Rear, Release & Monitor section
Mamoudou Sétamou
KP Also listed under Rear, Release & Monitor section
mamoudou.setamou@tam uk.edu
Glenn Wright Associate Research Scientist/Professor
KP University of Arizona 6425 W. 8th Street Yuma, AZ 95364
5. Critical Stakeholder Need & Long Term Goals
The presence of the HLB in Florida and most recently in Texas, and its pending spread to other states poses a substantial economic threat to all stakeholders in the US citrus industry. In Florida where HLB is endemic, 2006-2011 models estimate an economic loss of $3.6 billion due to the disease. Direct and indirect impacts of HLB on employment include a loss of 6,611 jobs, attributed to agricultural (48% lost), government (8%), health and social services (7%), and retail (7%) trades over this same period (Hodges and Spreen, 2012). Current HLB management practices are not considered sustainable and alternatives must be found; HLB management adds approximately $464 per acre to the costs of managing a Florida citrus orchard ($255 million if experienced across the 550,000 acre Florida industry). Depending on fruit yields, break-even costs for producing juice oranges increased from $.83 per pound solids in 2003/04 to $1.18 currently, an increase of 42% (Muraro, 2011).
See full description of Critical Stakeholder Needs beginning on page 12.
The long term goal of this project is to interfere with the spread of HLB within orchards where HLB is endemic and to interfere with the invasion of CLas into areas where ACP is established but HLB has not been detected.
See full description of Long Terms Goals beginning on page 12.
6. Outreach Plan
A public education campaign will be developed, focusing on the benefits to stakeholders of a modified psyllid (nuPsyllid). Citrus Extension teams are well-positioned to expand the extensive effort currently underway to provide outreach response to ACP and HLB in affected states.
See full description of the Outreach Plan beginning on page 31.
7. Economic, Social and Environmental Benefits
Dealing with HLB is a social (or collective) problem; individual growers cannot manage their way out of an HLB outbreak independently, creating an economically crippling scenario for growers and other stakeholders. The successful execution of the proposed plan will present an environmentally benign and low-cost solution to mitigate the threat of HLB and will decrease the use of pesticides. See also recap of economic impact at number 5 above; see more in-depth
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discussion of Social, Economic and Environment Benefits in Socio-Economics/Modeling beginning on page 26.
8. Stakeholder Engagement
Throughout this process, citrus stakeholders in Florida, Texas, Arizona and California have been consulted through Citrus Research and Development Foundation (CRDF) and Citrus Research Board (CRB) board meetings, grower meetings, advisory committees, and National Citrus Council communication.
Our Advisory Committee (consisting of Stakeholder Advisors, Science Advisors and a Regulatory Advisor) will play a critical role in the oversight of the project. The Advisory Committee will receive and review quarterly progress reports from each sector of the program and will participate in annual progress meetings. See full description of Stakeholder Engagement beginning on page 13.
See Appendix 12A for details on the creation of the Advisory Committee panel and the credentials of those serving.
9. Logic Model
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Over the past few years, the National Citrus Science and Technology Coordinating Group developed a logic model to coordinate research on HLB toward the ultimate strategic objective of maintaining a prosperous and productive citrus industry. (NCSTCG is a public-private partnership with the citrus industry that also includes expert representatives from USDA-APHIS, Citrus Health Response Program (CHRP), USDA-ARS, USDA-NIFA and the major US citrus research centers of expertise.) Outcome 2 of the NCSTCG is to slow or prevent the spread of ACP & HLB. This outcome is directly addressed by our project.
Project Plan Outcome: Slow or prevent the spread of ACP & HLB
Strategy Measurable
Indicators Means of
Verification Important
Assumptions Eliminate HLB as an economic threat to U.S. citrus production by blocking the ability of the Asian citrus psyllid to vector Candidatus Liberibacter asiaticus.
Eradication or reduction in spread of CLas; no HLB+ trees are detected in new plantings; no significant increase of HLB+ trees in existing plantings Growers continue to produce high quality fruit, juice processors remain profitable, citrus trees continue to be planted in commercial groves and the urban landscape
Trap & visual surveys show no or very low numbers of wtACP detections or HLB spread; nuPsyllid populations are stable and fecund; No new geographical spread of HLB where nuPsyllid is released HLB in infected areas decreases Crop and Juice production records – NASS; disease & pest surveys; stable market both domestic and international
At least one strategy of modifying psyllids is successful; nuPsyllid has high level of fitness and control efficacy in the natural environment; Growers & public accept release of nuPsyllid; nuPsyllid release meets regulatory approval in a timely manner
Focus Area Activity Indicators of Success Time Frame
Driver System • Development of transgenic ACPs by the introduction of a selfish DNA element that has the innate ability to “push” itself into the population
• Development of a virus vector that can be deployed in a manner in which it delivers genetic material to the ACP population
• Utilize endosymbiotic bacteria that reside
• Transformation is successful and colonies are stable
• Survey of naturally occurring viruses results in viable candidates
• Relationship between endosymbionts and ACP is determined
• Biology is developed within 1-3 years
• Group decides which driver system is most effective; pursues the best and eliminates activity on unsuccessful system(s)
• Years 4-5 are spent field testing modified psyllid colonies
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Focus Area Activity Indicators of Success Time Frame
within the ACP by either using its innate ability or genetically modify its genome to deliver the desired genetic material.
Effector Mechanism • Construction of a synthetic gene that encodes a protein or RNAi that blocks circulative movement of CLas within ACP
• Expression of antimicrobial peptides (AMPs) within the ACP to eliminate CLas before it can be transmitted
• Delivery of a cell- death suicide gene that is activated when CLas enters the cell and produces a protein that kills the cell
• Physical barriers of bacteria are verified
• Titer of Clas is reduced
• Activity of AMPs verified
• Cell death is verified
• Systems are developed within years 1-3
• Group decides which effector system is most successful; pursues the best and eliminates activity on unsuccessful system(s)
Rearing, Releasing, Monitoring
• Development and standardization of protocols that optimize the mass- rearing of nuPsyllid
• Effective means will be devised for transporting nuPsyllid to target sites
• Monitor total ACP populations
• Monitor and evaluate populations of released nuPsyllid
• Stable colonies of modified psyllid
• Established colonies that are capable of producing hundreds of thousands of modified psyllids
• Massive numbers of psyllids are successfully moved from the rearing facility to release site
• Modified psyllids are readily detected in field locations
• Colony evaluation occurs in years 3-5
• Mass rearing standardization occurs in years 4- 5
• Release of hundreds of thousands of modified psyllids in several geographic locations in year 5
Socio- Economics/Modeling
• Visualize the problem of ACP population dynamics across a very large spatial
• Collection data maintained in a central location
• Model output
• Data collection starts as soon as psyllids are released in small
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Focus Area Activity Indicators of Success Time Frame
scale; • Predict and study the
interaction dynamics of existing ACP populations with the released nuPsyllid;
• Evaluate economic implications
• Evaluate public reaction through surveys
informs nuPsyllid release strategy
• Survey analyses and model output inform outreach messages
• Surveys indicating understanding of benefits of nuPsyllid
field plots and continues beyond the duration of this project
• Preliminary scoping of social/actor structure in nuPsyllid initiative starts in year 1
Outreach • Evaluate messaging format that successfully educates public (message testing);
• Create outreach materials such as brochures, radio & TV ads, web pages
• Outreach blitz: multi media campaign when modified psyllid is released
• Positive response from grower and general public
• Message testing starts year 1
• Development of literature and web pages begins year 2
• Web pages, social media begins year 3
• Evaluate audience in a timely manner
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INTRODUCTION The applicant for the project is the Citrus Research and Development Foundation (CRDF). CRDF was established as a not-for-profit Corporation as a Direct Support Organization (DSOs) of the University of Florida. The University of Florida provides space and other resources and services for the CRDF, approves the appointment of Board of Directors, employs the CRDF Chief Operating Officer, has oversight of CRDFs expenditures, business practices and requires an annual financial audit to be submitted to the UF Board of Trustees. The purpose of CRDF includes sponsoring and managing research, education and extension to develop new technologies to combat biological threats to the citrus industry and advocating their rapid deployment through education, demonstration and extension. CRDF is working in close collaboration with the California Citrus Research Board (CRB), as well as seven experts with the USDA and several universities in Florida, Texas, Arizona and California in bringing together the necessary pieces to successfully find a solution to huanglongbing (HLB) disease.
1. Long Term Goals, Critical Needs of Citrus Industry, and Outreach Objectives Long Term Goals The primary long term goal of this project is to interfere with the spread of HLB within orchards where HLB is endemic and to interfere with the invasion of CLas into areas where ACP (the only known vector of the causal agent of HLB) is established, but in which HLB has not been detected, by strategically releasing a nuPsyllid population that is incapable of transmitting CLas. A further goal is to ensure the necessary adoption of the method by the social system of growers, and understanding and acceptance by consumers and the general public. Once established, this novel system of biological control would be operationally transferred to the USDA-APHIS, Citrus Health Response Program (CHRP). Other ongoing support, if necessary, will be provided by the stakeholder organizations (see Project Management Plan Appendix 12B).
Critical Needs of Citrus Industry There is nowhere in the world where HLB is both endemic and adequately controlled (Gottwald, 2010). The insect vector, ACP, feeds on multiple citrus plants and acquires/transmits CLas by feeding within the citrus phloem sieve elements. Symptoms of HLB include chlorosis of leaves, misshapen fruit, premature fruit drop, decreased yield, off-flavor fruit and juice, and tree death. Newly planted trees are at greatest risk because they may succumb to HLB infection prior to the 5-years it takes for them to come into production. As disease severity increases, yield is reduced, making orchard production uneconomical within 7 to 10 years after planting (Aubert et al., 1984; Aubert, 1990; Gottwald et al., 1991; Roistacher, 1996). Estimates indicate that HLB has resulted in an economic loss of $3.6 billion and caused a loss of over 6,600 jobs in Florida in a 5-year period from 2006-2011.
ACP was first detected in Florida in 1998. Shortly thereafter, ACP had spread to the point that eradication seemed implausible. HLB was discovered in Florida in August of 2005, is now present in all 34 citrus-producing counties. Alarmingly, the first detection of HLB in Texas was just reported on January 13, 2012. The vector ACP also has been detected and is being addressed in California and Arizona through attempts at eradication or suppression. An estimated 100 million trees are affected by HLB worldwide; HLB has already impacted the citrus industry with the loss of many small growers who could not afford the more costly management practices.
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In Florida, as well as all other regions of the world where HLB is endemic, the citrus industry must rely on the only proven management practice: control of ACP with insecticides, scouting and removal of HLB symptomatic trees, and replanting with disease-free trees. The state average infection rate increased from 1.6% for the 2007-08 season (Morris, Erick and Estes, 2009) to 21.9% in the 2010-11 season (Irey, Morris and Estes, 2011). HLB management is most feasible with large area-wide coordination, which is extremely challenging in the US citrus acreage because of the interface with urban and feral citrus. Indeed, most Florida citrus orchards have rates of infection (2-100%) that make current methods impractical. Optimal citrus nutrition combined with stringent chemical psyllid control may slow the decline in production under some field conditions but this approach is also unsustainable from a cost perspective.
Outreach Objectives Outreach objectives include determining the most effective means to deliver education to urban and grower communities, and helping them understand and accept the release of a modified psyllid (nuPsyllid). Keeping stakeholders apprised of updated information on reducing the common problem of HLB will be a major goal.
In summary, we believe current management practices are not sustainable, and in any event psyllid vector eradication has never been achieved, except on small islands. Alternative HLB- management approaches must be developed as a mid-term solution to the HLB problem. Without control measures in hand, citrus growers have no incentive to replace infected trees or to replant entire orchards. The uncertainties associated with HLB will undermine the stability of the industry in currently HLB-free areas. A mid-term solution is crucial to maintain a profitable industry until citrus lines with resistance to CLas or ACP can be developed and released. Therefore, we present a novel and more environmentally friendly alternative strategy, which we will convey to growers and the public.
2. Stakeholder Involvement
Identified stakeholders include citrus grower associations, citrus growers, consumers, homeowners, research universities, industries, businesses allied with citrus production and the general public. Different stakeholders have a different level and type of interest, all of which this proposal will address. Grower associations, educational seminars and grower meetings have long been valued for bringing together grower needs and the responses from research and extension. Grower supported research sponsors have cooperated among several states to best invest grower funds toward statewide and national needs; in Florida, through the Citrus Research and Development Foundation (CRDF), in Texas through the Texas Citrus Production Board (TCPB), and in California through the Citrus Research Board (CRB).
The National Citrus Science and Technology Coordinating Group (NCSTCG) selected the proposed method of HLB control by psyllid population replacement for further development. CRDF coordinated a meeting with a core team of research experts to discuss the scope and focus of a research plan to address intermediate solutions to psyllid transmission of HLB. This team constructed an outline of the technical and delivery steps necessary to develop, evaluate and monitor psyllid population replacement. A core team was then assembled to outline the project components, and to form the base of the citrus SCRI CAP team. Throughout this process, citrus stakeholders in each state have been consulted and updated through CRDF and CRB board meetings, grower meetings, advisory committees, and National Citrus Council communication. CRDF and CRB grower representatives have been consulted on the full project, the proposed approach, and the cost-share commitment.
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In addition to the continuing role of the project Stakeholder Advisory Committee, whose direct participation is detailed in Appendix 12A, the stakeholders from each state will be engaged and informed on progress, challenges and accomplishments. The regularly scheduled meetings of CRDF, CRB and TCPB (all grower stakeholders) will provide for consistent discussion, feedback and evaluation as the project progresses. Procedures for review of progress on projects directly funded by these grower groups will be employed to allow these stakeholders to review and evaluate the project goals and accomplishments.
This proposal is not merely supported by the industry, it is more accurately submitted on their behalf. Both of the lead and co-applicant organizations (CRDF and CRB) have diverse industry representation from fresh, processed and organic industry segments and are governed and managed by active professionals. Three of the four Project Director and co-Directors (Turpen, Polek, and Browning) of this proposal, work directly for these stakeholder organizations.
3. Body of Knowledge/Past Activities
The Florida, Texas, Arizona and California citrus industries continue to be challenged by a number of issues, foremost among them is the increasing rate of introduction of exotic pests and diseases and the associated costs of operations and yield loss. ACP and HLB are the most recent and severe threats to the long-term sustainability of citrus production in the US. These industries collectively represent 826,500 acres of bearing citrus trees (USDA, FASS, 2010). This acreage is already down from 954,200 acres in 2005, due largely to weather and disease losses.
Grower response to this disease has resulted in a mix of increased costs, modifications to long- successful production management systems, a diversion of marketing investment to research investment and acceptance of at least short-term yield and/or quality reductions. Total orchard loss is increasing as HLB spreads. Many predictive models suggest that without development of an adequate control strategy, commercial production will become economically unfeasible. The economy would suffer great repercussions if the economic benefit of citrus were lost or significantly diminished. Already, estimates indicate that HLB has resulted in an economic loss of $3.6 billion and caused a loss of over 6,600 jobs in Florida in a 5-year period from 2006-2011 (Hodges and Spreen, 2012). The real solutions to HLB and other challenges ultimately will come from aggressive research.
It is estimated that close to 100 million trees are affected by HLB worldwide. In the northern and eastern regions of Thailand, 95 percent of trees were affected as of 1981. In the Philippines, HLB reduced the citrus acreage by 60 percent between 1961 and 1970. In Java and Sumatra, 3 million trees were destroyed from 1960 to 1970, and Bali lost 3.6 million trees from 1984 to 1987. In the southwestern oases of Saudi Arabia, HLB had killed most sweet orange and mandarin trees by 1983. Reunion Island lost its entire citrus industry in the 1960s due to HLB. In São Paul State, Brazil, where HLB was recognized in March 2004, close to 3 million HLB affected sweet orange trees have been removed since mid-2004 in the HLB-control program (National Research Council, 2010).
4. Ongoing/Recently Completed Significant Activities
i. Driver System
Research will be conducted in parallel on three separate potential driver systems: Chromosomal, Viral and Bacterial. Chromosomal: For example, the participating Hay lab has created a gene drive system known as Medea, which is capable of spreading itself within Drosophila populations (Chen et al, 2007). They have also generated transgenics carrying components of a two-locus engineered underdominance system and are currently testing them for drive. Viral:
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The participating Falk lab is experienced in viral vector construction and use and recently utilized high-throughput nucleotide sequence and bioinformatic analyses to identify two viruses of the hemipteran insect, Homalodisca vitripennis and will apply the same methodology to ACP. Bacterial: The participating Pelz-Stelinski lab has recently begun to evaluate the distribution of endosymbionts, including Wolbachia, in different populations of Florida ACP. Initial results indicate that co-infection of ACP with CLas and Wolbachia occur at low rates within these populations.
ii. Effector Mechanism
Research will also proceed concurrently on two complementary approaches to identify effector mechanisms that will block the ability of ACP to vector CLas: Library and Genomic. Library Approach: The participating Hartung lab has constructed a phage display scFv antibody expression library containing 2 x 107 unique members generated against total protein extracts from CLas infected ACP and have used this to isolate scFv antibodies that bind to eight different CLas surface proteins. An ACP genome initiative originated by Drs. Hunter, Shatters and Hall has resulted in a first draft of the ACP genome that is available for researchers. Additionally, the participating Shatters and Hall labs have developed an artificial feeding system for ACP that allows uptake of biologically active macromolecules such as peptides and RNAi and a transmission bioassay based on excised citrus leaves. Genomic: The participating Brown lab is working on the comparative characterization of psyllid anatomy and the specific organs encountered during infection, multiplication, and circulation (Cicero et al., 2009; Cicero and Brown, in prep.) of Ca. Liberibacter solanacearum (CLsol), transmitted by the potato psyllid (PoP) Bactericera cockerelii (Sulc.) (Hansen et al., 2008) and the citrus greening-associated ACP-CLas system.
iii. Rear, Release, and Monitor Asian Citrus Psyllids (ACP)
Prior work in this area has focused on developing and evaluating sampling protocols necessary for estimating ACP population occurrence, abundance, and distribution in commercial orchards. We will build upon experience within the research team in rearing ACP.
iv. Socio-Economics/Modeling
The Integrated Plant Protection Center (IPPC) at Oregon State University (OSU) has developed and implemented more than 70 web-based temperature driven pest and crop development models which are connected to the most comprehensive network of weather stations in the USA, allowing pest development to be modeled at a range of spatial scales from local to national. The current socio-economic impact of HLB in the US is significant, and it will be critical in our project to expand current efforts to analyze the impact of this disease on commercial citrus growers, homeowners who enjoy backyard citrus trees, environmental groups and the public in general. As we develop and deploy this approach to mediate impacts of HLB, it will be important to prepare growers and the public to accept this alternative to pesticidal suppression of ACP, and to quantify the real and potential long-term benefits of the approach to sustainable production and public consumption of citrus and citrus products produced in the US.
Avoiding Duplication of Efforts CRDF manages a portfolio of over 100 research projects funded by the Florida citrus industry and hosts on-line progress reports for Florida (CRDF), California (CRB), and Texas (TCPB). California and Texas engage through CRB and TCPB, respectively, in a similar manner to fund research. The CRDF Industry Research Coordinating Committee annually updates national citrus research project inventories and links those projects to current research priorities, producing a Gaps analysis. Duplication of investment and effort is reduced through this national communication effort. Project PI’s within each of the research areas will organize meetings to
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update the current state of knowledge in each section, identify successful procedures and gaps in knowledge or technology. The PI will coordinate activities and communication between the team and PD. The PD will resolve differences of opinion, if any, in modifying the work plan after consultation with the Advisory Committee, Administrative Team and other Stakeholders.
5. Preliminary Data/Information
The following is a list of preliminary data and background information gathered by the teams listed under the heading “Program Staff” starting at page 3:
- A gene drive system was developed (Medea), to drive population replacement in Drosophila
(Chen et al., 2007) - Using viruses for insect biological control has been implemented in U.S. and world agriculture.
A specific example is the use of baculoviruses for lepidopteran control. Millions of hectares of soybeans have been treated in South America for velvet bean caterpillar control (Moscardi, 1999), and in North America various baculoviruses have been used for pest control in cotton, potatoes and even Douglas fir (Moscardi et al., 2011). Many dicistroviruses readily spread among the population via fecal oral transmission routes (Bonning et al., 2010). Small RNA viruses can be manipulated such that infectious viruses can be generated from cDNAs (Boyapalle, 2008), and foreign sequences can be inserted, expressed in hosts and retained (Bonning et al., 2010). There is recent precedence for using viruses to develop robust RNAi effects in the fruit fly, Drosophila. When a recombinant Sindbis virus was used to infect flies, rapid and systemic silencing resulted within the insect, even distal from the cells where Sindbis virus was replicating (Saleh et al. 2009).
- Transcriptomic sequences for other insects, including plant-feeding hemipterans, (e.g., the pea aphid, Acyrthosiphon pisum) are already available (Consortium, T.I.A.G., 2010).
- In laboratory studies with the pea aphid (a plant insect pest that feeds on phloem in the same manner as the ACP), peptide blocking of gut membrane-viral (Pea enation mosaic virus) pathogen interactions by feeding uptake of the peptide has been shown to reduce circulative movement of the virus within the aphid vector (Liu, 2010).
- Wolbachia is one of five known bacterial endosymbionts associated with ACP (Subandiyah et al. 2000). Wolbachia induces immune signaling and antimicrobial gene expression in Drosophila cell lines (Xi et al., 2008). Similar increases in the immune response of Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus occur as a result of Wolbachia infection, leading to inhibition of Dengue virus, filarial nematode and Plasmodium parasites, and West Nile virus (Bian 2010, Kambris et al. 2009, Hughes et al. 2011, Glaser and Meola 2010, Kambris et al. 2010). Infection of A. aegypti with the Wolbachia strain wMelPop conferred resistance to the pathogenic Gram-negative bacterium, Erwinia carotovora, and the Gram-positive bacterium Micrococcus luteus (Kambris et al., 2009).
- In the ACP- CLas interaction, it is accepted that the bacterium must move systemically through the ACP to the salivary glands after ingestion for it to be transmissible (circulative transmission) (Ammar et al, 2011 a and b, Cicero and Brown, submitted).
- Drs. El-Desouky Ammar, Robert Shatters and David Hall have developed and published on a nucleic acid FISH microscopy detection assay for monitoring movement and location of CLas in the ACP and in citrus (Ammar et al., 2011 a and b).
- Florida growers prefer to receive timely information about HLB in traditional ways such as field days and meetings, and they prefer to receive information from fellow growers, citing trust as a major barrier (Telg et al. 2010).
- Successful launch of biological control programs in California will require a blend of information-based advocacy and clearly defined public interest (Warner et al. 2011).
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RATIONALE & SIGNIFICANCE This proposal presents research targeting the elimination of HLB as an economic threat to US citrus production by blocking the ability of the ACP to vector the bacteria CLas, the causative agent of this disease. Our project directly and wholly addresses Focus Area #2 under “PART I— FUNDING OPPORTUNITY DESCRIPTION” of the RFA and the “FY 2012 Invited Coordinated Agriculture Project for Huanglongbing Disease of Citrus” request under the same Part. Focus Area #2 includes efforts to identify and address threats from pests and diseases, including threats to specialty crop pollinators. Sub-elements under this focus area that are targeted by this proposal include:
1. Create new scientific developments, technologies, and tools that will help reduce the incidence and impact of industry-critical insect and disease problems including, but not limited to: monitoring, control and management strategies; field based diagnostic tools; integrated management systems; and comprehensive strategies and mechanisms for eradication of newly introduced pests where eradication is a plausible strategy.
This proposal presents a systems-based, multi-state, trans-disciplinary approach to the elimination of HLB as a serious economic threat to the US citrus industry. The project includes integrated research, extension and outreach components. We will develop new scientific technologies, management and monitoring strategies targeted at the reduction of HLB as a threat. The proposed solution will greatly reduce the ability of the ACP to vector the HLB plant pathogen and thus manage the economic threat of this disease (HLB) in a cost-effective, timely and environmentally benign fashion. The proposal brings to agriculture a practical approach first pioneered by vector-born disease entomologists in the medical field.
2. Develop new integrated pest management tools, such as have been demonstrated with the integrated pest management - Pest Information Platform for Extension and Education (ipmPIPE) and other area-wide, integrated systems, that possess the potential for broad impact. Proposed projects should respond to stakeholder identified critical threats.
This proposed project provides a systems-based, multi-state, trans-disciplinary approach to create deliverables that will integrate with current area-wide management strategies, including CHMAs (Citrus Health and Management Areas), already in place to reduce the rate of HLB spread. The current practice involves heavy reliance on pesticide applications, including area-wide spray programs conducted cooperatively by federal, state, and grower organizations. Current heavy pesticide use is considered unsustainable due to the possible negative effects on non-targets, the environment, fruit contamination, resistance in the ACP population, and cost. Coordination of teams of research leaders for this effort will be used to integrate nuPsyllid replacement pest management strategies into current programs. As part of the Outreach Plan, beginning on page 31, significant extension and education components are built into the project for training purposes in line with this integration strategy, including a monitoring approach to gauge the success of the population replacement.
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APPROACH (For Coordinated Agricultural Projects) To achieve the goal of blocking HLB transmission, we propose a three-fold approach: 1. Develop a psyllid management strategy based on the development of psyllid populations
(nuPsyllid) incapable of transmitting CLas and strategically release the nuPsyllid population to displace current ACP populations (wtPsyllid) that have invaded the US.
2. Provide optimized orchard management strategies for integration of the proposed population displacement technique into current orchard management practices: a. Southeast and Southern U.S. (FL and TX) where both the ACP and CLas are endemic or
detected. b. Western U.S. (CA and AZ) where ACP is present and spreading while there is currently
no detection of HLB. 3. Integrate orchard and nuPsyllid management strategies with monitoring strategies to
continually assess effectiveness, and provide outreach education to the grower stakeholders and citizens about the control strategy.
1. Proposed Activities, Key Personnel, and Sequence of Activities
Proposed Activities and Key Personnel This CAP project will constitute a multi-faceted, multi-state, trans-disciplinary approach to provide a practical management tool for HLB. Ideally, the desired outcome would be to eliminate the psyllid that is the only known vector of the causal agent; however given the interface of suburban, feral and unmanaged citrus with commercial citrus, a more practical approach is to eliminate the psyllid’s ability to transmit the pathogen. Replacing existing wild- type pysllid (wtPsyllid) populations with a manipulated psyllid (nuPsyllid) population is a more realistic mid-term outcome than eliminating the psyllid altogether. There are five distinct areas in which we’ll be focusing activities (see team lists and contact information under the heading “Program Staff” starting at page 3):
1) Driver Systems: Determine method to develop a nuPsyllid population. 2) Effector Mechanisms: Define the molecular basis to block HLB transmission. 3) Rear, Release, Monitor: Develop and deploy nuPsyllid mass-rearing, release and monitoring
strategy. 4) Socio-Economics/Modeling: Optimization of the release mechanism strategy including
applied biological, social, and economic implications. 5) Outreach: Communication to stakeholders of nuPsyllid information and benefits; education
of public and citrus industry through existing university extension. See details in Outreach Plan, beginning at page 31.
These activities represent separate and distinct objectives of the project that are both independent of and interdependent on one another. Within each of these five areas are multiple approaches that will be discussed. Although there is a logical progression through areas 1 to 5 above, concurrent progress on all of them is necessary for timely implementation of the proposed solution. Regular meetings and/or conference calls among the area research leaders will be used to coordinate the integrated research/extension effort and manage the project adaptively in response to any emerging obstacles to progress and new priorities. Furthermore, outreach teams will be kept abreast of research progress to facilitate the creation of proper messaging and educational materials.
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Driver System We will develop a “driver system” to move new genetic material into wtPsyllids in a manner that allows the nuPsyllids to displace the wtPsyllid populations. The parallel lines of research that will be pursued within the driver system involve three technically independent approaches: 1) the development of transgenic ACPs with an engineered selfish DNA element that has the innate ability to “push” itself into the population; 2) the development of an ACP virus that can be deployed in a manner in which it delivers and expresses genetic material to the ACP population; and 3) the exploitation of Wolbachia endosymbiotic bacteria residing within the ACP - either to use its innate ability to suppress CLas transmission or to genetically modify its genome to deliver the desired genetic material.
Key Team Member Driver Area of Emphasis University/Organization
Dr. Bruce Hay Chromosomal California Institute of Technology Dr. Bryce Falk Viral University of California, Davis Dr. Kirsten Pelz-Stelinski Bacterial University of Florida
Effector Mechanism We will develop a genetic system that blocks the ACP’s ability to transmit or ‘vector’ CLas, the bacterial agent causing HLB. Parallel lines of research to identify an effector mechanism include: 1) construction of a synthetic gene that encodes a protein or dsRNA (for RNAi-based inhibition) that blocks the circulative movement of CLas through the ACP vector; 2) expression of antimicrobial peptides within the ACP to eliminate the bacterium before it can be transmitted and/or 3) delivery of an insect cell-death suicide gene that is activated by CLas. Genomics, transcriptomics, proteomics, bioinformatics, combinatorial peptide library and phage display recombinant antibody (scFv) library-screening research will be employed in this research area.
Key Team Member Effector Area of Emphasis University/Organization Dr. Robert Shatters Library Approach USDA (Florida) Dr. Judith Brown Genomic Approach University of Arizona
Rear, Release & Monitor Mass Rearing - The proposed research will lead to the development and standardization of protocols that optimize the mass-rearing of nuPsyllids. This will include optimizing key production factors such as selection of host plant species, density and sex ratio of ACP per host plant, duration of host-plant and psyllid rotation cycles, environmental factors, and host-plant cultivation.
Release - An effective means will be devised for transporting nuPsyllids to target sites (unsprayed refugia near orchard boundaries), and incorporating them in target populations. Key components of these procedures will be to reduce mortality during transport and to enhance successful establishment at the release site.
Monitor – Monitoring of the psyllids will be conducted using tap sampling, yellow panel sticky traps, and whole-tree fumigation. ACP populations tend to have aggregated distributions, both spatially and temporally. To reduce the variance among samples obtained from such populations, collections will be biased by sampling in areas with the most consistent psyllid densities, such as refugial boundaries with southern and eastern exposures to sunlight. The release and monitoring procedures will be integrated and procedures will be devised that will accurately assess the success of release and establishment. Independent of this proposal, several groups of scientists are working on developing better traps by identifying chemical cues or attractants. These improved traps will likely be available around the time that the nuPsyllids are ready to be
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released. As improved trapping technology becomes available, it will be integrated into the sampling protocols developed here to make them more effective.
Key Team Member Project Area of Emphasis University/Organization Dr. Joseph Patt* Mass Rearing, Release, Monitoring USDA-ARS (Texas) * Dr. Patt has assembled a team of top specialists within each of the specific areas of rearing, releasing and monitoring and will be responsible for coordinating activities among the groups and for integrating improvements in trapping technology from external projects.
Socio-Economics/Modeling The socio-economics/modeling infrastructure for the project has been designed for its diverse biological, social and economic objectives. To maximize cost effectiveness, the approach is based on proven capabilities for visualizing ACP population dynamics across a very large spatial scale, allowing the interaction dynamics between wtPsyllid and nuPsyllid to be predicted and studied, and surveying growers in citrus states to provide an evaluation of the economic implications of the planned nuPsyllid release program. In addition, as we develop and deploy this approach to mediate impacts of HLB, it will be important to prepare growers, environmental groups and the public to accept this alternative to pesticidal suppression of ACP, and to quantify the real and potential long-term benefits of the approach to sustainable production and public consumption of citrus and citrus products produced in the US.
Key Team Member Project Area of Specialty University/Organization Dr. Neil McRoberts Socio-Economics/Modeling University of California, Davis
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Sequence of Activities See Appendix 12B for the detailed timeline that identifies key personnel involved in various tasks during each time segment of the project. The appendix will identify short, medium and long term metrics that will be used in project evaluation, the expectations of each team member, a mechanism whereby progress metrics can be evaluated, and how the project will complement and/or link to existing programs or projects.
Year
1 2 3 4 5 Driver System
Development of Chromosomal, Viral, Bacterial Drivers X X X Refocus effort based on Driver research progress X Final selection of Driver System X
Effector Mechanism Genomic and Library approach to Effector identification X X X Refocus effort based on Effector research progress X Final selection of Effector X
Mass Rearing Assess and optimize current production capacity for wtPsyllid X X X Adapt and standardize protocol for nuPsyllid X X Implement nuPsyllid rearing process X X
Trapping Technology Integration/Releasing/ Monitoring Identify, evaluate and integrate trapping technologies X X X X X nuPsyllid evaluations in a caged environment X X nuPsyllid field trials X X
Socio-Economics/Modeling Develop, refine, and validate Phenology Model X X X X Develop, refine and validate Social/Actor and Economic Analyses X X X X Iterative refinement and integration with field data X X
Public Outreach Organize teams, message testing and web/social media campaigns X X X Educational materials developed, finalized, distributed X X Full media launch X
Regulatory Outreach Engage nuPsyllid team and regulatory staff in science and policy issues X X X X Apply and obtain regulatory permits X X
2. Methods & Feasibility
Below, we discuss Methods and Feasibility for each of the three Driver Systems, the two Effector Mechanisms, Rear, Release and Monitoring and Socio-Economics/Modeling.
Chromosomal Driver: Two chromosomal gene drive mechanisms will be developed: Medea (Chen, 2007 #1), and two-locus engineered underdominance (Davis, 2001 #133). Both systems have been successfully implemented in Drosophila (Chen, 2007 #1 and Dusinberre and Hay, unpublished), and the Medea system is being actively developed by the Hay lab as a gene drive vehicle for blocking molecules in multiple mosquito species. Fundamental to both systems is the ability to express molecules that disrupt psyllid development, as well as molecules that block
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these activities. Transcriptional profiling will be utilized to generate a list of psyllid genes, their splice forms, and their patterns of expression throughout development. Methods needed to carry out this work are well developed in the Hay and Brown laboratories. Promoters that drive maternal or early embryo-specific expression (the Medea system), or tissue-specific expression (underdominance), will be identified through tests in transgenics of specific fragments of DNA driving the expression of GFP. This, and the design of microRNAs that silence specific genes, are techniques that Dr. Hay’s laboratory has years of experience with. There are no identified technical barriers.
Transgenesis is required to build Medea, engineered underdominance or any other chromosomal- based drive mechanism, and has not been attempted in the psyllid. It has been achieved in many insects, using several different approaches, most involving injection of transposons and a source of transposase into early stage embryos, and thus has a high likelihood of success in the psyllid. This work will occur in collaboration with Dr. Paul Shirk and Dr. Alfred Handler (ARS, USDA, Gainseville FL), both experts in developing transgenesis methods in other insects of economic importance, and with access to psyllid colonies. Transgenesis will be useful for many other researchers who wish to study basic aspects of psyllid biology, or the roles of specific genes that facilitate the CLas life cycle in the psyllid, and is a foundational technology essential for the serious and long-term study and manipulation of any organism.
Viral Driver: Contemporary, high throughput nucleotide sequence analysis and bioinformatics will be used to identify viruses from a worldwide collection of the ACP. This approach has already proven successful for identifying similar insect and plant-infecting viruses and is the most accurate and efficient means to allow us to achieve our goals. Specific virus incidence will then be determined in D. citri populations using virus detection methods such as RT-PCR. Selected virus genomes will then be engineered using recombinant DNA technology so as to yield viruses for D. citri infection under controlled conditions (in the UC Davis Contained Research Facility). One or two viruses will be chosen and modified so as to accept and express desired sequences upon infection of D. citri. The Falk lab has done this with other plant- infecting viruses and will build from our expertise. Virus infection will result in systemic RNA interference and psyllid death, or expression of anti-microbial peptides, which negatively impact CLas thereby preventing its transmission to citrus by D. citri. Finally, population studies will be conducted to assess the efficacy of recombinant D. citri viruses for helping to control HLB.
Bacterial Driver: Recent results from the Pelz-Stelinski lab suggest that Wolbachia, a psyllid symbiont, differentially infects ACP populations, and psyllids are less likely to harbor CLas when Wolbachia is present. We propose to investigate this interaction by selecting naturally occurring ACP and by creating Wolbachia-infected and uninfected ACP colonies using microinjections to artificially infect ACP. Subsequently, we will evaluate the effects of native and foreign Wolbachia strains on transmission efficiency by ACP and the effects of this ACP symbiont on the replication of CLas within the psyllid, ACP fitness, and immune gene expression, using methodologies previously developed in our laboratories and in collaboration with the J. Brown lab (UA, Tucson). We anticipate the identification of Wolbachia strains that inhibit transmission of CLas by ACP. Our overall goal is to determine whether we can create ACP that are non-competent vectors of CLas by manipulating populations of their bacterial symbionts (Wolbachia). The Wolbachia strain with the greatest effector potential and that is able to outcompete the native Wolbachia strain will be selected for driving into D. citri populations.
This approach has been used successfully in the field of medical entomology to create mosquito lines that have a decreased ability to transmit human disease pathogens, such as malaria and Dengue virus (Bian 2010, Kambris et al. 2009, Glaser and Meola 2010, Kambris et al. 2010 Osta et al., 2004). The nature of this project, which involves an insect vector, a
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microbial symbiont, and a plant pathogenic bacterium, will require a trans-disciplinary approach among entomology, microbiology, and plant pathology scientists on our project team for conducting experiments and evaluating the success of Wolbachia in inhibiting the transmission of CLas.
Effector Mechanism - Library Approach: This research is designed to identify important macromolecules of either CLas or ACP origin that are essential for successful circulative movement of the bacterium within the ACP, and will employ several strategies that make no presumptions about which molecules to target (proteins, extracellular matrixes, i.e. carbohydrates) as potential components of the “CLas movement machinery.” We present a method of screening for inhibitors of systemic CLas movement within the ACP using both a series of overlapping and positional scanning peptide libraries as well as an already developed scFv recombinant antibody library constructed against CLas-harboring ACP. Expertise from Torrey Pines Institute for Molecular Studies (TPIMS) provides years of experience on peptide library screening. Dr. Hartung’s scFv library (containing 2 x 107 unqiue recombinant antibody clones against CLas and ACP epitopes) will be used for screening. Both screen systems will be used in a fashion similar to a method successfully used in aphid transmission research (Liu, 2010). A CLas/ACP membrane association bioassay will be used to identify peptide(s) and/or scFv antibodies that block specific association of CLas with either the digestive tract or salivary gland membranes and qPCR/ fluorescent in situ hybridization (FISH) detection of CLas will be used to quantitate blocking efficiency (Ammar et al., 2011). To improve efficiency of the assay, we will attempt to use ACP cell cultures derived from either digestive tract or salivary gland tissues following culture initiation techniques already developed (Marutani-Hert et al., 2009), but modified for use with digestive tract/salivary gland membranes. If cultures cannot be maintained, we will use membrane preparations directly from isolated ACP tissues. Scoring for effector efficiency will be based on ability to inhibit CLas membrane association/transmembrane movement and kinetics studies of the inhibitor binding. This will be verified using whole insect transmission studies with artificial diets (Hall et al., 2009) and an excised leaf transmission assay. The Shatters and Hall (USDA) laboratories have extensive experience with these bioassay systems. Ideally, a set of effector molecules will be identified from which a synthetic gene will be constructed and incorporated into the Driver System.
Effector Mechanism - Genomic Approach: For target identification we will employ comparative transcriptomics and RNAseq (Marioni et al., 2011) to profile ACP transcripts from infected and uninfected ACP and identify misexpressed genes that could interact directly as psyllid and CLas candidate effectors during the circulative infection cycle in the ACP host (J.K Brown, UA, Tucson and D. Gang, WSU, Pullman). Similarly, given CLas tropism to the ACP salivary glands (SGs) and guts (Ammar et al., 2011; Cicero et al., 2009; Cicero and Brown, in prep a,b), CLas - infected and uninfected SGs and guts transcriptomes will be sequenced and expression profiles compared to identify misexpressed transcripts to specifically target proteins in the organs known to participate directly in CLas infection and vector acquisition (Dinglasan and Jacobs-Lorena, 2005; Fedhila et al., 2006; Hartung et al., 2011; Jubelin et al., 2011). Transcripts will be mapped to the ACP genome for verification. Similarly, to identify prospective interacting CLas proteins- protein networks, a CLas cDNA library will be constructed, sequenced (Illumina), annotated, and mapped to the CLas genome (Duan et al., 2009). Based on bioinformatic annotations, the most ‘lucrative’ ACP and CLas targets will be subjected to protein network analyses (Noirot and Noirot-Gros, 2004).
Using the genome-mapped transcripts and project EST databases, the presence and fidelity of transcript sequences are verified by RT-PCR amplification followed by sequencing, cloning into the yeast di-hybrid vector system and screened as bait against the respective ACP or CLas library
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to interrogate the respective di-hybrid library for interactors using traditional (Wilson et al., 2004) and/or split-GFP reassembly (Jackrel et al., 2010). The ‘best ACP and CLas protein candidates’ are subjected to functional characterization. Each myc-labeled protein is tested in vivo for activity by immuno-precipitation from infected and uninfected psyllid protein (Yang et al., 2008). Resultant ACP- CLas interacting proteins are separated by protein electrophoresis and subjected to mass spectrometry and peptide (proteomic) identification, and subjected to protein modeling to visualize structural predictions (Arnold et al., 2006; Kiefer et al., 2009). For lucrative candidate effectors, expression levels in ACP (with and without CLas) will be assessed using quantitative polymerase chain reaction (qPCR) and localization by DNA/RNA fluorescent in situ hybridization (FISH) (Trask, 2002; Speicher & Carter, 2005). The most promising targets are subjected to analysis using reverse genetics (RNAi; Wuriyanghan et al., 2011) followed by bioassay as described above under Library Approach. If differential transcriptomic-protein network predictions fail to identify satisfactory effectors, traditional yeast-dihybrid screening will be employed as a backup means of identifying essential interactors using selected CLas genes to screen the entire ACP library.
Mapping the ACP (Hunter et al., in progress, pers. commun.) or CLas (Duan et al., 2009; Tyler et al., 2009) transcripts to the available reference genomes, respectively, will be carried out to place the coding regions in a genomic context and to map gene order, or ‘synteny’ (Soderlund, UAZ, Tucson) to facilitate downstream development of transgenic constructs and to provide promoter sequences. To avoid positional effects, alternate promoters will be required when transgenes are integrated into the same chromosomal location (Jasinskiene et al., 2003) and genome mining for the respective coding region will be made possible from the integrated CLas transcriptome- genome maps.
Mass Rearing: Gaps in knowledge about key rearing parameters will be addressed by conducting scale-appropriate, replicated experiments with inclusion of proper controls. These experiments will be conducted using standard experimental protocols with cages or similar enclosures in greenhouses, incubators, or walk-in environmental chambers. For example, tests will be conducted to compare production rate of ACP from a single-species of host-plant versus a binary mixture of host-plants. Similarly, tests will be conducted to determine the optimal number of psyllid pairs needed per host plant per rearing cage. Mass rearing parasitoids that attack ACP has already provided a body of knowledge about mass rearing ACP and we will utilize this information to optimize the process for mass rearing nuPsyllid. For mass rearing, the challenge will be to develop the capabilities to consistently produce nuPsyllid in the quantities needed. Assuming that modification does not incur large fitness costs, meeting this challenge will mostly entail optimization and standardization of the current practices being used to mass rear ACP for parasitoid production, thus the approach is feasible. Any fitness costs incurred by modification will need to be quantified so the fitness cost can be addressed by adapting rearing protocols such as using additional rearing units to compensate for fitness loss. As the first nuPsyllids become available, the mass rearing team will begin to work with others with ACP rearing experience (such as the USDA-APHIS-CHRP program) to optimize the rearing process to overcome potential fitness reductions in nuPsyllid and to obtain target production levels required to initiate wtPsyllid population replacement.
Releasing: ACP will be transported in small, ventilated containers to the release site. Tests will be conducted to determine optimal shipping conditions to enable nuPsyllid to survive in transit for up to 72 hours. Factors to be examined will include shipping container type, temperature range, and water and food provisioning methods. ACP will be emplaced in the target population by sleeving (the placement of the psyllids into mesh screen sleeves fitted onto the terminal shoots of host plants at the target release sites). Sleeving has the advantage of enabling the
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psyllids to transition from the shipping container to a host plant without the mortality associated with ‘explosive release’ from opened containers. Psyllids will be placed in the sleeves in the afternoon and the sleeves removed the next morning. Early in the project cycle tests will be conducted to determine the spacing and density of sleeves required for effective release and establishment of nuPsyllid within the release site.
Existing methodologies used to transport insects from insectaries to release sites will be used to determine the optimal shipping parameters. Procedures for sleeving and marking ACP with fluorescent powders (Nakata 2008) have already been developed. Scientists from the rearing research team and the monitoring research team will optimize procedures for transporting and emplacing nuPsyllid.
Monitoring: Because of the high degree of ecological heterogeneity within the target sites, sampling methods will be developed to provide a high degree of sampling accuracy, intensity and uniformity as a means of reducing the amount of variation between samples. This will include the establishment of ‘typical’ target sites and designation of ‘sentinel trees’ in which sampling will occur. Target site selection criteria will be devised to provide as much uniformity as possible between sites, and will include factors such as: proximity of site to commercial orchards, host-plant species composition, relative amount of site edge (perimeter), and site accessibility to researchers. Site edge is an important consideration because pronounced edge effects have been observed in the occurrence of both HLB (Gottwald 2010) and ACP (Sétamou et al. 2008). The selection criteria for identifying sentinel trees will include factors such as exposure level to sunlight, shading, and wind, care level, and dimensions (Sétamou et al. 2008, Hall 2010). The initial tests for optimizing sampling methods for nuPsyllids will be conducted with wtPsyllid populations, and will be conducted with the sampling methods currently used to measure ACP population densities and distribution: tap samples (gentle tapping of new shoots to dislodge psyllids) (Hall et al. 2007; Qureshi et al. 2009; Hall and Hentz 2010), visual scanning to quantify and collect nymphs (Pluke et al. 2008; Qureshi et al. 2009), and standard yellow sticky card traps (Hall 2009; Hall and Hentz 2010). The optimization process will involve determining the necessary number of sampling points (traps), optimal placement of sampling points within sentinel trees, their distribution within the target areas, and the frequency of sampling required to provide accurate estimates of population parameters (Hall 2009). To optimize sampling success, visual monitoring procedures will be conducted under weather conditions favorable for observing ACP; i.e., sunny, warm days (Hall 2009), and traps will be placed in only actively growing trees (Hall and Albrigo 2007). Sampling and analysis protocols, such as pooling samples (Ebert et al. 2010), may be adopted to improve the accuracy of population estimates. As the sampling methods are developed, their accuracy will be evaluated by brief periods of intensive sampling and later on, by information provided by modeling. Based on these evaluations, the sampling methods and protocols will be modified as needed. Adjustments will be made as needed as information on the fitness level and other physiological characteristics of nuPsyllid becomes available. Adjustment in the monitoring methods will also be made to take advantage of improvements in trapping technology (below).
Methods for measuring the dispersal ability of nuPsyllids will be developed using wtPsyllids at the beginning of the project. This will involve visually marking with colored fluorescent powders (Nakata 2008) and biochemical marking with sprayed applications of ‘marker’ proteins (i.e., casein and albumin) (Tiwari et al. 2010). These procedures will enable investigators to recognize the marked psyllids after they are captured on trapping arrays set at varying distances from the marking site (Tiwari et al. 2010). Fumigation of whole trees will be conducted to obtain absolute population size, to compare the efficacy of sticky traps and tap sampling with regards to estimating population size and penetrance. Laboratory assays will be developed for
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rapid identification of nuPsyllids collected from monitoring and dispersal tests. The methods described are the standard protocols for measuring the population abundance and distribution of ACP. The ecological complexity of the commercial orchard/urban citrus interface will make it challenging to sample at the intensive levels required to accurately estimate ACP population dynamics and dispersal patterns. However, it is expected that the demographic models devised in other sections of the proposed research will enhance the potential of sampling data to accurately estimate nuPsyllid population parameters and movement patterns.
Socio-Economics/Modeling: To better understand how to successfully release the nuPsyllid, we must understand the set of influences, barriers and constraints that may affect adoption of an altered psyllid within urban and grower communities. For education outcomes to be most effective, we will need to test information content and dissemination approaches that might be most salient and persuasive in influencing urban and grower communities at both early and later stages of knowledge and awareness of the release. The evolution of CHMAs in Florida over the past two years is a case study example of the large-scale success that can be achieved in pest and disease management that is successfully adopted by growers when it is delivered in a manner that increases their trust of university and government research and extension personnel (Telg et al., 2010). The methodology proposed for modeling the nuPsyllid release program integrates socio-economic analysis, field data collection, biological and environmental modeling, population genetics, GIS visualization. Modeling and analysis for both the psyllid dynamics and the socio-economic impacts will be iterative activities in which specific objectives are refined in response to feedback from the wider research/extension project team and key stakeholders. Furthermore, while the modeling approaches have been designed to provide a robust framework for integrating the empirical work on developing and deploying the nuPsyllid, they have also been designed in a flexible way, focusing on basic requirements in the early years of the project while the laboratory research develops the nuPsyllids, and ramping up in later years when the focus of the research is expected to shift from development to deployment. The proposed biological and socio-economic analyses are connected by their common use of monitoring data and an emphasis will be placed on focusing the modeling and economic analyses on the central issue of the cost-effectiveness of the nuPsyllid release and its impact on economic welfare.
Sociological research. A survey will be developed from previous survey tools and with research and advisory committee input. The questionnaire will include basic information about the project and goals; variables to measure respondents' knowledge of HLB, the Asian citrus psyllid, attitude towards insect modification, perception of risk to local and nationwide citrus industries, knowledge of sustainable agriculture concepts, control programs for ACP and the impact of these programs on HLB control. Data will be collected on how respondents would prefer to learn about control measures, and who they would usually learn from. Basic demographic, psychographic, attitudinal, preference, goals and actions data will also be collected. In addition, in order to understand the social network structure, respondents will be asked to name up to five other people they discuss ACP/HLB with. The questionnaire will be mailed to orchard owners and management companies in FL, TX, AZ and CA. Lists for the mailings will be obtained from citrus commodity associations. The survey will be distributed once in the initial year of the project, and repeated every year through year 5-project to allow us to develop an understanding of changes taking place in this dynamic environment. Including growers from FL, TX, AZ and CA will add to information relevant to stakeholder knowledge and attitudes when living in states of various ACP and/or HLB pressures. To have a representative and sufficient sample, a goal of 1000 responses from each state will be needed each from the urban and grower communities. Mailings will be sent, with follow-up reminders, to reach a response rate of 20%.
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The current socio-economic impact of HLB in the US is significant, and it will be critical in our project to expand current efforts to analyze the impact of this disease on commercial citrus growers, homeowners who enjoy backyard citrus trees, environmental groups and the public in general. As we develop and deploy this approach to mediate impacts of HLB, it will be important to prepare growers and the public to accept this alternative to pesticidal suppression of ACP, and to quantify the real and potential long-term benefits of the approach to sustainable production and public consumption of citrus and citrus products produced in the US.
Biological modeling. An ACP phenology model, driven by degree-day accumulation, will be developed based on published ACP development studies (e.g. Liu & Tsai, 2000; Pluke et al., 2008) and additional data from the project team. The phenology model will run on the USPEST server (http://uspest.org/risk/models) at Oregon State University, to provide a spatially-referenced, real-time index of potential ACP reproduction. The model will be validated by comparison with FL ACP monitoring data provided by the USDA-ARS Fort Pierce research team and, in later years, the monitoring data collected by the project team. We will compare the phenology model with ACP predictions currently generated by the USDA-ARS Fort Pierce team to make sure that modeled ACP population sizes and dynamics are robust. The phenology and population dynamics models together provide the basic population reproduction rate which will determine the rate of introgression of the nuPsyllid phenotype into the wild population. We will model the rate of introgression and determine any asymptotic results (e.g. final level of introgression achievable) using demographic population genetics models (Caswell, 2001). These models will be based on the known genetic behavior of the nuPsyllid traits (this being dependent on the particular driver mechanism in the nuPsyllid) in combination with the population dynamics. The projections of introgression will be validated against field monitoring data from the release and monitoring team. This tie between the models and field data closes the analytical loop between modeling and observation by providing ground-truth data on the ACP population structure; i.e. the proportions of wtPsyllids and nuPsyllids.
Economics. Introducing nuPsyllid will be an economic activity with costs and benefits to different sectors at different scales. Estimates indicate that HLB has resulted in an economic loss of $3.6 billion and caused a loss of over 6,600 jobs in Florida in a 5-year period from 2006-2011. The interplay between these costs and benefits, and their perception by different sectors, will set the overall socio-economic environment for the introduction program. We will make use of the GIS platform provided by the USPEST outputs to study the economic issues in a spatially explicit and semi-qualitative approach. Surveys will be used to compile costs and benefits along different economic dimensions for different sectors in different locations. These data will be used, with the simulated and projected insect population to produce economic welfare maps of the regions where the introductions of nuPsyllid are planned. In a complimentary approach we will use standard contingent valuation techniques to get at the economics of nuPsyllid for stakeholders in the affected areas. The baseline comparison for these valuations will be today’s production system, with the extra management burden imposed by ACP/HLB presence.
Monitoring economics of implementing sustainable HLB control. This portion of the project will economically evaluate the costs and benefits of rearing and releasing the nuPsyllid and model the impact on HLB spread in the presence or absence of nuPsyllid. Economic impact will be based on pesticide application rates, frequency of application, material and application costs, other identified production costs associated with HLB management, and yield changes. We will utilize a block-level HLB Gompetz function spread model (Gottwald et al. 2008) to depict the spread of HLB as a function of nuPsyllid population. Yield will be predicted in blocks of varying nuPysllid populations. Production costs associated with HLB management are expected to decline as the population of nuPsyllid is established over time. Economic benefits are expected
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from lower tree loss and greater fruit production as a result of introduced nuPsyllid populations and reduced infection rates.
The costs of rearing and releasing programs will be monitored in all states involved in the project. The first year (and potentially a portion of the second year) of rearing will provide a benchmark of these costs and allow us to project future costs going forward in time. Releasing costs can be modeled using variables such as nuPsyllid populations needed for a set area, number of areas, and methods of dispersion. The overall cost of rearing and releasing will include population monitoring and frequency of monitoring. A model will be developed that predicts the break-even costs and return on investment of rearing and releasing nuPsyllid. Output from these analyses will be used to guide messages delivered in the outreach program.
Dealing with a disease such as HLB is a social (or collective) problem; individual growers have difficulty in managing their way out of an HLB outbreak independently. For this reason, the highest priority for the socio-economic analyses is to analyze the social network and information structure associated with citrus production and to characterize the factors which will promote collaboration among growers and other stakeholders. The approach here will be to use survey- based data gathering and graph-theoretic statistical analyses and network modeling (Ortolani et al., 2010; Lubell et al., 2010).
The social network and socio-economic baseline surveys, together with the development and testing of the phenology model will be the focus of the first two years of work. In later years the modeling work will build on the basic tools developed in the first two years to explore different deployment approaches for the nuPsyllid and analyze their economic impacts. At this stage, standard economic valuation methods will be used to derive per hectare values for citrus to stakeholders with different utility evaluations for nuPsyllid presence in the environment, citrus sustainability, and different levels of pesticide use. Existing ACP population data combined with the economic analyses will allow us to carry out ex ante cost-benefits analysis of the nuPsyllid release program and to suggest optimization approaches.
All methods that will be used in the Socio-Economic/Modeling System of the research are well- validated within the disciplinary areas from which they come, indicating a high probability of feasibility, utility and success.
3. Expected Outcomes
Currently, an economically crippling plant disease (HLB), threatens all U.S. citrus production. By releasing a nuPysllid that is unable to vector the disease, we will reduce spread of HLB and begin the process of restoring the sustainability and securing the long term profitability of the citrus fruit and juice industries. Expected outcomes include: • Averting the loss of sustainable citrus industries in the US • Reducing current losses to HLB in Florida (and more recently, Texas) • Preventing HLB from expressing itself fully in other citrus producting states • Reducing need for reliance on pesticides for reducing vector psyllid populations • Providing novel solutions that have been developed, tested, and made ready for deployment
in multiple states • Establishing nuPsyllid rearing and monitoring systems that will have been advanced and
tested • Advancing regulatory considerations for adoption of the nuPsyllid concept • Assessing economic potential for nuPsyllid to mitigate loss from HLB
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4. Results Analysis Below, we discuss Results Analysis for each of the three Driver Systems, the two Effector Mechanisms, Rear, Release and Monitoring and Socio-Economics/Modeling.
Chromosomal Driver: Transgenesis, and the results of transcriptional profiling, will be used to carry out experiments designed to identify fragments of DNA with maternal, early embryo, or tissue-specific promoter activity. This information, in conjunction with analysis of maternally expressed genes, will be used to inform the synthesis of functional Medea elements and underdominance constructs. Simple crosses between transgene-bearing individuals and non- transgenics (wild type) will be used to determine if the constructs show drive and stability.
Medea and/or underdominance construct-bearing psyllids will be tested for drive through crosses with wild type individuals. Elements that show drive will be linked to effector genes and tested for effector activity and drive. Empty drive elements and drive elements carrying effectors (as they are developed) will be tested in more natural settings for drive and effector function. Drive- effector combinations that can spread and show efficacy in preventing HLB transmission are good candidates for field tests and regulatory consideration.
Viral Driver: RNA sequencing and bioinformatic analyses will be done on psyllids collected from various worldwide locations. We will identify D. citri viruses from these analyses and then confirm their incidence in specific populations. We will select specific virus(es) for further work including life history parameter effects, and create infectious cDNA copies of specific virus genomes. Wildtype and recombinant viruses will be evaluated and the latter used to express interfering RNAs and/or anti-microbial peptides or other effectors in D. citri maintained within the UC Davis Biosafety 3P Contained Research Facility. We will identify optimal targets and determine how best to deliver these to D. citri so as to induce effects at the population level.
Bacterial Driver: DNA sequencing and bioinformatics analyses will be conducted on psyllids collected from various locations to determine the strains of Wolbachia present and the proportion of psyllids infected within a population. Lines of psyllids naturally or artificially infected with Wolbachia will be evaluated via RNA sequencing and bioinformatic analyses to determine the effect of infection on gene expression. Concurrently, fitness and transmission assays will be conducted to evaluate the effect of Wolbachia infection on the life history of psyllids and the capacity of psyllids to transmit CLas. We will select strains of Wolbachia that best interfere with CLas transmission and psyllid fitness for establishment in a mass-reared psyllid colony. Psyllids carrying these Wolbachia will then be released into natural populations that lack Wolbachia. Spread of Wolbachia through the wild population will then be monitored.
Effector Mechanism - Library Approach: Experiments will target the identification of peptides or scFv that can block systemic movement of CLas within the ACP based on the use of bioassay screening. Results from two major assay systems will be analyzed: 1) In vitro CLas/ACP membrane/cell preparations. Base-line association of enriched CLas preparations with membranes/cell preparations of ACP will be monitored using Q-PCR and FISH detection of CLas with washed ACP cell/membrane preparations. Influence of peptides and scFvs on this association will be verified using the same CLas detection methods fluorescent in situ hybridization (FISH) detection of intact CLas cells and specific observations of ACP cellular uptake of CLas will be conducted using the FISH protocol (Ammar et al., 2011). Typical statistical analysis of Q-PCR data and quantitative analysis of CLas cells observed by FISH techniques will be employed. 2) ACP feeding uptake and transmission bioassays (feeding late instar nymphs on artificial diet and transfer emerged adults to excised citrus leaves) will be analyzed by Q-PCR of CLas within ACP that have fed on artificial diets containing enriched
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CLas preparations and then transferred to non- CLas containing medium to flush gut contents. Specific movement into the salivary glands will be observed by Q-PCR of isolated salivary glands (Ammaret al., 2011) and results will be verified by FISH. Transmission of CLas to citrus from these ACP will be quantified by Q-PCR detection of citrus leaves after ACP feeding for 3 weeks.
Effector Mechanism - Genomic Approach: The ACP transcriptome data sets will be used to inform vector-pathogen interactions integral to the CLas transmission pathway. We will aim for genes involved in infection, gut-establishment, mobility (hemolymph), entrance into/exit from the salivary glands, and salivary components. Comparative transcriptomic analysis provided an informative platform to initiate these studies by revealing a plethora of misexpressed unitrans. The proposed experiments will complete the data sets essential to identifying prospective determinants in ACP using ACP EST and RNAseq data sets that will profile expression of genes in salivary glands and guts in the presence and absence of CLas. This will aid in identifying barriers to infection (gut), migration, salivary gland entry and utilization that will be characterized in functional terms, and identify those that are truly key determinants of transmission. Promising targets validated using dsRNA bioassay (artificial feeding or microinjection), qPCR, and in transmission tests will be considered top candidates for effector constructs.
An ideal effector would have no negative effect on psyllid fitness and should not be overcome by a simple single mutational event. A straight-forward a priori hypothesis would be that targeting microbial molecules would have the least probability of affecting psyllid fitness. Conversely, targeted microbial sequences would potentially have the highest probability of incurring a mutation to overcome the blocking strategy because of the presumed higher mutation rate of the microbe and because of the selective pressure placed on the microbe. For this reason, our research focuses on targets that could be of either ACP or CLas origin. Ideally, we would recognize two targets for each or at least one from each of CLas and ACP. Candidate effectors will be selected based on the best binding kinetics and activity that reduces systemic movement of the bacterium through the psyllid and/or blocks transmission in leaf bioassays and ultimately in whole plant bioassays. We also propose to compare these results with those from other research being conducted on antimicrobial peptides, AMPs. If AMPs are discovered and shown to be active against CLas, they will be incorporated into the next phase of testing. There are no identified potential hazards to personnel.
Rear, Release and Monitor: Rear - results of experiments testing key parameters will be analyzed with standard parametric tests; i.e., ANOVA with appropriate means separation tests. In instances where fewer replicates are obtainable, then appropriate non-parametric tests will be used; i.e., Kruskal-Wallis test. As the entire process becomes optimized, then production norms will be established to enable the planned deployment of nuPsyllid. Release - results of tests of key transport conditions and experiments used to determine sleeve distribution and densities will be analyzed with standard parametric tests. Monitor - statistical protocols currently used to analyze ACP population parameters will be used to compare data sets generated in the proposed studies (Hall et al. 2008; Flores et al. 2009; Ebert et al. 2010; Hall 2010; Tiwari 2010). The mark-recapture data used to determine dispersal ability will be analyzed with Jolly-Seber methodology (Gall 1985).
Socio-Economics/Modeling: Biological Modeling: Results will be analyzed through use of the expertise at Oregon State University Integrated Plant Protection Center (developer of the USPEST system). The outputs from USPEST will be analyzed in GRASS format (an opensource GIS) and combined with overlays of other variables facilitating linkage to our other
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objectives. Results from the survey will be used to develop an understanding of baseline knowledge of ACP management and release of altered insects, the HLB problem, potential treatments to mitigate HLB, risk perceptions of growers, and information networks for education. Data will be analyzed using a number of approaches including multivariate analyses and univariate mixed models.
Socio-Economic Modeling: Surveys will be used to compile costs and benefits along different economic dimensions for different sectors in different locations. Results will be analyzed through the economic welfare maps from the regions where the introductions of nuPsyllid are planned. In addition, the results of stakeholder economics will be analyzed to develop a baseline comparison to be used to communicate to growers and other stakeholders, the extra management burden imposed by ACP/HLB presence.
5. Use of Results
Research directed toward functional Driver System and Effector Mechanisms will proceed in parallel over the first phase of the project. Based on a critical assessment of research results, one or more Driver Systems and several Effector Mechanisms will be advanced based on technical feasibility. The results from the Driver System research will be combined with the results of the Effector Mechanism research to deliver several candidate nuPsyllids for further comparative analysis in growth chambers and greenhouses. Candidate nuPsyllids will be analyzed for fitness, drive potential and the ability to block transmission of CLas. Technical feasibility is the obvious prerequisite for success but many other considerations such as the pathway for regulatory approvals will be required for the ultimate adoption of this technology for disease management. Therefore, one or more nuPsyllids that will be the result of this combined effort will then be made available for the rear, release and monitor phases.
Once optimized and standardized, the mass-rearing system will be capable of producing required numbers of nuPsyllid for release into target areas during scheduled release periods. The release results will be used to inform directly the procedures and methods used in transporting nuPsyllid from rearing facilities to target sites and successfully emplacing them at the site. Improvements in monitoring will provide the capability to sample and assess ACP population parameters and movement and distribution patterns in highly complex landscapes. This will permit accurate assessment of the emplacement, establishment, and penetration of nuPsyllids into wtPsyllid populations at target sites. This will also have the benefit of cutting costs to agencies and growers for monitoring wtPsyllids.
For socio-economics/modeling, through use of the Geographic Information Systems (GIS) platform, data will be used, with the simulated and projected insect population to produce “economic welfare” maps of the regions where the introductions of nuPsyllid are planned. In a complimentary approach using the standard contingent valuation techniques, the baseline comparison for these valuations will be today’s production system, with the extra management burden imposed by ACP/HLB presence.
6. Outreach Plan
The primary goal of this project’s outreach plan is to help the public to understand and accept the release of a modified psyllid. To develop the message, the outreach team would focus on the method of psyllid modification that is ultimately successful, and build the messaging strategy accordingly. We acknowledge the challenges of acceptance of a genetically modified solution (Frewer et al 1998) and will build the message as informed by sociological surveys we will conduct as described. Messaging points would emphasize the harmlessness of this insect and the potential for its release to reduce the chemical management of psyllids. It is essentially a ‘green’
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way of psyllid management and can be thought of as a way to biologically control the disease. The modified psyllid is not part of the plant and so it poses no risk to consumers of citrus. It is also compatible with biological control agents such as the ectoparasitoid Tamarixia radiata and other microbial control agents that provide for some reduction in psyllid populations but are not effective in stopping HLB spread. To be effective, outreach programs must be guided by advocacy for the public interest (Warner et al. 2011). Education of the public and citrus industry will be achieved using the existing infrastructure of e-Extension, university cooperative extension and train the trainer programs. Normal routes of media outreach have been used successfully in AZ, CA, FL, and TX for educating the rural and urban sectors on the dangers of exotic pests and diseases and the benefits of deployed control strategies. We will utilize this existing framework including radio and television interviews, newspaper and magazine articles, social media, and web sites for messaging. An example of the approach intended is the media blitz currently underway to publicize the release of Tamarixia radiata as a biological control for psyllids in California by Mark Hoddle, University of California, Riverside (UCR). The Assistant Chancellor and key industry representatives conducted the first release on the UCR campus. University and commercial media entities photographed, videotaped the release, and conducted interviews. In preparation for a major release in residential areas in Los Angeles, an interview with Dr. Hoddle was aired on the California Report, a program of National Public Radio. Karen Ross, California Secretary of Agriculture has been invited to conduct this release. Radio and television stations will be present for this event as well.
As the initial research is ongoing, research, outreach and advisory board team interaction will be critical so that the most successful message strategy can be developed and deployed. We will interact with state and federal agencies to share our strategy, seek advice, and benefit from synergistic outreach components. We intend to follow the model currently being used in California to educate the public and industry about the dangers of the Asian citrus psyllid and the disease it vectors in all citrus producing states. Importantly, outreach began in California prior to the first detection of ACP. Industry, the University of California, the California Department of Food and Agriculture joined efforts and agreed on a unified message. The public received brochures at their homes, booths were set up at Home & Garden Shows and manned by experts to target the public for one-on-one education. Master Gardeners were trained statewide. Industry officials, growers, state regulators, and university scientists participated in annual media tours. There were over nineteen million radio and TV public service announcements in both English and Spanish, broadcasted in 2011 alone. Written literature is translated into numerous languages and ethnic events are targeted. Such a proactive timeline, messaging outlets, and partnerships are central components of urban outreach strategies deployed in states currently battling ACP/HLB. In a similar fashion, the outreach activities for the proposed research will occur prior to the release of the nuPsyllids.
In addition to the urban campaign, the land grant Universities of Florida-IFAS, California, Arizona and Texas A&M extension team members will plan and launch a grower/producer education campaign. For this, we intend to follow the outreach model currently utilized in Florida to deploy area-wide pesticide control strategies for the Asian citrus psyllid; the Citrus Health and Management Area (CHMA). The CHMA structure was recommended by the National Research Council as a priority mechanism to reduce the spread of HLB. CHMAs were developed with CRDF sponsorship and deployed to approximately 4/5 of the Florida commercial citrus growing acreage by the University of Florida-IFAS extension team, and now managed in partnership with the Florida Department of Agriculture’s Department of Plant Industry. CHMAs are grower-driven collectives governed by growers, making them excellent outlets to successfully reach the grower/producer sector. We will begin by informing this segment of the population about project goals and objectives, explain the method of psyllid modification, and
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discuss the benefits of the strategy and potential risks, if any. Our success in developing educational materials for exotic diseases (www.crec.ifas.ufl.edu/extension) will be built upon to provide printed fact sheet materials, laminated cards, memory aids, social media, DVDs and video presentations to inform growers. The CHMA structure has been developed in Texas (TX Pest Management Areas) and is currently being developed in AZ and CA. We anticipate that the development of CHMAs in these states will be advanced by the time this project has progressed to make this local structure an excellent mechanism of outreach for our information. In addition to the above mechanisms, we will take appropriate actions to target growers through existing national, state and regional organizations and events. California holds the Citrus Showcase and Grower Conferences; Florida holds the Citrus Expo, Citrus Show and Grower Meetings; Texas Citrus Mutual hosts the annual Citrus Show and Grower Meetings, the Universities and county extension programs conduct educational seminars. In Arizona, the citrus extension program targets the commercial grower, the Master Gardener and the general public. In the case of the modified psyllid, outreach activities would include educational meetings and seminars, English and Spanish language brochures and other publications, interviews with the media and development of an improved ACP/HLB website.
For the proposed project, the outreach efforts of each state would be studied and expanded on,
unifying information and sharing outreach tactics. In addition to printed materials and media events we will also utilize the World Wide Web. We intend to hire a web designer to assist with web page content that will be incorporated into existing websites throughout the nation (USDA – www.saveourcitrus.org, www.aphis.usda.gov/hungrypests, Florida DACS DPI – www.freshfromflorida.com, www.citrusrdf.org, California – www.Californiacitrusthreat.org, www.ipm.ucdavis.edu, ucanr.org/sites/kacditrusentomology, cisr.ucr.edu, Texas - www.texascitrusgreening.org, Arizona – www.azda.gov/PSD/acphtm, Louisiana - www.lsuagcenter.com, National Plant Disease Network, and e-Extension. Dr. Grafton-Cardwell has extensive experience developing on-line courses for growers and will create a course that explains how nuPsyllids were modified and how they are used. This course will be available to a worldwide audience in both English and Spanish. These outreach efforts using different media would also include the nuPsyllid. At the onset of our education effort, we will reach out to other states (currently LA, GA, SC) impacted by ACP and/or HLB to share our information, and project objectives and goals, so that the national ACP/HLB community is aware of our efforts and will benefit from its outcome.
Because of her experience with the successful California outreach program, Dr. Beth Grafton- Cardwell has agreed to be the National Coordinator for Outreach for this project. Her role will be to oversee the generation of outreach materials and provide them to her colleague extension specialists in each state that will be releasing the modified psyllid.
Our public outreach will consist of the following team leaders: Outreach Area Personnel National Coordinator Beth Grafton-Cardwell Florida Michael Rogers Texas Mamoudou Sétamou Arizona Glenn Wright California Matt Daugherty
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7, 8, & 9. Potential Pitfalls, Limitations & Hazards Below, we discuss Potential Pitfalls, Limitations and Hazards for each of the three Driver Systems, the two Effector Mechanisms, Rear, Release and Monitoring and Socio- Economics/Modeling.
Chromosomal Driver: The potential pitfall is the possibility of not being able to get transgenesis to work in the psyllid, which would prevent transgenesis-based blockers and drive mechanisms such as Medea and underdominance from being developed, though the likelihood of failure is low as transgenesis has already been achieved in many insects of economic importance. The proposed developments are limited by the quality of data that can be gathered from transgenesis based experiments required for promoter identification, blocker testing, and generation of full Medea or underdominance-bearing transgenics. The greatest source of limitation would be that transgenesis occurs at a low frequency, which would limit our ability to test multiple hypotheses and reagents in a timely manner. There are no identified potential hazards to personnel.
Viral Driver: The potential pitfall is the possibility of not finding a suitable virus for the work described, though highly unlikely due to the abundance of viruses. There are no identified limitations at this time. Some aspects of the work will require using hazardous chemicals that will be handled according to University protocols using stringent safety equipment and protection. The viruses used are specific to insects and do not pose a threat to personnel. We will generate new recombinant DNAs, and have University approved use authorizations and will follow all appropriate guidelines. Finally, we will require rearing the ACP, D.citri, for our work. As this is a restricted pest, we will perform our research within the UC Davis Biosafety 3P Contained Research Facility and no live organisms will be removed from the facility.
Bacterial Driver: The proposed work depends on the potential for a Wolbachia strain to be isolated or created to elicit immune or other changes in the psyllid that interfere with transmission. Although this approach has successfully identified such effects in other vector species, it is possible that D. citri will not exhibit a similar response. The greatest limitation of this objective is that immune gene expression or other changes elicited by Wolbachia may reduce transmission, but not to a degree that would be useful for mitigation of transmission in the field. There are no identified potential hazards to personnel.
Effector Mechanism – Library Approach: A potential limitation to this work is the development of a robust bioassay where we can detect and quantify the association of CLas with whole cell or membrane preparations from specific ACP tissues. To avoid limiting our research to the success of this one interaction, we will also develop a functional bioassay where the peptides and scFvs are screened in a whole insect feeding system using an artificial diet (Hall et al., 2009) and a leaf-based bacterial transmission assay we have developed. Another potential pitfall of a broad library screening technique is that non-specific interactions confound the ability to distinguish specific inhibitors. Although this can often be avoided through optimization of the bioassay conditions, binding kinetic studies will be conducted to show specificity of the interactions. There are no identified potential hazards to personnel.
Effector Mechanism – Genomic Approach: The potential pitfall within the genomics work is that no candidate effectors will be identifiable using the proposed functional genomics-proteomics approach, however, the likelihood of that is very low. The most potentially difficult aspect is the pull-down assay, however the combined use of yeast-2 hybrid (and split GFP assay, as needed), FISH localization, and protein-network predictions will provide robust corroboration. Several methods are available to study protein–protein interactions in vitro and in vivo, including the two
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most trusted, yeast two-hybrid analysis (Y2H) and split protein complementation assays. The Y2H library screen relies on interactions between ‘known’ bait and ‘unknown’ prey complexes that upon binding restore transcription factor activity. This is an indirect indicator of gene expression, and the assay can lead to a false positive owing to auto-activation. Additionally, weak interactions can be lost in follow-up co-immuno-precipitation experiments aimed at confirming Y2H results. The split-GFP system is useful because this assay produces a direct interaction and is not prone to false positives at this stage of the assay, and is compatible in many cell types. However, it too has some potential limitations most notably, weak expression that can be mistaken for lack of interaction. We will use split-GFP reassembly (Park et al., 2007) as a complementary approach to achieve comprehensive, robust results. The UA-Institutional Biosafety Committee IBC oversees use and regulation of chemical, biological, and other hazardous materials; each PI must receive IBC approval for all protocols prior to proposal submission.
Effectors will be selected from peptides, scFvs or RNAi that block systemic movement of CLas within the psyllid or block other important protein-protein interactions directly or indirectly required for transmission. The potential pitfall is that no candidate effectors will be identified. Because of the parallel and comprehensive approach, this is highly unlikely. However, if we have not been able to identify enough targets, we will develop a control strategy based on CLas- infected cell-specific induced suicide. This strategy involves the use of a CLas induced promoter, discovered through the RNAseq research that induces the production of a protein that kills the CLas infected cell. We propose to use a ribonuclease-encoding gene show to work in other systems (Barnase). It is understood that genes induced by CLas may also be induced under other circumstances and so our focus will be to identify induced genes related to defense to avoid those that may also play a developmental or other basic metabolic role.
Rearing, Releasing and Monitoring: Rearing: Procedures may need to be modified to meet the limitations of available facilities and conditions at each rearing site. Since no artificial diet is available for rearing ACP, successful mass-rearing will depend upon the availability of a sufficient inventory of CLas-free host-plants. ACP cultures are susceptible to infestations of tiny parasitoids (i.e., Tamarixia spp.) and infection by pathogenic fungus (i.e., Isaria spp.). These pests can be controlled by screens and maintaining proper humidity and temperature levels. There are no identified potential hazards to personnel.
Releasing: ACP mortality has the greatest chance of occurring during transportation from the rearing facility and following release from the shipping container. Existing technologies can be adopted for use in developing optimal shipping methods, and sleeving procedures have already been devised for emplacing ACP at target sites. If modification alters the pysllid’s ability to withstand shipping conditions or sleeving procedures, then adjustments and modifications will need to be made to ensure acceptable levels of survivorship during transit and release. The use of containers for shipping ACP and their relative intolerance to cold conditions may restrict shipping times to 48 to 72 hours before unacceptable levels of mortality occur. There are no identified potential hazards to personnel.
Monitoring: The ecological complexity of the commercial orchard/urban citrus interface will make it challenging to sample at the intensive levels required to accurately estimate ACP population dynamics and dispersal patterns. Current sampling methods (i.e., yellow sticky card traps) will require intensive sampling efforts to accurately estimate nuPsyllid population abundance, distribution, and penetrance. The selection of ‘typical’ target sites will help reduce variation in sampling data. However, the use of both sentinel trees and ‘typical’ target sites for
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sampling still may not produce data sufficient for accurately estimating population parameters and dispersal patterns because of the complexity of the overall landscape and the tendency of ACP to aggregate. If the proposed sampling protocols are inadequate, then the sampling intensity will be adjusted to overcome resolution problems caused by high variance levels. The proposed modeling and expected improvements in trapping technology will also enhance utilization of data for devising estimates. Modifications will need to be made in the sampling procedures to accommodate differences in regional environments; however, the procedures must remain uniform enough to enable comparisons to be made across all treatment areas.
The ability to accurately estimate ACP populations in complex landscapes is limited by the capability to intensively sample with the available technology such as yellow sticky traps. Sampling capability will be enhanced by expected improvements in sampling technology, which will attract greater numbers of psyllids from the local population. These improvements will be integrated into the sampling protocols as they become available. In addition, sampling capability will be improved by information provided by modeling, which will inform the deployment and distribution of sampling points necessary to provide accurate estimates of population parameters. There are no identified potential hazards to personnel.
Socio-Economics/Modeling: As an exercise in making theoretical predictions about nuPsyllid dynamics and economic impacts, there are no significant limitations to the socio- economics/modeling work. The involvement of industry research bodies in the research should also ensure a high penetration of the social network data surveys into the grower communities and a high rate of return. As a vehicle for integrating the biological research, the value of the proposed work depends entirely on the success of the various empirical areas of research. If any of those have significant problems it will inhibit the practical usefulness of the modeling. Although none of the individual pieces of modeling work depends on novel approaches, their synthesis into an overall analysis is likely to be technically demanding. The topic leader has 15 year’s experience leading interdisciplinary multi-site projects and is well equipped to ensure progress and integration. The proposed research is limited by the quality of data collected from the field. There are no identified potential hazards to personnel. Planned grower and public outreach efforts should effectively address and manage the concerns of those communities.