Source: UNIVERSITY OF FLORIDA submitted to NRP
BT TOXIN-BASED STRATEGIES FOR MANAGEMENT OF DIAPHORINA CITRI AND CITRUS GREENING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1012952
Grant No.
2017-70016-26755
Cumulative Award Amt.
$2,476,099.00
Proposal No.
2017-03289
Multistate No.
(N/A)
Project Start Date
Feb 1, 2017
Project End Date
Jan 31, 2022
Grant Year
2017
Program Code
[CDRE]- Citrus Disease Research and Extension Program
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
ENTOMOLOGY AND NEMATOLOGY
Non Technical Summary
When an insect, the Asian citrus psyllid (ACP) feeds on the sap of a citrus tree, it can transmit a pathogen to the tree that causes citrus greening or Huanglongbing (HLB). As a result of this disease, citrus yields in Florida have dropped by 50% since 2008, and citrus in Texas and California is now threatened. Effective means for limiting the spread of ACP are the first line of defense against the spread of HLB into new areas in Texas and California and into new plantings in Florida. To manage HLB, citrus growers have repeatedly applied chemical insecticides to control ACP. Unfortunately, these pesticides preclude the use of biological control methods that use beneficial insects (natural enemies) to manage ACP populations and the elimination of insect natural enemies has led to an increase in populations of insects not previously known to be pests on citrus. It is clear that a more sustainable, less costly, and more specific control method is urgently needed to control ACP.Toxins from a soil-dwelling bacterium called Bacillus thuringiensis (Bt) have been used successfully to control some insect pests. On ingestion, the Bt toxin damages the gut tissue and kills the insect. Bt toxins are specific and provide a sustainable approach that can be used in combination with the best insecticide programs or with biological control agents. Having identified Bt toxins that have toxicity against ACP and having developed a method to further improve toxicity against such sap-sucking insects, the goal of this project is to take the next step toward practical use by developing multiple delivery systems for the ACP-active toxins. To be ingested when ACP feeds on the citrus tree, the toxins must be in the plant sap. The sap delivery systems to be tested include microorganisms (bacteria, plant virus) that reside within citrus trees, and modification of the citrus plant itself to produce the toxin. The economic impact of these Bt toxin-based strategies for ACP control will be evaluated. Information about these new management approaches will be delivered through multiple sources to growers and to the public, and feedback solicited to facilitate adoption of Bt-based ACP control tools by citrus growers.
Animal Health Component
75%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110115015%
2124010202015%
2124030202015%
2110999202020%
9030999303025%
6010999301010%
Knowledge Area
601 - Economics of Agricultural Production and Farm Management; 212 - Pathogens and Nematodes Affecting Plants; 211 - Insects, Mites, and Other Arthropods Affecting Plants; 903 - Communication, Education, and Information Delivery;

Subject Of Investigation
0999 - Citrus, general/other; 4010 - Bacteria; 3110 - Insects; 4030 - Viruses;

Field Of Science
2020 - Engineering; 3010 - Economics; 1150 - Toxicology; 3030 - Information and communication;
Goals / Objectives
The U.S. citrus industry has an annual economic impact of more than $11 billion. Fruit production levels have been significantly reduced in Florida however due to the effects of citrus greening or huanglongbing (HLB). The causative agent of this disease is a bacterium, Candidatus Liberibacter asiaticus (CLas) that is vectored by the Asian citrus psyllid (ACP), Diaphorina citri. The damage caused by HLB is threatening commodity price stability and affordability of citrus products and increasing the economic and environmental costs for insecticide-based management programs. New, efficient, and long-lasting tools are urgently needed to control the insect vector, D. citri, toward mitigation of this disease. Preventing D. citri transmission of the bacterium CLas is key to curtailing the spread of citrus greening. Based on our recent critical breakthrough that makes Bacillus thuringiensis (Bt) toxins effective against phloem-feeding insects our long-term goal is to enable an environmentally benign, Bt toxin-based approach for citrus growers to control ACP that works within an integrated pest management (IPM) strategy and minimizes psyllid resistance.To accomplish this, the specific goals of the proposed project are to: 1) further create ACP-active Bt toxins that suppress psyllid populations and thereby curtail transmission of HLB (resulting in increased effectiveness and reduced likelihood of resistance), and 2) develop delivery approaches suitable for each of three citrus-growing states (Florida, Texas, and California). These first two goals align with the practical needs of growers in developing appropriate toxin delivery systems for use in IPM. We will examine and test four distinct approaches for efficient Bt toxin delivery. Three delivery methods used with non-transgenic citrus are the CTV vector, phloem-inhabiting bacteria, and a transgenic trap plant. The fourth delivery approach will use transgenic citrus plants. Further goals are to: 3) evaluate the economic impact of the Bt toxin-based delivery strategies, and 4) deliver information to stakeholders throughout the course of the project. Grower attitudes toward adoption of these strategies will be positive due to targeted and sound insecticide application schedules that rely on an integrated approach and allow for other control measures, such as biological methods. The result will be a reduction in the environmental consequences of intense insecticide applications and direct economic benefits to growers by reducing the number of required annual sprays for ACP management and slowing the spread of HLB.The objectives are to:1. Identify and optimize additional ACP-active Bt toxins. We hypothesize that we can isolate and optimize wild-type Bt toxins with even greater toxicity against ACP than those identified to date. The use of multiple toxins will reduce the likelihood of psyllid resistance;2. Evaluate toxin delivery strategies (CTV, phloem-inhabiting bacteria, and trap plants with non-transgenic citrus; and Bt toxin delivery via transgenic citrus plants). We will develop delivery approaches suitable for the three primary citrus-growing states by evaluating Citrus tristeza virus (CTV) vectors, phloem-inhabiting bacteria, transgenic trap plants, and transgenic citrus for their efficacy of Bt toxin delivery against D. citri. We hypothesize that different toxin delivery systems will be appropriate in different states.3. Evaluate the economic impact of Bt toxin-based strategies using mathematical modeling and economic analyses to assess the impact of different delivery systems in different states. We hypothesize that Bt toxin use in ACP management programs will result in economic benefit to citrus growers.4. Deliver information to stakeholders and seek input through outreach activities. We hypothesize that needs-based outreach activities via multiple channels will facilitate the adoption by growers of Bt-based technologies for suppression of D. citri.
Project Methods
Objective 1: Identify and optimize additional ACP-active Bt toxinsIsolation of D. citri active toxins Up to an additional 60 Bt strains will be screened in bioassays with D. citri for toxicity. Amplified strains will be checked for stability, crystal proteins purified and proteolytic profiles characterized. ACP toxicity assays will be conducted by membrane feeding assay using established procedures. The psyllids will be examined daily to monitor survival over a 7-day period. Five replicate bioassays with five ACP per treatment will be conducted. Relative toxicity of toxin combinations from individual strains will be estimated by comparing mortality rates of ACP exposed to Bt to mortality rates of ACP feeding on control diet, using one-way ANOVA, Tukey's test, and logistic regression analysis (Proc Logistic; SAS version 9.4; SAS Institute, Cary, NC).Purify individual toxins from Bt strains with toxicity to psyllids and test individual toxins for ACP toxicity. Purification of individual toxins is a multistep process involving solubilization of toxins and gel filtration column purification. Fractions will be analyzed on SDS-PAGE gel, and those containing a single toxin will be pooled. Purified protein will be stored at -20°C. The median lethal concentration (LC50) values for purified individual Bt toxins will be determined by conducting bioassays as described above. Purified toxins with ACP toxicity will be separated by SDS-PAGE and stained with Coomassie Blue. Stained toxin bands from the gel will be cut out used for toxin identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Coding sequences for specific toxins will be identified by analysis of the genome of the relevant bacterial strain with reference to peptide mass fingerprinting results.Modify toxins identified with ACP gut-binding peptide for enhanced efficacy. We will add an ACP gut-binding peptide to selected toxins to further increase efficacy against ACP. Selected toxins will be modified with ACP gut-binding peptide 15 previously isolated in the Bonning lab. Modified toxin constructs will be expressed and purified for testing in ACP bioassays.Objective 2: Develop and evaluate toxin delivery strategiesHaving identified two wild-type Bt toxins with ACP activity, we will test two delivery options for efficient delivery of toxins to ACP (CTV, transgenic citrus) and develop delivery systems based on phloem-inhabiting bacteria and trap plantsCTV vector - CTV constructs containing different Bt toxin derivatives will be introduced into citrus cultivars, and their impact on ACP survival, reproduction, and ability to transmit CLas assessed using techniques established in the Dawson lab. We will test the hypothesis that CTV delivery of an ACP-active toxin will efficiently suppress ACP populations on non-transgenic citrus. We will determine the positions from which to express the Bt toxins for both optimal activity against psyllids and optimal stability of the vector. We will utilize the CTV vector to quickly assess the efficacy of different Bt-toxin constructs and the value of the vector as a means to rapidly deploy effective Bt-toxin constructs in the field.Phloem-inhabiting bacteria-We propose to deliver ACP-active Bt toxin to citrus trees via phloem-inhabiting bacteria. We will 1) conduct proof-of-concept experiments using a bacterial pathogen of citrus (Spiroplasma citri) for Bt toxin delivery, and 2) develop delivery systems using non-pathogenic bacteria isolated from phloem-enriched citrus tissues.Transgenic Indian curry leaf plant - The Indian curry leaf plant (Murraya koenigii) is an alternative host for ACP. Transgenic M. koenigii plants expressing the Bt toxin will provide an additional tool for management of ACP on the basis that trap plants can be of considerable value for IPM. We will follow published reports of Indian M. koenigii propagation for production of transgenic plants, but using plants derived the U.S.Transgenic citrus plant - We propose to use Agrobacterium tumefaciens-mediated transformation of juvenile stem explants for production of transgenic Duncan grapefruit expressing Bt toxins using well-established procedures.Objective 3: Evaluate the economic impact of Bt toxin-based strategiesThe economic analysis of Bt toxin-based psyllid control strategies will build on current modeling efforts and will involve three steps: 1) a farm-level analysis of how each effective Bt toxin and delivery method affects the costs to produce citrus for the grower, 2) an area-wide analysis of how each effective Bt toxin and delivery method affects the rate of spread of HLB and regional methods to manage HLB, and 3) a market analysis of how changes in citrus production and costs affect final market prices and quantities. From these data, the net benefits to consumers and producers can be estimated.Objective 4: Deliver information to stakeholders and seek input through outreach activitiesOur outreach efforts will focus on dissemination of project information through meetings with multi-state audiences, statewide biannual meetings and field days, and small informal and more frequent visits to key stakeholders and industry decision-makers identified in our social network analysis. We will develop web pages to disseminate new information on our existing websites. Building on our success in educational materials development for HLB, we will provide printed materials, laminated cards, DVDs, and video presentations at each meeting and on request. We will organize at least two annual meetings with our respective grower organizations or present our results at regularly scheduled annual grower meetings to receive feedback on our progress and advice on how to proceed.

Progress 02/01/17 to 01/24/22

Outputs
Target Audience:The target audience for this project is comprised of pest management researchers, and stakeholders associated with the citrus industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the final year of the project, undergraduate students Pierre Ghobriel, Serena Wang, Lucia Navia, Vanessa Gonzalez, graduate students Junjun Dong and Jahon Amirebrahimi, and postdoctoral researchers Ruchir Mishra and Rahul Banerjee received training, professional development opportunities and mentorship from project investigators Bonning and Jetter. How have the results been disseminated to communities of interest?Results have been disseminated at in person and virtual meetings and webinars to growers, production managers, industry personnel and academics in Florida and elsewhere. Reports and publications resulting from this project are available online. Information is also available at the Science for Citrus Health web site https://ucanr.edu/sites/scienceforcitrushealth/ What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Identify and optimize additional ACP- active Bt toxins 1) Major activities Bioassays were conducted to screen mixtures or individual pesticidal proteins to assess toxicity against ACP. Genome sequences of 18 strains were determined. Mpp51Aa modified with peptide GBP15 were tested in bioassay. Additional ACP gut binding peptides were identified, gut binding confirmed, and gut binding partners identified. 2) Data collected The mortality of adult ACP fed on native or modified pesticidal proteins was recorded. ACP gut binding peptides and peptide binding partners were identified. 3) Summary statistics and discussion of results Of 22 pesticidal proteins for testing, 15 were tested for ACP toxicity. GBP15 modification of Mpp51Aa1 did not increase toxicity. 4) Key outcomes or other accomplishments realized Eight proteins drawn from five different structural groups are toxic to ACP. Two novel gut binding proteins have been characterized for engineering of Cry and Mpp proteins. Objective 2. Deliver Bt toxin to citrus phloem via phloem inhabiting bacteria. 1) Major activities A GFP-labeled version ofthreePantoea sp.strains, and mCherry-labeled versions ofCurtobacterium sp. andCupriavidus sp. strains, isolated from citrus in Florida, were created. Citrus trees were inoculated with the labeled bacteria through different inoculation methods. 2) Data collected Tissue localization of the labeled bacteriain citrus was determined by microscopy. 3) Summary statistics and discussion of results Pantoea sp., Curtobacterium sp. and Cupriavidus sp. strains were able to colonize citrus xylem. 4) Key outcomes or other accomplishments realized Pantoea vaganswas identified as a potential candidate for delivery of Bt toxin to citrus phloem/xylem. Objective 2. Develop and evaluate toxin delivery strategies: Citrus tristeza virus vector delivery of modified Bt toxins 1) Major activities CTV constructs were made for delivery of Cry1Ba or Mpp51Aa1 in citrus. Protein expression and CTV stability were evaluated. ACP population bioassays were conducted on plants infected with modified and control CTV. Psyllids fed on CTV-delivered Mpp51Aa1 were examined by TEM. 2) Data collected The stability of CTV constructs and protein expression levels were assessed. ACP population levels on test and control plants were monitored. TEM images were generated. 3) Summary statistics and discussion of results Following CTV construct optimization, bioassay data showed an inverse correlation between Mpp51Aa1 expression level and psyllid numbers. Damage to the gut epithelium was evident in ACP fed on test plants. 4) Key outcomes or other accomplishments realized CTV constructs for expression of Cry1Ba were unstable due to insert size. The guts of psyllids fed on CTV-delivered Mpp51Aa1 were damaged with associated decreased population numbers relative to control treatments in population bioassays. Objectives 2. Develop and evaluate toxin delivery strategies: Bt toxin delivery via transgenic citrus plants and transgenic trap plants 1) Major activities Transgenic citrus plants (Duncan grapefruit and Valencia sweet orange) and Indian curry were generated for expression of Cry1Ba1 and Mpp51Aa1. Protein expression was assessed by western blot. Selected plants were used in whole plant bioassays with ACP. The guts of ACP fed on test and control plants were examined by TEM. 2) Data collected Pesticidal protein expression levels were determined by western blot. Psyllid survival was monitored in whole plant bioassays. Electron micrographs showing gut physiology of test and control ACP were collected. 3) Summary statistics and discussion of results Significant ACP mortality was observed on Cry1Ba transgenic plants, with associated gut damage apparent by TEM. Mpp51Aa1 had minimal impact on adult ACP survival. 4) Key outcomes or other accomplishments realized Transgenic plant delivery of Cry1Ba resulted in gut damage and associated ACP mortality. Objective 3. Evaluate the economic impact of Bt toxin-based strategies 1)Major activities a.Compared trends in citrus acreage, production and farm prices for the U.S., Florida, Texas and California. b. Compared retail prices and sales for juice (orange, grapefruit) by GMO label (with and without label), and retail prices and sales for fresh oranges by whether organic or conventionally grown.c.Developed programs to automatically update citrus market data (grower production, prices, imports, exports, retail consumption and prices, etc.) from the National Agricultural Statistics Survey, California Agricultural Commissioners, USInternational Trade Commission, and National Health and Nutrition Examination Survey data. d.Developed a farm level simulation model of the benefits of using grafted mCTV citrus to deliver modified Bt-protein to navel orange groves in CA. 2)Data collected Farm statistics for oranges in FL and CA. Farm level production data for CA fresh oranges was used in the simulation model.These data were also used to update the Sample Costs of Production for Oranges in the San Joaquin Valley crop budget. 3)Summary statistics and discussion of results a.Both dollar sales and production of fresh oranges in CA have declined, though the overall production of citrus is about the same as growers removing older orange groves and replanting with tangerines.b. The economic simulation model estimated the number of years the modified Bt-protein would need to prevent the entry and spread of HLB for the benefits to a grower of investing in this technology to be greater than the costs. The simulation model compared the break-even number of years assuming different yields, grower prices, grove life and rates of spread. The results show that for scenarios with a longer orchard life (10, 15, & 20 years), the Bt-protein technology needs to only delay the entry and spread of HLB by one to two years under all yield, price and spread scenarios. It is only when the grove has a life expectancy of 5 years that the benefits are not greater than the costs. For a grove expectancy of less than 5 years the changes in yields following the entry and spread of HLB are too small to make the benefits outweigh the costs. 4)Key outcomes or other accomplishments The farm simulation model is one component of the bio-economic model. It links the changes in yields from the biological model to the market model. The market model is where the benefits and costs are estimated for consumers and growers as an industry. Objective 4. Deliver information to stakeholders and seek input through outreach activities 1) Major activities Citrus industry personnel were surveyed. Educational outreach was implemented through multiple media outlets to address knowledge gaps regarding aspects of genetic engineering and use of pesticidal proteins derived from Bt as pest management tools.New material was added to the Science for Citrus Health (SCH) outreach website and content updated monthly. Multiple extension presentations were given to groups of growers, production managers, or industry personnel in Florida and elsewhere. 2) Data collected Survey responses were collected. 3) Summary statistics and discussion of results Survey responses revealed unbiased support for these new technologies, whether or not responding growers were currently managing their groves for HLB. The SCH website serves as a repository for up-to-date new information on Bt, molecular-based and other emerging technologies for citrus management that is easily accessible for consultation by stakeholders. 4) Key outcomes or other accomplishments realized Progress was made in filling knowledge gaps related to Bt technology. There is overall support for implementation of Bt technology for HLB management by the U.S. citrus industry. Awareness of the SCH website as an outreach tool was raised among the citrus growing community. Key advances for use of bacterial pesticidal proteins as tools for ACP management in citrus were made.

Publications

  • Type: Other Status: Published Year Published: 2021 Citation: Kallsen CE, Douhan GW, Jetter K, Stewart D, Sumner DA. 2021. Sample costs to establish an orchard and produce oranges. Navels, San Joaquin Valley South, Low Volume Irrigation. UC ANR and UC Davis Department of Agricultural and Resource Economics. Davis, CA. https://coststudyfiles.ucdavis.edu/uploads/pub/2021/08/12/2021orangessjvsouth.pdf.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Flavia Campos Vieira and M. Caroline Roper. 2021. Colonization of citrus trees by native bacteria with biocontrol potential. Poster presentation at the International Society for Molecular Plant Microbe Interactions, December 1-2, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Bonning, BC. 2021. Bacterial pesticidal proteins for psyllid control. Presentation in Secretaries of Ag Forum symposium on Issues in Pest Management: Citrus Greening, February 19, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Bonning, BC. 2021. Bacterial pesticidal proteins for management of Asian citrus psyllid. Presentation in Webinar: Emerging Technologies to Manage HLB, February 24, 2021.


Progress 02/01/20 to 01/31/21

Outputs
Target Audience:The target audience for this project is comprised of pest management researchers, and stakeholders associated with the citrus industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this reporting period, undergraduates Jordan Vann, Emily Vu, Pierre Ghobriel, Serena Wang, graduate students Junjun Dong and Jahon Amirebrahimi, and postdoctoral researchers Flavia Campos Vieira, Freddy Ibanez, Ruchir Mishra and Rahul Banerjee received training, professional development opportunities and mentorship from project investigators Bonning, Borneman, Roper, Jetter, Stelinski. How have the results been disseminated to communities of interest?Results were disseminated initially at in person meetings, and subsequently via online meetings (due to the Covid-19 pandemic) to growers, production managers, and industry personnel in Florida and elsewhere. What do you plan to do during the next reporting period to accomplish the goals?During the final 6 months of the project, we plan to complete the following: Objective 1 (Bonning): Individual proteins with hemipteran activity will be tested against ACP. Toxicity to adults and nymphs will be compared using the newly developed, long term nymph bioassay protocol. Gut binding of newly isolated gut binding peptides will be assessed. Peptide modified Mpp51Aa1 will then be tested for efficacy against ACP. Objective 2 (Dawson): Bioassays with CTV-delivered Mpp51Aa1 and gene silencing RNAs will be repeated. Down regulation of targeted psyllid genes will be assessed. (Orbovic): Southern blot analysis will be used to assess gene insertions in selected transgenic Cry1Ba and Mpp51Aa1 plants. Whole plants will be used to assess the impact of Mpp51Aa1 on ACP populations. Objective 3 (Jetter): Update simulated farm analysis for oranges, complete simulated farm analysis for tangerines and lemons in California. Complete market analysis. Objective 4 (Stelinski): Education of growers and the general public will continue based on results from our primary research.

Impacts
(N/A)

Publications

  • Type: Other Status: Published Year Published: 2020 Citation: Sankovitz, M., Alonso, B. Rivera M, Stelinski L., Garc�a-Figuera S., Lemaux P.G., Grafton-Cardwell B. HLB Research featured on website: Science for Citrus Health. https://citrusindustry.net/2020/10/06/hlb-research-featured-on-website/


Progress 02/01/19 to 01/31/20

Outputs
Target Audience:The target audience for this project is comprised of insect pest management researchers, and stakeholders associated with the citrus industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the current reporting period, graduate students Beth Peacock, Turksen Shilts, Christina Korb, and postdoctoral researchers Pavan Kumar, Flávia Campos Freitas Vieira,Seyed-Ali Ravanfar, Kjersti Nes, Jo Ann Lee received training and professional development opportunities and mentorship from project investigators Borneman, Roper, Orbovic, Keesling, Jetter and Bonning. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest via presentations at stakeholder conferences (Citrus Expo, International Research Conference on Huanlongbing) and via the web (e.g. Science for Citrus Health). What do you plan to do during the next reporting period to accomplish the goals?Objective 1 (Bonning): The toxicity of Cry51Aa1 modified with GBP15 against ACP will be determined. Additional individual toxins derived from effective toxin mixtures will be assessed for toxicity against ACP. Western blot analyses to assess toxin expression in transgenic plants and CTV-infected plants generated in objective 2. Objective 2 (Dawson): CTV inoculum from "source" citrus trees will be inoculated into propagated cuttings of transgenic plants expressing the Bt toxin to test for survival of ACP targeted by both RNAi and Bt toxins. (Roper): The localization of two additional Pantoea strains labeled with GFP within citrus tissues will be assessed. Bacterial Illumina rRNA ITS analyses of ACP collected in the field in Florida and California will be analyzed. (Orbovic): Analysis of transgenic plant expression of Cry1Ba1 or Cry51Aa1 by western blot will continue. Whole plants will be used to assess the impact of these toxins on ACP populations. Objective 3 (Keesling): Continue with modeling efforts on 1) the effect of infestation on production levels: Model the progress of citrus greening disease and the specific effect of CLas on the development of symptoms, and 2) the progress of citrus greeing in Florida from its inception. (Jetter): The objective of the final year is to develop an integrated bioeconomic model using dynamic programming. Objective 4: We plan to 1) Continue educating growers and the general public on results that have become available from our primary research. 2) Gather additional data to evaluate attitudes toward acceptance and implementation of genetic engineering tools as attitude changes may occur given project progress. 3) Continue analysis of survey data to identify the most influential citrus growers for targeting with instructional information as project goals near completion. 4) Uncover relationships between knowledge gaps, current practices and attitudes toward implementation of Bt technology in the future.

Impacts
(N/A)

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Fernandez-Luna, M.T., Kumar, P., Hall, D.G., Mitchell, A.T., Blackburn, M.B., Bonning, B.C. 2019. Toxicity of Bacillus thuringiensis-derived pesticidal proteins Cry1Ab and Cry1Ba against Asian citrus psyllid, Diaphorina citri (Hemiptera). Toxins pii: E173. doi: 10.3390/toxins11030173
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: BC Bonning 2019 Charles Chesley Doane Distinguished Lecture, University of Wisconsin, Madison. Orange juice, psyllids and Bt toxins. (Presentation)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Pavan Kumar, M. Teresa Fernandez-Luna, David Hall, Michael Blackburn, and Bryony C. Bonning. 2019. Bt toxins for management of Asian citrus psyllid (Diaphorina citri, Hemiptera). International Research Conference on HLB. Riverside, CA (Presentation)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: BC Bonning 2019. Bt toxins for suppression of the Asian citrus psyllid. Citrus Expo, Fort Myers, FL. (Presentation)


Progress 02/01/18 to 01/31/19

Outputs
Target Audience:During this reporting period, a total of 43 stakeholders completed the survey with the respondent group largely comprised of citrus grove owners and managers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the current reporting period, graduate students Kjersti Nes, Jo-Ann Lee, Ross Ptace and Beth Peacock, and postdoctoral researchers Pavan Kumar, Rahul Banerjee, Alex Blacutt and Flavia Campos received training and professional development opportunities and mentorship from project investigators Jetter, Keesling, Borneman, Roper and Bonning. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Objective 1: (Bonning) Bt toxin mixtures derived from additional Bt strains will be screened for toxicity against ACP. The toxicity of Cry51Aa1 against nymphal ACP will be assessed. The toxicity of GBP15-modified Cry51Aa1 against ACP will be determined. Western blot analysis will be conducted to assess toxin expression in transgenic plants and in CTV-infected plants generated in objective 2. Objective 2: (Dawson) CTV inoculum from "source" citrus trees will be used to inoculate additional trees that will then be used to test for survival of ACP on exposure to plant delivered Cry51Aa1. CTV constructs will be built to test for synergy between Bt toxins and RNAi against psyllids: CTV-delivered gene silencing RNAs will be delivered from transgenic plants expressing a Bt toxin. (Roper) FISH methods will be refined to verify that the citrus P. vagans strain(s) inhabit the phloem or xylem. We will also inoculate citrus with the GFP-labeled P. vagans stain to confirm localization. On confirmation of phloem localization, we will engineer this strain for production of Bt toxin. The efficacy of this bacterium-delivered toxin against ACP will then be tested in vitro and in planta. We will also isolate more of the putative citrus phloem inhabitants that we have identified and verify localization using FISH and bacterial labeling. Xylem inhabitants of citrus will be identified and localization verified by FISH and bacterial labeling. (Orbovic) Analysis of transgenic Indian curry and citrus expressing Cry1Ba will be completed, including optimization of protein detection protocols. The impact of transgenic plants on ACP survival will be tested using detached leaf bioassays. Plants with promising results from detached leaf bioassays will be propagated. These plants will be used in conjunction with CTV-delivered silencing RNAs to test for synergism between the two modes of action. Transgenic citrus expressing Cry51Aa1 will be produced. Objective 3: (Keesling) Biological model: Work is planned to produce a model that will assist in determining the effectiveness of the use of Bt-toxin technology together with other appropriate management practices. 1) Model tree bagging and CUPS programs to protect trees from infestation by ACP. The trees need this protectionat least through the first two or three years of development. Follow groves that have used tree bags and are now ready to remove them. 2) Model effect of infestation of groves by ACP on production levels. Work with Chris Vincent, CREC to use seven years of data from a Florida grower on ACP infestation levels. Combine these data with production levels to measure the direct effect on production. 3) Refine the biological model by using it to understand the history of psyllid infestation and HLB infection in Florida in collaboration with Susan Halbert and Alicia Lawrence (IT) Florida DPI. (Jetter) Economic model: Complete the market model for the U.S. orange, grapefruit, lemon and tangerine markets. Interview farm advisors about current citrus management practices due to ACP/HLB in Florida, California and Texas. Link the biological model being developed to analyze the tree bagging, CUPS, and production data to the economic model. Objective 4: We plan to 1) Initiate educating growers and general public on results that have become available from our primary research, 2) Gather further data to evaluate attitudes toward acceptance and implementation of genetic engineering tools as attitude changes may occur given project progress, 3) Continue analysis of survey data to identify the most influential citrus growers for targeting with instructional information as project goals near completion.

Impacts
What was accomplished under these goals? Objective 1. Identify and optimize additional ACP- active Bt toxins 1) Major activities completed / experiments conducted: Bioassays were conducted to test the efficacy of individual toxins and of toxin mixtures derived from selected Bt strains against ACP. Bioassays were conducted to determine the LC50 against for three individual toxins. The cDNAs for nine versions of Cry51Aa with GBP15 inserted at different sites were constructed. 2) Data collected: The mortality of adult ACP fed on individual toxins and on toxin mixtures was recorded. 3) Summary statistics and discussion of results: Lethal concentration bioassays indicate that each of the three individual toxins are toxic to adult ACP. 4) Key outcomes or other accomplishments realized: Of the three ACP-active toxins identified, Cry51Aa1 holds the most promise for use against ACP due to small size and tractability. Objective 2. Deliver Bt toxin to citrus phloem via phloem inhabiting bacteria. 1) Major activities completed / experiments conducted: Citrus phloem-inhabiting bacteria were identified from multiple states by optimizing a phloem-enriched isolation method and using an Illumina bacterial rRNA ITS analysis to examine phloem-enriched samples. A GFP-labeled version of the most promising bacterium, Pantoea vagans was created. 2) Data collected: Bacterial Illumina rRNA ITS datasets of citrus phloem from Florida and California were generated. Fluorescence localization data were generated. 3) Summary statistics and discussion of results: P. vagans may be a non-pathogenic bacterium that inhabits citrus phloem in Florida and California. As other P. vagans isolates have been genetically manipulated, our citrus strains should be tractable for engineering for expression of Bt toxin in the citrus phloem for suppression of ACP populations. 4) Key outcomes or other accomplishments realized: Pantoea vagans was identified as a potential candidate to deliver Bt toxin to the citrus phloem. Objective 2. Develop and evaluate toxin delivery strategies: Citrus tristeza virus vector delivery of modified Bt toxins 1) Major activities completed / experiments conducted: To test CTV for its ability to deliver Bt toxin into the phloem of a citrus plant, from which it can be ingested by ACP, various constructs of CTV were made. Four constructs built for expression of Cry51Aa1 were agro-inocluated in N. benthamiana. Western blot analysis was used to test for Cry51Aa1 expression. 2) Data collected: Sequence data for the CTV constructs were collected to confirm correct incorporation of toxin and signal sequences. Westrn blot data was used to assess whether Cry51Aa1 was expressed in N. benthamiana. 3) Summary statistics and discussion of results: Virions for the four new CTV constructs expressing Cry51Aa1 were produced. Expression of Cry51Aa1 by CTV in N. benthamiana was confirmed by western blot.. 4) Key outcomes or other accomplishments realized: Four new CTV constructs have been made two of which express Cry51Aa1 well. Objectives 2. Develop and evaluate toxin delivery strategies: Bt toxin delivery via transgenic citrus plants and transgenic trap plants 1) Major activities completed / experiments conducted: Transgenic Indian curry plants for use as a trap plant were produced and transcript levels determined. Transgenic citrus plants expressing Cry1Ba were produced, relative Cry1Ba1 transcript levels, and protein expression determined. 2) Data collected: Optimal conditions for growth media and for rooting of Indian curry shoots were identified. Cry1Ba1 transcript levels in transgenic Indian curry and in citrus plants were determined. Western blot data were collected to assess levels of Cry1Ba1 expression. 3) Summary statistics and discussion of results: Although transcription of Cry1Ba1 by transgenic Indian curry and citrus has been confirmed, Cry1Ba protein has not been detected by western blot thus far. As Cry1Ba1 was found to have a potential negative effect on plants, efforts have now shifted to transgenic expression of Cry51Aa1. 4) Key outcomes or other accomplishments realized: Methodology for production of transgenic Indian curry has been optimized. Transcription of the Cry1Ba1 transgene in both transgenic citrus and Indian curry was confirmed. Objective 3. Evaluate the economic impact of Bt toxin-based strategies 1) Major activities completed / experiments conducted: Farm management research was reviewed and techniques are undergoing refinement. A vertical citrus market model was developed that takes into account the farm sector, imports and exports, and the retail sector, and market segments. 2) Data collected: Data at the farm level (acreage, production, prices) and import and export data (quantities and prices) have been collected. 3) Summary statistics and discussion of results: Significant progress was made toward the baseline bioeconomic model. Florida growers are primarily concerned with 1) how to keep production up so that processors stay in business, 2) how to maintain the health of current groves, and newly planted orchards. California growers are primarily concerned with keeping ACP out of the Central Valley and keeping HLB out of all commercial citrus. 4) Key outcomes or other accomplishments realized: Substantial progress was made on both the economic and biological modeling efforts. Objective 4. Deliver information to stakeholders and seek input through outreach activities The goal of this objective is the delivery of needs-based outreach activities via multiple channels to facilitate the adoption by growers of Bt-based technologies for ACP suppression 1) Major activities completed / experiments conducted: A follow-up survey was conducted at the largest citrus grower meeting in Florida to measure changes in knowledge and perception of genetic engineering in response to outreach efforts. 2) Data collected: Forty-three stakeholders completed the survey with the respondent group largely comprised of citrus grove owners and managers. 3) Summary statistics and discussion of results: The clientele overestimates the level of effort invested in research on Bt technology as compared with the level of education they are receiving in this area. In cases of other general topic areas, growers felt generally more knowledgeable and their knowledge level was more congruent with the perceived level of IFAS investment in those areas as compared with the topic of Bt crops for pest management. 4) Key outcomes or other accomplishments realized: The interaction of HLB and hurricane Irma damage was blamed as a causal factor for significant change in clienteles' occupations and their livelihood. Participants admitted to knowing much less about Bt technology compared with other topics in pest and disease management/crop production.

Publications


    Progress 02/01/17 to 01/31/18

    Outputs
    Target Audience:During this reporting period, atotal of 112 stakeholders completed the survey with the respondent group largely comprised of citrus grove owners and managers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the current reporting period, graduate students Kjersti Nes and Jo-Ann Lee, and postdoctoral researchers Pavan Kumar and Alex Blacutt received training and professional development opportunities and mentorship from project investigators Jetter, Keesling, Bonning, and Roper, respectively. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Bt toxin mixtures derived from additional Bt strains will be screened for toxicity against ACP. The toxicity of Cry51Aa1 against nymphal ACP will be assessed. Optimal conditions for expression of Cry1Ba1 will be determined. Objective 2: CTV virions extracted from infected N. benthamiana will be used to inoculate small citrus trees. Inoculum from these "source" trees will be used to inoculate additional trees that will then be used to test for survival of ACP on exposure to plant delivered Cry1Ba1. For production of transgenic Indian curry, factors required for successful shoot growth will be further defined. Transgenic citrus plants will be assessed for levels of Cry1Ba1 expression. For delivery of Bt toxin to the ACP feeding site via phloem-inhabiting bacteria, a spiroplasma (Spiroplasma citri) that infects citrus trees will be tested for delivery of Bt toxin as proof of concept. Non-pathogenic bacteria isolated from citrus trees will be examined to identify which inhabit the phloem as candidates for delivery of ACP-active toxins. Objective 3: Following completion of budget information as it pertains to Florida citrus, we will gather similar information from Texas for use in the model. We will continue to model movement of CLas within a tree (in collaboration with Silvio Lopes) and assess how this might relate to symptom development. We plan to the test the bioeconomic model for citrus in Florida. Objective 4: We plan to 1) Continue analysis of survey data to identify the most influential citrus growers for targeting with instructional information as project goals near completion. 2) Gather further data on initial attitudes toward acceptance and implementation of genetic engineering tools in order to measure behavioral and attitude changes as the project progresses. 3) Continue providing educational resources as primary research data become available for sharing with the general public. The start of this project was delayed by 4 months following the transfer of the award from Iowa State University to University of Florida in conjunction with the relocation of the Project Director. All objectives are on track to meet Year 1 milestones by June 1, 2018.

    Impacts
    What was accomplished under these goals? Objective 1. Identify and optimize additional ACP- active Bt toxins 1) Major activities completed / experiments conducted: Bioassays were conducted to test the efficacy of individual toxins and of toxin mixtures derived from selected strains of Bacillus thuringiensis against the Asian citrus psyllid (ACP), Diaphorina citri. The sequence encoding the toxin Cry1Ba1 (previously identified by our group as having basal activity against ACP), was modified by addition of coding sequence for a short peptide (GBP15) that binds to the gut epithelium of ACP. This gut binding peptide is expected to provide an additional anchor for toxin binding to the ACP gut, thereby increasing toxicity against ACP. 2) Data collected: The mortality of adult ACP fed on individual toxins and on toxin mixtures, along with the mortality of ACP in control treatments was recorded. The sequences of modified Cry1Ba1 were determined to examine correct incorporation of GBP15 sequence. 3) Summary statistics and discussion of results: Bioassays indicate that Cry51Aa1 is toxic to adult ACP. Several toxin mixtures from specific Bt strains also showed toxicity against adult ACP in membrane feeding assays. Seven constructs with GBP15 inserted into different sites within Cry1Ba were made. 4) Key outcomes or other accomplishments realized: The toxin Cry51Aa1 holds promise for use against ACP. The identification of specific strain-derived toxin mixtures with toxicity to ACP provides new leads for identification of additional ACP-active toxins. Objective 2. Develop and evaluate toxin delivery strategies: Citrus tristeza virus vector delivery of modified Bt toxins 1) Major activities completed / experiments conducted: To test CTV for its ability to deliver Bt toxin into the phloem of a citrus plant, from which it can be ingested by ACP, various constructs of CTV were made. CTV vectors were engineered for expression of Cry1Ba1 (optimized for expression by a citrus plant) with or without a secretory signal to test whether such a signal is needed for movement of the toxin out of the phloem cell into the sieve element, which is the site of ACP feeding. The toxin sequence was inserted into the CTV vector in two different positions, which are expected to result in differences in stability of the vector and Cry1Ba expression level. CTV constructs were delivered to Nicotiana benthaminia for production of CTV virions, which will then be used to infect citrus plants. 2) Data collected: CTV constructs were sequenced to confirm correct incorporation of toxin and signal sequences. 3) Summary statistics and discussion of results: Virions for three of the four CTV constructs expressing Cry1Ba1 have been produced. 4) Key outcomes or other accomplishments realized: Four CTV constructs have been made. These constructs will be used to assess 1) the optimal site for insertion of Bt toxin sequence into the CTV vector, 2) whether a secretory signal is necessary for movement of the toxin to the site of ACP feeing within a citrus plant, 3) whether sufficient toxin is available to ACP on feeding to cause mortality in bioassays. Objectives 2. Develop and evaluate toxin delivery strategies: Bt toxin delivery via transgenic citrus plants and transgenic trap plants 1) Major activities completed / experiments conducted: To be able to use Indian curry expressing a Bt toxin as a trap plant for ACP control, conditions were optimized to allow for the genetic engineering of this plant species. Experiments were conducted to identify the optimum medium that promotes growth of curry shoots. Experiments were conducted for production of transgenic citrus expressing Cry1Ba, using Duncan grapefruit, Carrizo citrange and Valencia sweet orange. 2) Data collected: Two recipes for growth media that promoted the growth of Indian curry shoots were identified. 3) Summary statistics and discussion of results: A total of 23 transgenic plants of Duncan grapefruit, Valencia sweet orange, and Carrizo citrange were produced and moved to small pots. The transformation rate of citrus plants was unexpectedly low, suggesting that Cry1Ba1 may have a negative effect on the plants. 4) Key outcomes or other accomplishments realized: Key steps toward accomplishment of objective goals have been achieved, specifically identification of media that support shoot growth of Indian curry, and transformation of three types of citrus plant. Objective 3. Evaluate the economic impact of Bt toxin-based strategies 1) Major activities completed / experiments conducted: A bioeconomic model will be developed that will provide the framework in which the costs and benefits of different ACP and HLB management scenarios can be estimated. Project personnel met with growers and other stakeholders in Florida in June 2017 so that practices, costs and yields in Florida can be introduced into the model. Meetings or discussions were also held with UF, UC and USDA scientists on how the presence of HLB affects growers, and factors that would help determine differences in disease progression between Florida and Brazil. 2) Data collected: Information was collected on practices, costs and yields from citrus growers in Florida. 3) Summary statistics and discussion of results: Significant progress was made in developing the baseline bioeconomic model. Florida growers are primarily concerned with 1) How to keep production up enough so that processors stay in business and there is still a foundation upon which to rebuild the Florida citrus industry when science or nature develops an HLB resistant plant, 2) How to protect or maintain the health of current groves, and newly planted orchards. 4) Key outcomes or other accomplishments realized: Substantial progress was made on both the economic and biological modeling efforts. Much information was learned about the choices Florida growers have that will be used to develop the baseline simulations. These choices include options for managing both newly planted and existing groves. Progress was made in the biological modeling by incorporating additional variables such as temperature into the model's parameters. Objective 4. Deliver information to stakeholders and seek input through outreach activities The goal of this objective is the delivery of needs-based outreach activities via multiple channels to facilitate the adoption by growers of Bt-based technologies for ACP suppression 1) Major activities completed / experiments conducted: A survey was conducted to assess respondent knowledge of- and attitudes toward- genetic engineering. A Research Snapshot was written about the current project and posted on the Science for Citrus Health web site http://ucanr.edu/sites/scienceforcitrushealth/ This web site describes the ACP/HLB situation and provides resources for growers to better understand the techniques that are being developed to battle the disease. 2) Data collected: A total of 112 stakeholders completed the survey with the respondent group largely comprised of citrus grove owners and managers. 3) Summary statistics and discussion of results: About 50% of current citrus industry professionals participate in extension events and use a broad array of instructional methods to gather information. "Insecticide control" was considered the method most likely to impact HLB management, although there was willingness to accept a diversity of possible technologies for HLB management. Public perception was a primary concern for use of Bt technologies in citrus production. 4) Key outcomes or other accomplishments realized: The first survey of grower knowledge regarding genetic engineering technologies and Bt technology is complete. Initial gathering of information regarding grower influence on industry decision-making is nearing completion. Outreach has been established through the Science for Citrus Health website highlighting the initiation of this project and what it may bring to fruition.

    Publications

    • Type: Websites Status: Published Year Published: 2017 Citation: E. Grafton-Cardwell, P.G. Lemaux, L. Stelinski. 2017. Research Snapshot: A new, Bt toxin-based strategy for suppression of the Asian citrus psyllid vector of HLB. Science for Citrus Health web site. http://ucanr.edu/sites/scienceforcitrushealth/