BT TOXIN-BASED STRATEGIES FOR MANAGEMENT OF DIAPHORINA CITRI AND CITRUS GREENING

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 of limiting the spread of ACP is 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 systems to deliver the Bt toxins to the sap 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.

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. Resulting 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.