Progress 02/01/19 to 01/31/24
Outputs
Target Audience:Citrus growers, citrus industry, citrus nerseries, citrus extension, field advisorsand citrus researchers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided excellent training opportunities to postdocs, graduate students, and undergraduate students. Dr. Chien-Yu Huang, who is the postdoc and project manager in the Jin lab has got a tenure-track faculty position in Luisanna State University in 2023. She started her own lab 9 months ago. The graduate students associated with this project havelearnedmolecular biology techniques and citrus growth and sampling.Ms. Araujo in the Godfrey group was provided training on the propagation of transgenic citrus using cuttings. The cuttings from transgenic citrus can be difficult to root and made to thrive. Researchers in the Folimonova lab have mastered the skills in engineering virus-based expression vectors and in techniques used for virus inoculation and detection. In Mauck's lab, The project served as a basis for undergraduate research to understand 1. plant resistance effects on psyllid feeding behavior and survival, 2. Ca. Liberibacter-psyllid interactions, and 3. SAMP effects on ACP performance and behavior. One student performed work on an honors thesis project addressing the first research area. A second student performed research for area 1 for course credit. A third student performed research on area 2 for course credit, and a fourth student performed research on area 3 for course credit. Two of these students successfully obtained fellowship funding for their projects. One is now applying to graduate school in plant pathology, and another is working for the USDA-ARS in the field of entomology. These successes are directly linked to the training these students received in connection with this project. How have the results been disseminated to communities of interest?We have disseminated the findings to communities and public through multiple ways: The PI Hailing Jin has been interviewed on the findings of the novel antimicrobial peptides by a dozen News outlets, including National Public Radio, LA Times, Progressive Crop Consultant Magazine, Western U.S. newspaper, The Packer, the produce industry newspaper, the Australian Broadcasting Corporation's national rural affairs TV program Landline, etc. She has also given several talks in multiple international conferences, many universities and institutions,to the Citrus Research Development Foundation Inc (CRDF) in Florida and gave a webinar to the citrus industry on Antimicrobial Peptides organized by CRDF. The project manager Chien-Yu Huang in Jin group has joined a virtual conference to present our work to researchers at 12thJapan-US Seminar in Plant Pathology. Huang has joined virtual conferences to present our work at Plant Biology 2021 Worldwide Summit, North San Diego California Rare Fruit Growers (CRFG) event and San Diego Botanic Garden Monthly Volunteer Meeting. We also published two peer reviewed manuscripts on this finding in highly respected journals: Huang CY, Niu D, Kund G, Jones M, Albrecht U, Nguyen L, Bui C, Ramadugu C, Bowman K, Trumble J, and Jin H. 2020. Identification of citrus defense regulators against citrus Huanglongbing disease and establishment of an innovative rapid functional screening system. Plant Biotechnol J. https://doi.org/10.1111/pbi.13502 Huang CY, Araujo K, Sánchez JN, Kund G, Trumble J, Roper C, Godfrey KE, and Jin H. 2021. A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing. PNAS. (Released on Feb 1st 2021, featured on the issue cover ) Dissemination of results has primarily been through outreach efforts of the extension personnel (Tracy Kahn and Georgios Vidalakis). Fielded questions from public and growers on the article "UC Riverside discovers first effective treatment for citrus-destroying disease New license agreement commercializes innovative, safe technology". We communicated with people who work with citrus. James J. Shanley, President Shanley Farms: Commercial Grower of Australian Finger limes. They are extracting the juice vesicles and oil as well as selling the fruit. Susan Von Zabern, Manager for Non-profit management board Citrus State Historic Park in Riverside. She wanted to start educating public about this "solution" to HLB Tom Spellman, board member Citrus State Historic Park and Nurseryman at Dave Wilson Nursery Vincent Moses, Ph.D. Citrus historian who gave presentation to Osher class at UCR and wanted to tell public about this discovery as part of his presentation. Helped answer questions on ACP for "By the Numbers Citrus Greening Disease" for Jules Berstein Contributed to UC ANR article "UC: researchers at work finding solutions for huanglongbing disease to save California citrus" September 2020 UC ANR https://ucanr.edu/sites/QBELab/files/336704.pdf Reviewed article soon to come out entitled "Jewel in the Crown - The UCR Givaudan Citrus Variety Collection sits at the forefront of global agricultural innovation" by Holly Ober which discusses this project. The co-PI Mauck also assisted UCR Science News personnel several times throughout 2020 in developing entomologically correct educational materials on HLB research at UCR, including pieces covering the work in this project. The co-PI Dewdney participated in meeting with a group of Florida citrus industry representatives to explain the goals of the project and what progress had been accomplished to date. 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. The SAMP-treatedWashington Navel, Lisbon lemon and Tango mandarin were all growing better and hadlower CLas titer than mock-treated trees. 'Washington Navel' orange trees on Carrizo rootstock were exposed to ACP (contain the San Gabriel strain of CLas) for 3 weeks, incubated for 1 week, and then subjected to SAMP injection (4 weeks after ACP exposure). Due to the pandemic, the plant sampling is delayed the subsequent treatment is changed to foliar spray once per 2 months. After 18 months of infection, the result indicates the SAMP-treated trees have a significantly lower infection rate (23%) compared to mock (buffer only) treated trees (78.5%). 'Lisbon' lemon trees on Carrizo rootstock were treated similarly. The result indicates the SAMP-treated trees have a lower infection rate (50%) compared to mock (buffer only) treated trees (100%). This is the first test of micro-needle injection. Some of the trees died after 2-3 weeks of treatment due to the injured of this method. The method is improved by more practice. The SAMP-treated trees were growing better than mock-treated trees and with lower CLas titer. 'Tango' mandarins on Carrizo rootstock were treated in the same way and showed that the SAMP-treated trees have a lower infection rate (14.2%) compared to mock (buffer only) treated trees (75%). The SAMP-treated trees were growing better than mock-treated trees and with lower CLas titer. Objective 2:Pre-exposure foliar sprays of SAMP and SAMPH2 induces priming and shows promising protection against infection. 1. Total of 24'Washington Navel' were sprayed with SAMP, SAMPH2 or buffter respectively, then were exposed to hot ACP.Nymphs and F1 adults developed in 2 SAMP, 5 SAMPH2, and 5 buffer treated trees. Eggs were laid, but did not develop into nymphs on 1 SAMP, 1 SAMPH2, and 0 buffer treated trees. After 10 months of infection, the result indicates the SAMP-treated trees have a significantly lower infection rate (23%) compared to mock (buffer only) treated trees (100%). The SAMH2-treated set had a lower infection rate (57%) compared to mock-treated trees but not as low as the SAMP-treated set. The trees were kept monitoring trees until 18 months post-infection. 2. Total of 30 Tango mandarin trees weretreated similarly.Nymphs developed on 6 SAMP, 5 SAMPH2, and 2 buffer-treated trees. Eggs were laid but did not develop on 1 SAMP, 1 SAMPH2, and 2 buffer-treated trees. After 10 months of infection, the result indicates the SAMP and SAMPH2-treated trees have a lower infection rate (50 and 62.5%, respectively) compared to mock (buffer only) treated trees (90%). The trees were kept monitoring trees until 18 months post-infection. 3. Total of 28'Lisbon' lemon were treated. Eggs and nymphs were found on 4 SAMP, 4 SAMPH2 and 4 buffer-treated trees. When transferred from the lab to the greenhouse, 1 SAMP, 2 SAMPH2, and 1 buffer-treated tree died. After 10 months of infection, the result indicates the SAMP and SAMPH2-treated trees have a lower infection rate (25 and 71%, respectively) compared to mock (buffer only) treated trees (83%). The trees were kept monitoring trees until 18 months post-infection. Objective 3.Citrus tristeza virus (CTV)-based vector mediated delivery (Folimonova Lab) Folimonova Lab generated CTV vector containing a nucleotide sequence encoding the peptide SAMP, which could express SAMP in citrus by infection. The CTV-SAMP1 virus inoculum was amplified by introducing the respective construct into Nicotiana benthamiana plants. CTV-SAMP1 inoculum was used for bark-flap inoculation of citrus plants (Citrus marcophylla) to test the viability of this virus vector expressing SAMP1 and to initiate virus infection. In total, 3 separate attempts, including 10 plants in each, 30 plants total, were used in a series of inoculation experiments. Around three months post inoculation, infection of 20 C. macrophylla plants was confirmed using ELISA with a CTV-specific antibody. Presence of a gene encoding SAMP1 in the CTV genome was confirmed via RT-PCR. These plants expressing SAMP1 from a CTV vector were maintained in the insect-proof greenhouse to be used as the CTV-SAMP1 budwood source for further propagation of additional citrus plants that would express SAMP1. We then verified the expression of the SAMP1 mRNA in plants infected with CTV-SAMP1 vector. The CTV-SAMP1-infected citrus plants were maintained in the insect-proof greenhouse for additional 3 months to ensure that the plants will become uniformly infected with the CTV-SAMP1 vector. Using the budwood from these initially produced plants, we propagated additional citrus plants of Citrus macrophylla and sweet orange Madam Vinous varieties. Some of these plants we keep in our greenhouse as the source "mother" plants. Other CTV-SAMP1-containing plants were exposed to hot CLas-infected psyllids (psyllids developed on the HLB-infected plants were bagged onto young flushs of CTV-SAMP1-infected plants) for subsequent inoculation with CLas. The following CTV-SAMP1-infected plants were exposed to the hot-psyllid greenhouse:10 sweet orange Madam Vinous plants infected with CTV-SAMP1, 13 sweet orange Madam Vinous plants infected with CTV-GFP as control plants, 6 Citrus macrophylla plants infected with CTV-SAMP1, 6 Citrus macrophylla plants infected with CTV-GFP as controls. These plants are now being monitored for symptoms and infection by CLas. In the following months - August 2024, September 2024, December 2024, which correspond to the 2-, 3-, and 6-months post psyllid exposure, respectively. Objective. 4- Evaluate SAMPs as tools to disrupt HLB transmission by ACP vectors.? We quantified effects of AP applications on induction and release of chemical cues used by psyllids during host selection. Effects of APs were compared to those of a known elicitor of psyllid defenses (Acibenzolar-S-methyl [ASM]), mock-treated controls (carrier only) and controls receiving no treatment. Experiments were performed with Lisbon Lemon and Valencia orange. We then enclosed plants in large glass headspace sampling containers and pumped filtered air in. Headspace was resampled through an adsorbent trap to collect volatiles. Samples were taken over the first 24 hours, 24-48 hours, and 48-72 hours, to track induction of volatiles and changes in release over time. Volatiles were quantified and identified using GC-MS. Determine whether APs can function directly to inhibit CLas colonization of ACP vectors. We found that SAMPs had no significant effect on the reproduction of female ACP confined to flush on Valencia oranges. There was a trend toward lower titers in ACP feeding on SAMP-treated C. macrophylla in the first replication.Although SAMP treatments did not significantly reduce CLas titers in adult ACP, there were some negative effects of SAMP treatment on ACP survival. In all cases, fewer ACP survived to the recovery date (12 days) on plants treated with SAMP. SAMP treatment did not have a strong effect on the development of the offspring population for C. macrophylla. For C. medica, SAMP induced a slight acceleration in development, with fewer nymphs in the third instar stage and more in the fourth instar stage relative to the DMSO treatment. However, there also were fewer nymphs total on SAMP-treated C. medica plants (100 across all SAMP plants compared to 221 across all DMSO plants). This could have influenced nymph competition, with less competition for resources on the more sparsely colonized treatment leading to faster development. Recently, We treated Lisbon Lemon plants in flush with APs.SAMP weaklysuppressed reproduction of female ACP, fewer eggs and nymphs show in the new flush with SAMP treatment.Overall, these results suggest that SAMPs may not directly alter CLas titers in ACP. SAMP effects on ACP likely are indirect (plant defense mediated) and mostly affect the adult stage by reducing survival, especially of individuals harboring CLas infection.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Chien Yu Huang, DongDong Niu, Gregory Kund, Mike Jones, Ute Albrecht, Lincoln Nguyen, Christine Bui, Chandrika Ramadugu, Kim D. Bowman, John Trumble, and Hailing Jin*: Identification of citrus immune regulators involved in defense against Huanglongbing using a new functional screening system. Plant Biotechnology Journal, 2020, doi: 10.1111/pbi.13502.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Chien-Yu Huang, Karla Araujo, Jonatan Ni�o S�nchez, Gregory Kund, John Trumble, Caroline Roper, Kristine Elvin Godfrey, and Hailing Jin*: A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing. 2021, DOI: 10.1073/pnas.2019628118
Journal Articles 2021
|
Progress 02/01/22 to 01/31/23
Outputs
Target Audience:Students, growers, industry personnel, nurserymen, end-users of commodity. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The graduate student in Jin lab is learning molecular biology techniques of sample testing and introduced the project in Dr. Khan's class (NASC093) which focused on citrus research and the industry. Ms. Araujo in the Godfrey group was provided training on the propagation of transgenic citrus using cuttings. The cuttings from transgenic citrus can be difficult to root and made to thrive. Researchers in the Folimonova lab have mastered the skills in engineering virus-based expression vectors and in techniques used for virus inoculation and detection. In Mauck's lab, The project served as a basis for undergraduate research to understand 1. plant resistance effects on psyllid feeding behavior and survival, 2. Ca. Liberibacter-psyllid interactions, and 3. SAMP effects on ACP performance and behavior. One student performed work on an honors thesis project addressing the first research area. A second student performed research for area 1 for course credit. A third student performed research on area 2 for course credit, and a fourth student performed research on area 3 for course credit. Two of these students successfully obtained fellowship funding for their projects. One is now applying to graduate school in plant pathology, and another is working for the USDA-ARS in the field of entomology. These successes are directly linked to the training these students received in connection with this project. How have the results been disseminated to communities of interest?Dissemination of results has primarily been through outreach efforts of the extension personnel (Dr. Vidalakis and Dr. Kahn). Dr. Kahn provided fruit from the UCR Givaudan Citrus Variety Collection (GCVC) for Chien-Yu Huang's research. The PI Hailing Jin has given numerous talks to the universities, international conferences, general public and growers. The project manager Chien-Yu Huang in Jin group has present our work at California Rare Fruit Growers (CRFG) event in December 2022. The co-PI Dewdney met with group of Florida citrus industry representatives to explain the goals of my part of project and our findings to date and wrote a short outline of the project that was presented as part of a booklet at the Florida Citrus Expo. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: The greenhouse experiments are almost complete for this objective. Dewdney group is working on the field test in FL. Any new trees that are suspected to have HLB will have leaf and root samples tested with qPCR using the Wang et al. (2006) protocol. Objective 2A: The greenhouse experiments are almost complete for this objective. We will continue experiments with exposures and treatments and collect the data for the result analysis. Dewdney group is working on the field test in FL. Folimonova group will proceed further analysis of SAMP1 expression in the citrus trees and its effect on the challenge with HLB. Jin group is characterizing the SAMP cisgenic citrus. Objective 2B: Experiments are complete for most objectives. We have some remaining samples to process from the experiments in the BSL3 and some remaining data analysis to complete after sample processing. Data analysis of volatile data from SAMP induction experiments will be completed. We will write a manuscript on the transgenic AP-expressing potato work, publish an article in the Citrograph magazine to update stakeholders, and produce a manuscript covering AP effects on psyllid-host interactions. Objective 3: Dr. Kahn will continue to provide plant material from the GCVC and address questions about the project from those who contact her. Dr. Kahn a co-organizer for the UCR Citrus Day for Industry which will be held March 10, 2022. There will be 5 to 7 talks in the field related to the Huanglongbing citrus disease and Hailing Jin will be one of the presenters. Objective 4: Our industry partner, Invaio Science is consulting the EPA registration and preparing the data of SAMP safety testing. Dr. Babcock is evaluating the application of our produce in FL and CA.
Impacts
What was accomplished under these goals?
Objective 1. Identify the most effective SAMPsolutions and delivery methods for curing CLas-positive trees of different genetic backgrounds. (1) For curing the infected tree, we mainlyfocus on the pneumatic trunk injection method with very sick trees (6-10 months after infection with obvious symptoms) in the first year. In the second year, we have tested the micro-needle trunk injection method in the early infected tree (4 weeks after ACP exposure) and foliar spray. In the third year, we have completed the most of treatments at CRF, UCD: (all the citrus scion is grafted onto Carrizo rootstock) (2) Dewdney group started field trials to test if SAMP can remediate an HLB-affected young citrus grove. This experiment is ongoing in the Ridge region of Florida on the deep sandy soils. Pre-treatment data was collected in 2020 and reported on in the previous year's report. The trial was laid out as a randomized complete block design with five blocks and five replicates per block. The treatments in this trial were an untreated control and bimonthly foliar application. Some trees were affected by the hurricane. The test is still under monitoring and expects to evaluate at the end of 2023. Objective 2A: Examine the priming effect of SAMPs on healthy citrus seedlings. 2A.1. Pre-exposure foliar sprays of SAMP and SAMPH2 treatment for protectant development - The following plants were sprayed with either a SAMP, SAMPH2 spray, or a buffer containing a spray before being exposed to CLas-positive ACP. The plants were sprayed 5 days before exposure to ACP. Foliar spray application of SAMP, SAMPH2 and buffer were applied to trees at two-month intervals for a total of three treatments after insect inoculation. A bark and midrib tissue sample for each tree was collected at two-month intervals. 1.'Washington Navel' on Carrizo rootstock- treated and exposed in June 2021.After 10 months of infection, the result indicates the SAMP-treated trees have a significantly lower infection rate (23%) compared to mock (buffer only) treated trees (100%). 2.Tango mandarin on Carrizo rootstock- treated and exposed April, 2021. The result indicates the SAMP and SAMPH2-treated trees have a lower infection rate (50 and 62.5%, respectively) compared to mock (buffer only) treated trees (90%). 3.'Lisbon' lemon on Carrizo rootstock- treated and exposed in September 2021. The result indicates the SAMP and SAMPH2-treated trees have a lower infection rate (25 and 71%, respectively) compared to mock (buffer only) treated trees (83%). 2A.2. Dewdney group has started a field trial to determine if newly planted trees could be protected from HLB by SAMP for at least two years was planted on October 15, 2020 in Lake Alfred, FL. The trees were planted next to a grove block that has a substantial psyllid population. The block has no insect vector control. The test is ongoing. We have observed the trees with SAMP pre-treatment have a lower declining rate (16%) compared to no treatment trees (21%). 2A.3 Citrus tristeza virus (CTV)-based vector mediated delivery (Folimonova Lab) Folimonova group has propagated CTV-SAMP1 in citrus plants.10 sweet orange Madam Vinous plants infected with CTV-SAMP1, 13sweet orange Madam Vinous plants infected with CTV-GFP as control plants,6 Citrus macrophylla plants infected with CTV-SAMP1, 6 Citrus macrophylla plants infected with CTV-GFP as controls. These plants are now being monitored for symptoms and infection by CLas. 2A.4 Test the HLB tolerance/resistance of SAMP expression citrus (Jin and Godfrey) Propagation of transgenic Carrizo received in 2019 and 2022 continued - Cuttings from three transgenic lines constructed by David Tricoli at the Ralph M. Parsons Foundation Plant Transformation Facility at UC Davis and 2 additional lines received in 2022 from Dr. Jin were successfully propagated. We continue to make cuttings from the source plants and have a few scion types successfully grafted onto both transgenic and wild-type Carrizo rootstock. For scions grafted on wild-type Carrizo, we have 20 Tango mandarins, 1 Lisbon lemon, and 16 Washington navels. Objective. 2- Evaluate SAMPs as tools to disrupt HLB transmission by ACP vectors. 2B.1 Quantify effects of APs on ACP behaviors mediating CLas acquisition and inoculation. Effects of APs on psyllids are being evaluated in two systems - citrus-ACP and tomato/potato with potato psyllids. This allows us to work with two pathosystems in parallel - the citrus-CLas-ACP system (which has certain restrictions and must be evaluated under BSL3 level containment) and the CA-native Solanaceae-CLso-potato psyllid system (which can be easily and quickly manipulated outside of containment). During the 2022 project period, we continued evaluating psyllid responses to APs expressed in transgenic solanaceous crops (mainly potato) as well as effects of transgenic AP expression on susceptibility to the Ca. Liberibacter solanacearum (CLso) bacterium. 2B.2 Determine the molecular basis of AP effects on ACP behavior. We quantified effects of AP applications on induction and release of chemical cues used by psyllids during host selection. Effects of APs were compared to those of a known elicitor of psyllid defenses (Acibenzolar-S-methyl [ASM]), mock treated controls (carrier only) and controls receiving no treatment. Experiments were performed with Lisbon Lemon and Valencia orange. We then enclosed plants in large glass headspace sampling containers and pumped filtered air in. Headspace was resampled through an adsorbent trap to collect volatiles. Samples were taken over the first 24 hours, 24-48 hours, and 48-72 hours, to track induction of volatiles and changes in release over time. Volatiles were quantified and identified using GC-MS. 2B.3 Determine whether APs can function directly to inhibit CLas colonization of ACP vectors. In 2022, we continued running repetitions of experiments run the first time in 2021 in the BSL3 facility to obtain additional biological repeats. We quantified the effects of AP delivery to CLas-infected psyllids on psyllid CLas titer and psyllid performance metrics as well as effects of treatment of infected plants with APs on CLas acquisition by psyllid nymphs. We treated Citrus medica and Citrus macrophylla plants in flush with APs at a concentration of 10 micromolar, or with a solution of carrier only (mock). Age standardized CLas+ ACP (San Gabriel strain) were allowed to feed on the plants for 12 days and were then removed for testing to measure CLas titer in insects fed on AP-treated plants relative to mock-treated plants. Additionally, nymphs were counted on all plants and the distribution of instars quantified. We also performed experiments with CLas infected plants and uninfected ACP. Infected plants were treated with APs and then uninfected ACP were allowed to oviposit on these plants five days later. Nymphs from this oviposition bout were recollected after developing into adults to measure CLas titer. Overall, these results suggest that SAMPs do not directly alter CLas titers in ACP. SAMP effects on ACP likely are indirect (plant defense mediated) and mostly affect the adult stage by reducing survival, especially of individuals harboring CLas infection. Objective 3: Dr. Kahn and Dr. Vidalakis interacted with Citrus growers and nursery owners for the California Citrus Industry, public and UCR undergraduate students. More detail of outreach activities related to this project is list in the question: How have the results been disseminated to communities of interest? Objective 4: Dr. Backcock are performing an economic analysis of the difference of prospect on HLB management between FL and CA. Our industry partner Invaio has set up 29 injection trials on four different varieties, including fruit-bearing and new transplant trees in FL and Tx preparing the data and consulting for EPA registration.
Publications
|
Progress 02/01/21 to 01/31/22
Outputs
Target Audience:International Plant Pathology Scientific Community International Citrus Research Community Citrus Growers Citrus Research Board in CA and FL Industry parters who are interested in our invention Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The graduate student in Jin lab is learning molecular biology techniques of sample testing and introduced the project in Dr. Khan's class (NASC093) which focused on citrus research and the industry. Ms. Araujo in the Godfrey group was provided training on the propagation of transgenic citrus using cuttings. The cuttings from transgenic citrus can be difficult to root and made to thrive.Researchers in the Folimonova lab have mastered the skills in engineering virus-based expression vectors and in techniques used for virus inoculation and detection. The project served as a basis for undergraduate research in the Mauck group to understand 1. Plant resistance effects on ACP feeding behavior and survival, and 2. ACP use of alternative (non-Rutaceous) hosts. One student performed work on an honors thesis project addressing these two research areas and another performed research for course credit. One student wrote and successfully obtained a small research grant based on her project. How have the results been disseminated to communities of interest?Dissemination of the research findings were published in high impact journals, which have been broadly read by not only the scientific communities, agricultural industry partners, but also the citrus growers. The PI Dr. Jin has been interviewed by many domestic and international news outlets, such as LA times, NPR news, Australian Broadcasting Corporation's national rural affairs TV program Landline, to introduce this finding. She was filmed as part of an episode of the Australian television show Landline focused on HLB and how the peptide from Australian finger limes was a potential tool to combat this devastating disease (Citrus Greening: Protecting Australian farms from a devastating citrus disease - Landline (abc.net.au)). The PI Hailing Jin has given more than ten talks on this antimicrobial peptide for controlling citrus HLB at international conferences, universities, and to citrus industry (CRB, CRDF, and companies) and citrus growers. The project manager Chien-Yu Huang in Jin group has joined virtual conferences to present our work at Plant Biology 2021 Worldwide Summit, North San Diego California Rare Fruit Growers (CRFG) event and San Diego Botanic Garden Monthly Volunteer Meeting. The outreach activities have primarily been carried out by the extension personnel (Dr. Vidalakis and Dr. Kahn). Dr. Kahn provided fruit from the UCR Givaudan Citrus Variety Collection (GCVC) for Chien-Yu Huang's research and fruit for the image selected as the cover of PNAS Volume 118 Issue 6 and fruit of Bergera koenigii to Kerry Mauck to enable her to continue to rear Asian Citrus Psyllids on seedlings of Bergera koenigii. Dr. Kahn Introduced Chien-Yu Huang (Jin group) to Carolyn Buchman, Program Chair North San Diego Chapter CRFG for April 9, 2021, presentation to the California Rare Fruit Growers. Dr, Kahn provided updates to Hailing Jin on articles in March 10 Ag Alert and Riverside Historical Society Journal that describe this project. She also spoke individually to growers at the California Citrus Conference Oct. 19, 2021, in Visalia CA organized by the Citrus Research Board and to nursery owners at the California Citrus Nursery Society annual meeting in Riverside Nov. The co-PI Dewdney met with group of Florida citrus industry representatives to explain the goals of my part of project and our findings to date and wrote a short outline of the project that was presented as part of a booklet at the Florida Citrus Expo. She was also filmed as part of an episode of the Australian television show Landline focused on HLB (Citrus Greening: Protecting Australian farms from a devastating citrus disease - Landline (abc.net.au)). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We are currently increasing tree production for micro needle delivery of the antimicrobial peptide. Lisbon lemons, Washington navels, and Tango mandarins are being produced by grafting onto Carrizo rootstock and by rooting scion directly onto coco coir or peat moss pellets. A re-graft attempt on the remaining transgenic Carrizo to determine if transgenes will cross onto healthy grafted scion. To determine if the antimicrobial peptide has any effect on CLas titer in adult ACP that have fed on a treated plant, or on ACP life history, Volkameriana lemons grown from seed will be divided into three treatments (control (no treatment), buffer treated, and SAMP treated). Seedlings will be exposed to either CLas positive (San Gabriel, Type 2) ACP or CLas negative ACP for 21 days using sleeve cages. The adults originally placed on the plants (called parental adults) will be collected at the end of the exposure period and the titer of CLas in each insect will be determined. If the parental insects oviposited on the plant, the F1 progeny will be allowed to develop. Samples will be taken of late instar nymphs and F1 adults and the titer of CLas in each insect will be determined. Remaining F1 adults will be caged on healthy plants and allowed to feed for 21 days to estimate transmission of CLas. The flush tip from which the F1 adults were reared will also be assayed to determine the titer of CLas in the tip. Collecting data on the number of eggs laid, number of nymphs and F1 adults produced, and the titers of CLas in the various insect and plant samples should provide some insight as to any impact of the peptide on ACP or its life history. Dewdney group is working on the field test in FL. Any new trees that are suspected to have HLB will have leaf and root samples tested with qPCR using the Wang et al. (2006) protocol. Objective 2 A: We will continue experiments with exposures and treatments and collect the data for the result analysis. Dewdney group is working on the field test in FL. Folimonova group will proceed further analysis of SAMP1 expression in the citrus trees and its effect on the challenge with HLB. Jin group will generate SAMP cisgenic transformed citrus. B. Determine the molecular basis of SAMP effects on ACP behavior. For transgenic SAMP effects on psyllids, reduced performance (oviposition/nymphs) seem to be evident for psyllids carrying CLso only. We will determine if transgenic SAMP expression reduces CLso titers in psyllids adults and nymphs. On the plant side, we will evaluate expression of defense genes in SAMP-expressing lines fed upon by CLso+ and CLso- psyllids.If behavioral effects are detected following treatment of citrus varieties with APs, we will perform similar evaluations of defense gene expression. Objective 3: Dr. Kahn will continue to provide plant material from the GCVC and address questions about the project from those who contact her. Dr. Kahn a co-organizer for the UCR Citrus Day for Industry which will be held March 10, 2022. There will be 5 to 7 talks in the field related to the Huanglongbing citrus disease and Hailing Jin will be one of the presenters. Objective 4: Our industry partner, Invaio Science is consulting the EPA registration and preparing the data of SAMP safety testing. Dr. Babcock is evaluating the application of our produce in FL and CA.
Impacts
What was accomplished under these goals?
Objective 1. Identify the most effective SAMP(AP in proposal, now we give the name as stable antimicrobial peptide, SAMP) solutions and delivery methods for curing CLas-positive trees of different genetic backgrounds and growth stages. For curing the infected tree, we mainly focused on the pneumatic trunk injection method with very sick trees (6-10 months after infection with obvious symptoms) in the first year. In the second year, we have tested the micro-needle trunk injection method in the early infected tree (4 weeks after ACP exposure) and foliar spray. In the past year, we have completed the following application of treatments at CRF, UCD: (all the citrus scion is grafted onto Carrizo rootstock) (1) To test if SAMP can eliminate CLas at the early infection stage. 'Washington Navel' orange trees on Carrizo rootstock--Healthy trees were exposed to ACP (contains the San Gabriel strain of CLas) for 3 weeks, incubated for 1 week, and then subjected to SAMP injection (4 weeks after ACP exposure). Due to the pandemic, the plant sampling was delayed and the subsequent treatment was changed to foliar spray once per 2 months. After 18 months of infection, the result indicates the SAMP-treated trees have a significantly lower infection rate (23%) compared to mock (buffer only) treated trees (78.5%). The SAMP-treated trees were growing better than mock-treated trees and with a lower CLas titer. 'Lisbon' lemon trees on Carrizo rootstock - Healthy trees were exposed to ACP (contains the San Gabriel strain of CLas) for 3 weeks, incubated for 1 week, and then subjected to SAMP micro-needle injection (4 weeks after ACP exposure). Due to the pandemic, the subsequent treatment was changed to foliar spray once per 2 months. After 18 months of infection, the result indicates the SAMP-treated trees have a lower infection rate (50%) compared to mock (buffer only) treated trees (100%). This is the first test of micro-needle injection. A couple of the trees died after 2-3 weeks of treatment due to the injury caused by this method. The method has been improved by more practice. The SAMP-treated trees have been growing better than mock-treated trees and with a lower CLas titer. 'Tango' mandarins on Carrizo rootstock - The treatment is the same as the previous 'Lisbon' lemon. After 18 months of infection, the result indicates the SAMP-treated trees have a lower infection rate (14.2%) compared to mock (buffer only) treated trees (75%). The SAMP-treated trees were growing better than mock-treated trees and with a lower CLas titer. (2) Dewdney group has started a field trial to test if SAMP can remediate an HLB-affected young citrus grove. This experiment is ongoing in the Ridge region of Florida on the deep sandy soils. We have located a four-year-old commercial 'Hamlin' sweet orange grove in the Lake Wales region. The trial was laid out as a randomized complete block design with five blocks and five replicates per block. The treatments in this trial were an untreated control and bimonthly foliar application. The CLas titer was measured in the trees prior to trial initiation and six months later. After one SAMP treatment, there was not a substantial difference among the treatments at either time point. There was a reduction in titer observed at the second time point, but this is consistent with the seasonal decline in titer observed in other studies with a seasonal component. All trees were found to be infected with CLas as expected. We will keep monitoring the trees for at least 2 years. Objective 2: Examine the priming effect of SAMPs on healthy citrus seedlings and determine the minimum dosage/solution and treatment frequency needed to protect healthy citrus seedlings and young trees from CLas infection. We have tested the protective effect on 2 sets of 'Madam Vinous' sweet orange trees and published in Huang et al., 2021 in PNAS. In these 2 tests, one set is infected by ACP, another is by grafting with disease budwood. We further tested in commercial varieties including 'Washington Navel' sweet orange and 'Lisbon' lemon trees in CRF, UCD. To test if SAMP treatment affects ACP feeding, in the 3 sets of tests, the trees were foliar spray with SAMP solution and exposed to ACP by choice feeding. Interestingly, all the 3 sets of trees have a low infection rate in both mock and SAMP-treated trees after 18 months of exposure. This is an unexpected result, which may indicating that SAMP can reduce the titer of Clas in the gut. Thus, in the following test, we still used non-choice feeding with ACP as an inoculation method. Starting last year, we tested if the active motif of SAMP, the truncated SAMPH2 is sufficient to protect trees from CLas infection in greenhouse trial. In addition, Dr. Muck and Dr. Godfrey are designing experiments to test the effects of SAMP on ACP. Dewdney group has started a field trial to determine if newly planted trees could be protected from HLB by SAMP for at least two years was planted on October 15, 2020 in Lake Alfred, FL. The experiment was laid out in a completely randomized design with three treatments and sixty trees per treatment. The treatments are 1. Untreated control, 2. Treatment in the nursery 1 week prior to planting and field applications (10 µM in 158 ml/tree) every 2 months and 3. No nursery treatment with field applications every 2 months starting 10 days post-planting. It is expected that the treatment volume will increase as the trees grow over the next two years. When the titer of CLas was measured in December 2021, there were no differences among the average titers. There was more variation in the titer for the field application treatment than the other treatments. The variability in the CLas titer may reflect the time the trees were infected, and it is assumed that most of the infections occurred since June 2021. The trees were planted next to a grove block that has a substantial psyllid population. The block has no insect vector control. We hope to observe whether any treatment thrives more than others despite infection in the following year. Citrus tristeza virus (CTV)-based vector mediated delivery (Folimonova Lab) A Citrus tristeza virus (CTV)-based vector containing a nucleotide sequence encoding the peptide SAMP1 was generated in the previous year. The resulting construct was verified to contain the SAMP1 gene. The construct was named as CTV-SAMP1. During the current funding period, we amplified the CTV-SAMP1 virus inoculum by introducing the respective construct into Nicotiana benthamiana plants. CTV-SAMP1 inoculum was used for bark-flap inoculation of citrus plants (Citrus marcophylla) to initiate virus infection. At two months post-inoculation, infection of 20 C. macrophylla plants was confirmed using ELISA with a CTV-specific antibody. The presence of a gene encoding SAMP1 was confirmed via RT-PCR. These plants expressing SAMP1 from a CTV vector are now ready for further challenge with the HLB bacterium. Objective 3: Dr. Kahn and Dr. Vidalakis interacted withCitrus growers and nursery owners for the California Citrus Industry, public and UCR undergraduate students. More detail of outreach activities related to this project is list in the question: How have the results been disseminated to communities of interest? Objective 4: Dr. Backcock are performing an economic analysis of the difference of prospect on HLB management between FL and CA. Our industry partner Invaio are preparing the data and consulting for EPA registration.
Publications
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Progress 02/01/20 to 01/31/21
Outputs
Target Audience:International Plant Pathology Scientific Community International Citrus Research Community Citrus Growers Citrus Research Board in CA and FL Industry parters who are interested in our invention Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided excellent training opportunities to graduate students, postdoctoral fellows and early career researchers.They have had the opprotunities not only to do the research in the lab, but also to present the project and results in international and national scientific meetings and to communicate with the citrus industries and growers. It also provided training opportunity in management to the project manager Dr. Huang. She has done an excellent job on organizing the annual team meeting at the end of Jan, 2021 among advisory board members and co-PIs. She is also organizing all theexperimental plans and data analysis among all the groups. The Covid-19 pandemic significantly limited opportunities for professional development in most regards (cancelled meetings, few opportunities for in-person talks, and undergraduates banned from working in on-campus labs). However, with some creative thinking, we were able to develop remote projects for two undergraduate researchers in 2020. Godfrey group is learning new molecular biology techniques to improve testing of samples. This was done by working with other scientists at UC-Davis. Jin group have a new graduate student join the project. One student in Mauk group performed experiments evaluating effects of priming agents on psyllid behavior. The other is performing psyllid surveys to identify transiently used ACP hosts outside of the Rutaceae, and to collect various psyllid samples from Central California which would provide information on Liberibacter diversity among other psyllid species in CA. A postdoc was scheduled to come to Ft. Pierce to participate in experiments in FL. All agency applications for this visit were approved and security clearance had been granted, then the pandemic hit. Currently no outside visitors may come to the USHRL and all of the original approvals have expired. Although limitations on allowing students to work with ACP outside of the lab precluded more direct professional development activities, both of these projects closely relate to the ongoing work of this grant and are contributing key information of relevance for control of HLB in CA and FL. We will continue to look for creative professional development opportunities involving undergraduate researchers and opportunities for key personnel to engage in mentoring despite pandemic restrictions. How have the results been disseminated to communities of interest?We have two scientific publications: Huang CY, Niu D, Kund G, Jones M, Albrecht U, Nguyen L, Bui C, Ramadugu C, Bowman K, Trumble J, and Jin H. 2020. Identification of citrus defense regulators against citrus Huanglongbing disease and establishment of an innovative rapid functional screening system. Plant Biotechnol J. https://doi.org/10.1111/pbi.13502 Huang CY, Araujo K, Sánchez JN, Kund G, Trumble J, Roper C, Godfrey KE, and Jin H. 2021. A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing. PNAS. DOI:10.1073/pnas.2019628118. Dissemination of results has primarily been through outreach efforts of the extension personnel (Tracy Kahn and Georgios Vidalakis). Fielded questions from public and growers on article "UC Riverside discovers first effective treatment for citrus-destroying disease New license agreement commercializes innovative, safe technology". Below is a list of some of the public and growers I spoke to for your information but not necessarily for report: James J. Shanley, President Shanley Farms: Commercial Grower of Australian Finger limes. They are extracting the juice vesicle and oil as well as selling the fruit. Jim Churchill, Churchill Farms citrus grower Susan Von Zabern, Manager for Non-profit management board Citrus State Historic Park in Riverside. She wanted to start educating public about this "solution" to HLB Tom Spellman, board member Citrus State Historic Park and Nurseryman at Dave Wilson Nursery Vincent Moses, Ph.D. Citrus historian who gave presentation to Osher class at UCR and wanted to tell public about this discovery as part of his presentation.as Helped answer questions on ACP for "By the Numbers Citrus Greening Disease" for Jules Berstein Contributed to UC ANR article "UC: researchers at work finding solutions for huanglongbing disease to save California citrus" September 2020 UC ANR https://ucanr.edu/sites/QBELab/files/336704.pdf Reviewed article soon to come out entitled "Jewel in the Crown - The UCR Givaudan Citrus Variety Collection sits at the forefront of global agricultural innovation" by Holly Ober which discusses this project. The PI Hailing Jin has been interviewed on the findings of the novel antimicrobial peptides by a dozen of News outlets, including National Public Radio, LA Times, Progressive Crop Consultant Magazine, Western U.S. newspaper, The Packer, the produce industry newspaper, the Australian Broadcasting Corporation's national rural affairs TV program Landline, etc. She has also given several talks to the Citrus Research and Development Foundation Inc (CRDF) in Florida, and gave a webinar to the citrus industry on Antimicrobial Peptides organized by CRDF. The project manager Chien-Yu Huang in Jin group has joined virtual conference to present our work to researchers at 12th Japan-US Seminar in Plant Pathology. The co-PI Mauck also assisted UCR Science News personnel several times throughout 2020 in developing entomologically correct educational materials on HLB research at UCR, including pieces covering the work in this project. The co-PI Dewdney participated in meeting with group of Florida citrus industry representatives to explain the goals of the project and what progress had been accomplished to date. What do you plan to do during the next reporting period to accomplish the goals?Objective 1:We will continue experiments with exposures and treatments and collect the data for the result analysis. The varieties in CRF under testing are important vatieties for CA. Co-PI Dr. The SAMP we are using is the full length version. We have find out the 22 residue bacteria killing functional domain in the SAMP by in vitro viability assay with Liberibactercrescens, the closest culturable stain to CLas. Using the shorter version of SAMP could greatly reduce the cost of peptide production. Jin group and Godfrey group will start to screening the formula base on short version of SAMP. We are currently increasing the densities of the CLas-positive (San Gabriel strain) ACP colonies in preparation for upcoming insect inoculation experiments. Due to space limitations, we will replicate each study through time, inoculating between 20 - 25 trees per replicate. The planned studies include treating: 1. 75 Tango mandarin on Carrizo rootstock trees - 25 treated with buffer (mock), 25 treated with SAMP (2nd version) and 25 treated with SAMPH2 (a genetically shortened version of SAMP peptide) using foliar application ; 2. 60 Washington navel on Carrizo rootstock trees - 20 trees treated with buffer; 20 trees treated with SAMP, and 20 treated with SAMPH2; 3. 48 Lisbon lemon on Carrizo rootstock trees - 16 treated with buffer; 16 treated with SAMP, and 16 treated with SAMPH2. All will be exposed to CLas-positive (San Gabriel strain ) ACP. For the trees above, the insect inoculation will be done using sleeve cages to identify the inoculation point on the plant. The ACP used for the inoculations will be collected after a 21 day exposure and assayed individually for the presence of CLas. The number of eggs and nymphs will be counted on each tree. The trees will be treated with either buffer, SAMP, or SAMPH2 by foliar application. Dewdney group is working on the field test in FL. Dewdney group plans to collect data on the number of new shoots in the 3rd year, make a visual HLB rating biannually, and monitor for ACP at least every two weeks.Any new trees that are suspected to have HLB will have leaf and root samples tested with qPCR using the Wang et al. (2006) protocol. McCollum group is working on a therapeutic experiment using foliar spray with 60 citron cuttings those produced from CLas-infected stock plants. All of the cuttings were unambiguously HLB symptomatic prior to the application of treatments. Were sprayed either with buffer or buffer + peptide four times at two week intervals between applications. Following treatment the plants were held for evaluation of HLB symptoms. Plant samples were collected for CLas diagnostics, but have yet to be analyzed due to the abbreviated work schedule. Objective 2A: We will continue experiments with exposures and treatments and collect the data for the result analysis. McCollum group is working on protection test with Valencia. Dewdney group is working on the field test in FL. Folimonova group will proceed with inoculating the CTV-SAMP1 construct into the citrus plants, which will be followed by further analysis of SAMP1 expression in the citrus trees and its effect on the challenge with HLB. Objective 2B: Evaluate SAMPs as tools to disrupt HLB transmission by ACP vectors. 2B.1 Quantify effects of SAMPs on ACP behaviors mediating CLas acquisition and inoculation. We will complete development of a LC-MS/MS-based method for detecting the AP peptide and validate movement of the peptide into plant tissue and psyllids. This method will be more sensitive than western blot and amenable to use with individual or small pools of psyllids. We will complete EPG experiments to evaluate effects of APs on resistance of different citrus varieties to ACP (outside of containment - EPG cannot be used in containment). Parallel experiments will continue to evaluate anti-psyllid effects of transgenic vs. foliar applications of SAMPs in Solanum species alongside anti-pathogen effects (e.g., quantifiable differences in psyllid survival, reproduction, infection rates, symptom severity, and pathogen titer). As a component of this, we will test delivery of SAMPs via different foliar methods that include commercially available "helper" compounds (e.g., nanoparticle formulations) that facilitate translaminar uptake of larger molecules. 2B.2 Determine the molecular basis of SAMP effects on ACP behavior. Phenotypes observed in transgenic SAMP-expressing potato will be pursued directly to evaluate molecular mechanisms of AP effects on psyllid survival, oviposition, and CLas pathogen infections. Phenotypes observed following optimization of foliar SAMP delivery will likewise be explored. Methods will include expression of key defense genes following applications over a time course and measurement of plant metabolites relevant for psyllid behavior. 2B.3 Determine whether SAMPs can function directly to inhibit CLas colonization of ACP vectors. This work was delayed previously by the pandemic restrictions on BSL-3P project initiation (no work in the facility was possible until June) and the need to acquire updated paperwork to allow Co-PD Mauck to work in the facility independent of PI Jin (all completed by December 2020). CLas-infected psyllids will be established in the UCR BSL3 facility and we will perform evaluations of titer reductions due to adult and nymphal feeding on artificial diets containing SAMPs. We will also evaluate effects of AP acquisition by infected psyllids on subsequent inoculation to susceptible hosts and local parent-to-offspring transmission. Finally, we will test whether applications of SAMPs to citrus will block parent-to-offspring CLas transmission facilitated by locally inoculated CLas pathogen pools in flush host tissue - the main mode by which nymphal psyllids become infectious. Parallel work outside the BSL3 facility will continue to evaluate direct SAMP effects on CLso titers and transmission using the potato-psyllid/tomato system. Objective 3: Due to Covid-19 we can expect most of the meeting will be canceled start from April, 2020. We will mainly focus on the web based outreach activity next year. Dr. Kahn will continue to provide plant material from the GCVC and address questions aboutthe project from those who contact her. We may have a UCR Citrus Day with speakers online in 2021 since we can't have an in person one due to COVID-19 pandemic and campus closure to public.Dr. Kahn isone of the hosts for the Chancellor's Celebration" Living the Promise: The Campaign for UCR" February 18-20 Mixology event. She is planning to come up with trivia questions aboutSAMP. We will have a blub below it on Instagram account for the Citrus Variety Collection: https://www.instagram.com/citrusvarietycollection/?hl=en, Once regular citrus events reopen, Dr. Kahn could present at one of them: LREC Citrus Tasting, CA Citrus Mutual Citrus Showcase, 2022 UCR Citrus Day. Dr. Kahn is also on the board for the California Citrus Nursery Society. Deborah Pagalacci, the manager is looking for speakers for this year's online events which we may join the activity to introduce SAMP for citrus nursery. Objective 4: Our industry partner, Invaio Science isconsulting the EPA registration and preparing the data of SAMP safety testing.
Impacts
What was accomplished under these goals?
What was accomplished under these goals? Despite of the negative impact of the pandemic, we still managed to make significant progress on this project. We have published two peer-reviewed papers related to this project in the past year: Huang CY, Niu D, Kund G, Jones M, Albrecht U, Nguyen L, Bui C, Ramadugu C, Bowman K, Trumble J, and Jin H.* 2020. Identification of citrus defense regulators against citrus Huanglongbing disease and establishment of an innovative rapid functional screening system. Plant Biotechnol J. https://doi.org/10.1111/pbi.13502 We illustrated how we identified defense regulators against citrus HLB including SAMP form HLB-tolerant vairties in this publication; Huang CY, Araujo K, Sánchez JN, Kund G, Trumble J, Roper C, Godfrey KE, and Jin H.* 2021. A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing. PNAS. DOI:10.1073/pnas.2019628118 Characterization of SAMP, five sets of citrus plants tested with SAMP at CRF and mode of action of SAMP is included in this publication. Objective 1. Identify the most effective SAMP(AP in proposal, now we give the name as stable antimicrobial peptide, SAMP) solutions and delivery methods for curing CLas-positive trees of different genetic backgrounds and growth stages. For curing the infected tree, we major focus on trunk injection method in the first year. At the second year we have test the micro-needle trunk injection method and foliar spray. In the past year. we have completed the following application of treatments at CRF, UCD: (all the citrus scion is grafted onto Carrizo rootstock and are seedling plants) (1) Pneumatic injection treatment (Gardner Custom Trunk Injection System)- We have tested different concentration of SAMP solutions on 8 CLas-positive Citrus marophylla and monitor the CLas titer for 20 months. Test with 14 CLas positive 'Madam Vinous' sweet orange. The seedlings is grafted infected after 10 months and start to decline. Test with 7 CLas positive 'Lisbon' lemon. The seedlings is grafted infected after 6 months and start to decline. The result of test a-c was published in Huang et al., 2021 in PNAS. (2) Micro-injection treatment - we have test a new injection method that can be a easierinjection method be used in the field. Injection of 15 trees with SAMP and 15 trees with buffer have been completed for Lisbon lemon and Tango mandarin. All of the trees have been exposed to CLas-positive ACP adults that contain the San Gabriel strain. These plants are in the greenhouse and sampling of leaf material was planned to begin at the end of April 2020. Due to the pandemic, the plant sampling is delayed the subsequent treatment is changed to foliar spray. We are still monitoring the plant. Objective 2A: Examine the priming effect of SAMPs on healthy citrus seedlings and determine the minimum dosage/solution and treatment frequency needed to protect healthy citrus seedlings and young trees from CLas infection. (1) Pre-exposure foliar sprays of antimicrobial proteins treatment for protectant development - The following number of plants were sprayed with either an SAMP spray or a buffer containing a spray before being exposed to CLas-positive ACP. The plants were sprayed 5 days before exposure to ACP. a. For testing the protection effect of SAMP, we have a foliar sprays test on 20 one-year-old health 'Madam Vinous' trees at CRF. The plants were subjected to ACP infestation (no choice feeding test) after foliar spray with buffer or SAMP. The plants with SAMP treatment have fewer infection plants and lower CLas titer after infection for 12 months. The result of test was published in Huang et al., 2021 in PNAS. b. Another foliar sprays test on 18 one-year-old health 'Madam Vinous' trees at CRF. The plants were subjected to budwood grafting inoculation of CLas. The plants with SAMP treatment have lower infection rate and lower CLas titer after infection for 10 months. The result of test was published in Huang et al., 2021 in PNAS. Dewdney group has started a field trial to determine if newly planted trees could be protected from HLB by SAMP for at least two years was planted on October 15, 2020 in Lake Alfred, FL.The experiment was laid out in a completely randomized design with three treatments and sixty trees per treatment.The treatments are 1.Untreated control, 2.Treatment in the nursery 1 week prior to planting and field applications (10 µM in 158 ml/tree) every 2 months and 3.No nursery treatment with field applications every 2 months starting 10 days post planting.It is expected that the treatment volume will increase as the trees grow over the next two years.An additional treatment from an additional funding source is to look at the peptide as a biannual injection treatment.To date, two foliar treatments have been applied. (2) Citrus tristeza virus (CTV)-based vector mediated delivery A nucleotide sequence encoding the AP1 peptide (SAMP1) was cloned into the insertion site of the Citrus tristeza virus (CTV)-based vector at the 3' end of CTV genome, under the promoter of the natural CTV coat protein (CP) subgenomic RNA. The resulting construct was verified to contain the SAMP1 gene. The construct was named as CTV-SAMP1. The lab experiments were interrupted by to a COVID-19 university lock-down in late spring of 2020. The CTV-SAMP1 DNA was introduced into Agrobacterium tumefaciens EHA105 by heat shock. The resulting transformants were selected by antibiotics resistance and used to generate bacterial cultures for further infiltration into the Nicotiana benthamiana plants. Objective. 2B - Evaluate SAMPs as tools to disrupt HLB transmission by ACP vectors. 2B.1 Quantify effects of SAMPs on ACP behaviors mediating CLas acquisition and inoculation. This work requires generation of insecticide-free trees of the proposed varieties. Rootstocks were grown during the prior project period and in 2020 we grafted all of the proposed varieties to generate insecticide-free citrus trees for full experiments. Access to buildings due to COVID-19 restrictions delayed grafting by approximately three months, but we were able to complete generating insecticide-free plants of all relevant varieties. Lisbon lemon plants are now of sufficient size for experiments. Other varieties will be ready within 3-6 months. In January 2020, we initiated experiments to use electrical penetration graphing (EPG) and peptidomics to evaluate effects of SAMPs on ACP feeding behavior on Lisbon lemons. Ongoing EPG experiments are evaluating changes in probing and feeding behaviors associated with successful host use and pathogen inoculation. Complementary peptidomics will measure SAMP uptake by the insects from treated plants. 2B.2 Determine the molecular basis of SAMP effects on ACP behavior and 2B.3.Determine whether SAMPs can function directly to inhibit CLas colonization of ACP vectors. This work was recently initiated. Objective 3: Dr. Kahn provided training on Australian Finger Limes at the CCNS webinar: On November 24, personnel from theUSDA Citrus Germplasm Repositoryand theUC Riverside Citrus Variety Collectionwill present a virtual tour of the finger lime trial located at UCR. Seeds of highly pigmented Australian finger limes were received by the USDA and are being cooperatively evaluated by USDA and UCR personnel.Finger limes in the trial are diverse in color and size and may be of interest to the California citrusnursery industry. Thevirtual tour will present information on the acquisition of the material, development of the evaluation criteria, and the processfor release andbeing made available to the industry. Objective 4: We have tested the SAMP can be digest by human pepsin shortly and have low allergenic possibility by in silicon prediction. We are preparing the data and consulting for EPA registration.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Chien-Yu Huang, Karla Araujo, Jonatan Ni�o S�nchez, Gregory Kund, John Trumble, Caroline Roper, Kristine Elvin Godfrey, and Hailing Jin*: A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing. 2021, DOI: 10.1073/pnas.2019628118
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Chien Yu Huang, DongDong Niu, Gregory Kund, Mike Jones, Ute Albrecht, Lincoln Nguyen, Christine Bui, Chandrika Ramadugu, Kim D. Bowman, John Trumble, and Hailing Jin*: Identification of citrus immune regulators involved in defense against Huanglongbing using a new functional screening system. Plant Biotechnology Journal, 2020, doi: 10.1111/pbi.13502.
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Progress 02/01/19 to 01/31/20
Outputs
Target Audience:International Plant Pathology Scientific Community International Citrus Research Community Citrus Growers Citrus Research Board in CA and FL Industry parters who may be interested in our invention Changes/Problems:We don't have any changes on the project, but we may expect some delays because the normal research and field activities have been on hold due to the COVID19 shut down. What opportunities for training and professional development has the project provided?This project provided excellent training opportunities to graduate students, postdoctoral fellows and early career researchers. They have had the opprotunities not only to do the research in the lab, but also to communicate to the citrus industries and growers. It also provided training opportunity in management to the project manager Dr. Huang. She has done an excellent job on organizing the annual team meeting among advisory board members and co-PIs. She is also organizing all the experimental plans and data analysis among all the groups. How have the results been disseminated to communities of interest?We have a the grant annual meeting in Riverside, CA at Jan 30th 2020 with advisory broad member invited and provide us important advised. The meeting agenda are attached. Dr. Mauck have incorporated lessons on HLB and ACP into introductory biology and introductory entomology courses taught on campus. The project manager Chien Yu Huang have a presentation at department seminar to introduce the HLB disease and our work to students. We have joined conference meeting to present our work to researchers and growers including: oral presentation at UCR Citrus Day for Industry. Riverside, CA at Feb 2019 which is host by Co-PI Dr. Kahn; oral presentation by zoom meeting to California Citrus Nursery Board at Feb 2019; Poster presented at International Research Conference on Huanglongbing VI. Riverside, CA (Co-PI Vidalakis is the major host); 2019 IS-MPMI XVIII Congress. Glasgow, Scotland. Dr. Jin and Dr. Chien-Yu Huang attended the meeting, and both gave a presentation. Dr. Huang exclusively talked about the new progress of this project, and her talk was very well received by the international plant pathology community. What do you plan to do during the next reporting period to accomplish the goals?We will continue all the tests as proposed, although there might be some delays during to the COVID19 shut down.
Impacts
What was accomplished under these goals?
Objective 1. Identify the most effective SAMP(AP in proposal, now we give the name as stable antimicrobial peptide, SAMP) solutions and delivery methods for curing CLas-positive trees of different genetic backgrounds and growth stages. For treating the infected tree, we've mainly focused on trunk injection method in the past year. We have finalized the concentration of SAMP and application frequency for treatments. To date, we have completed the following application of treatments at CRF, UCD: (note that all the citrus scion is grafted onto Carrizo rootstock and are seedling plants) (1) Pneumatic injection treatment (Gardner Custom Trunk Injection System)- a. We have tested different concentration of SAMP solutions on 8 CLas-positve Citrus marophylla and monitor the CLas titer for 20 months (Figure 1A). We inject the SAMP solution every 2 months. We observed the CLas titer decrease with 2 trees have almost no CLas detection (Figure 1B). After 2nd injection, we observed the CLas titer will increase after 6 months without treatment. Then we injected SAMP solution again and find out the CLas titer decreased. According to this test result, we finalized the testing SAMP solution and application frequency as 10 μM and 2 months interval for the following test. b. Test with 14 CLas positive Madam Vinous sweet orange. The seedlings is grafted infected after 10 months and start to decline. After SAMP treatment the seedlings have lower CLas titer and growing better compared to mock treatment plants (Figure 2). The result we get so far the CLas titer is suppressed compared to mock treatment. The tree could be too sick but recovering slowly. We are still monitoring this set of dample. c. To test if SAMP can eliminate CLas at early infection stage. 30 Washington navel trees have been exposed to ACP (contain the San Gabriel strain of CLas) for 3 weeks and subjected to SAMP injection at 3 weeks after the end of ACP exposure. 15 trees were injected with antimicrobial protein and 15 trees, with buffer. These plants are in the greenhouse and sampling of leaf material will begin at the end of April. (2) Micro-injection treatment - we have test a new injection method that can be a easier injection method be used in the field. Injection of 15 trees with antimicrobial protein and 15 trees with buffer have been completed for Lisbon lemon and Tango mandarin. All of the trees have been exposed to CLas-positive Asian citrus psyllid (ACP) adults that contain the San Gabriel strain (Type 2; prophage type 1 CLas). The insects used for these exposures are awaiting analysis. These plants are in the greenhouse and sampling of leaf material will begin at the end of April. Objective 2A: Examine the priming effect of SAMPs on healthy citrus seedlings and determine the minimum dosage/solution and treatment frequency needed to protect healthy citrus seedlings and young trees from CLas infection. (1) Pre-exposure sprays of antimicrobial proteins treatment for protectant development - The following number of plants were sprayed with either an antimicrobial protein spray or a buffer containing a spray before being exposed to CLas-positive ACP that contain the San Gabriel strain of CLas. The plants were sprayed 5 days before exposure to ACP. a. For testing the protection effect of SAMP, we have started a test on 20 one-year-old health Madam Vinous trees at CRF. The plants were subjected to ACP infestation (no choice feeding test) (Figure 3A) after foliar spray with buffer or SAMP. We have monitored the expression of defense marker genes and collected tissue for CLas titer detection. The trees growing better with SAMP spraying after 12 months of infection (Figure 3B). We have observed the SAMP treatment plants have fewer ACP feeding marks, eggs and nymphs after 2 weeks ACP exposure (Figure 3C). The plants with SAMP treatment have fewer infection plants and lower CLas titer after infection for 12 months (Figure 3C). b. Washington navels - 20 plants - 10 with antimicrobial protein and 10 with buffer. It was noted that the buffer treated plants had slightly higher numbers of flushing tips and ACP eggs. It was noted trees dropped a significant amount of leaves after spray application of antimicrobial peptide and buffer. The buffer and antimicrobial sprayed plants were placed in equal numbers inside exposure cages and the ACP adults could choose which plant to settle, feed, and oviposit on. For the buffer treated plants, 4 out of 10 plants had flush tips and 10 healthy ACP eggs and 1 first instar ACP nymph was found on the flush tips. For the antimicrobial protein treated trees, 1 out of 10 plants had flush and 6 healthy ACP eggs were found on that flush tip. These trees have returned to the greenhouse, 4 out of 20 trees did not recover from the stress of leaf loss and insect feeding totaling to 8 antimicrobial treated plants and 8 buffer treated plants. Leaf tissue samples were collected in October 2019 and December 2019 and sent to Jin lab at UCR for analysis. Due to a lack of symptoms and CLas detection, these plants were re-exposed to CLas positive ACP using sleeve cages from February 7-28, 2020. c. Lisbon lemons - 2 studies - In the first study, it was noted that for the first 4 days after the introduction of ACP adults into a cage, that the ACP settled first on the buffer treated plants before finally moving to plants treated with antimicrobial proteins. This was a choice study using 10 trees sprayed with buffer and 10 trees sprayed with antimicrobial protein. For plants treated with buffer, 7 out of 10 plants had flush tips and 13 ACP eggs and 13 first instar nymphs were found on 14 flush tips. For antimicrobial protein treated trees, 5 out of 10 plants had flush and 36 healthy ACP eggs and 9 dead ACP eggs were found on 8 flush tips. It was also noted that some of the flush tips without ACP eggs were dying. To date, 3 out of 10 plants treated with antimicrobial peptide have expressed mild leaf curl or other leaf deformities, 4 out of 10 plants have expressed slight yellowing of midrib, and 6 out of 10 plants have not expressed symptoms of citrus greening disease. For buffer treated plants, 5 out of 10 plants have expressed moderate leaf curl or other leaf deformities and mild yellowing of midrib, 1 out of 10 has expressed moderate mottling of leaves, and 2 out of 10 plants appear asymptomatic. These plants were sampled the months of September 2019, October 2019, December 2019, February 2020, and April 2020. The very low level of CLas is detected at Feb 2020. We will keep on sampling the plants.
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