Deployment of a Spectrum of Bactericides to Cure and Prophylactically Treat Citrus Huanglongbing

DEPLOYMENT OF A SPECTRUM OF BACTERICIDES TO CURE AND PROPHYLACTICALLY TREAT CITRUS HUANGLONGBING

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

TERMINATED

Funding Source

OTHER GRANTS

Reporting Frequency

Annual

Accession No.

1011807

Grant No.

2017-70016-26053

Project No.

CA-R-PPA-5139-CG

Proposal No.

2016-10981

Multistate No.

(N/A)

Program Code

CDRE

Project Start Date

Jan 15, 2017

Project End Date

Jan 14, 2023

Grant Year

2017

Project Director
Roper, C.

Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521

Performing Department
College of Nat & Agr Sciences

Non Technical Summary
Our research objectives are to design and identify bactericides that can cure/suppress or prophylactically treat Huanglongbing (HLB), and to target these bactericides to the phloem where the associated bacterium, Candidatus Liberibacter asiaticus (CLas), resides. We will develop two classes of bactericides, the first based on silver and sulfur nanoparticles, the second rooted in natural product discovery, mining anti-CLas compounds produced by microbes that inhabit HLB survivor trees in Florida. The difficulty with any anti-HLB formulation is optimizing its delivery to the phloem. In all cases, delivery of the bactericides to, and within, the phloem sieve tubes to kill CLas will be of paramount importance. Thus, we will perform detailed analyses of the phloem transit routes that a given bactericide takes when introduced through common application methods (trunk injection, foliar application or root applications). We will also continue to develop a promising, new petiole/branch delivery system for use in field trees. We will explore the chemistry of bactericides to optimize uptake by the sieve tubes. Because significant amounts of phloem plugging occurs in CLas-infected trees, we will evaluate bactericide transit pathways at the whole-plant level at varying stages of infection. This work on HLB-phloem transit routes will provide important information for us and for others in the HLB research community who are evaluating delivery of materials to the phloem. We will integrate this research with a robust extension and outreach program that will be coupled with an economic cost-benefit analysis structured around adoption of these treatments into commercial citriculture.

Animal Health Component

Research Effort Categories

Basic

55%

Applied

40%

Developmental

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
205	0999	1100	75%
206	0999	1020	25%

Knowledge Area
206 - Basic Plant Biology; 205 - Plant Management Systems;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1100 - Bacteriology; 1020 - Physiology;

Keywords

Goals / Objectives
The goals of this proposal are to facilitate phloem uptake and transport of bactericides that target the bacterium associated with citrus Huanglongbing disease, Candidatus Liberibacter asiaticus.To accomplish this, we will obtain and use knowledge of citrus phloem architecture/transport and source/sink pathways in connection with developing nanoparticle and microbial natural product based bactericides. This project addresses priority number one outlined in the Request for Proposals: Development of therapies to kill or suppress Candidatus Liberibacter asiaticus (CLas) within trees or prevent CLas infection of healthy trees. In the larger scope it lies within the system components (page 5 of RFP): production- crop management; reduce environmental footprint; and food security and overlaps with consumers and markets: vitality of rural communities; impacts on urban systems.A major challenge in developing an effective anti-HLB formulation is optimizing its mobility and delivery to the phloem tissue where the HLB-associated bacterium, CLas, resides. Efficient transport of bactericides to specific, appropriate locations within the tree, especially the roots, is a crucial aspect of a functional anti-CLas delivery system. However, there is little detailed knowledge available regarding the fate of phloem mobile materials once they are introduced into trees using common delivery methods that are being used, such as trunk injection, soil or foliar applications. Although, one very recent (August, 2016) manuscript investigated retention rates of oxytetracycline in citrus trees following trunk injection. We will conduct florescent tracer experiments that map phloem transport pathways when materials are introduced through these application methods. This will yield important information about the routes bactericides travel when administered through delivery methods currently being utilized by growers and other funded SCRI CDRE projects (foliar or soil applications and trunk injections) as well as a novel petiole/branch feeding technique developed in this project.There is presently no cure for HLB or any suitable genetic sources of resistance to HLB. Management of HLB and its psyllid vector is difficult and expensive. Inspections, testing, tree and inoculum removal, biological and chemical psyllid control all contribute to the cost of production. The socio-economic effects of HLB are linked to the current unsustainable management strategies, such as constant insecticide sprays. This is economically unsustainable and harmful to human health and the environment. This project will contribute to the long-term profitability and sustainability of citrus production by providing novel, effective and sustainable strategies to manage Huanglongbing (HLB). To disseminate the fundamental discoveries and deliverable solutions created by this project, we will fully integrate the research with a robust array of extension and outreach components.Accordingly, our objectives are:Objective 1: Optimizing formulations of chemical bactericides to facilitate phloem mobilityObjective 2: Harnessing the power of bactericides produced by citrus-inhabiting microbesObjective 3: Testing bactericides for phloem mobility and as HLB therapiesObjective 4: Integration of Research and Extension

Project Methods
Objective 1: Optimizing formulations of chemical bactericides to facilitate phloem mobility. Initially, we will map citrus phloem transport patterns in detail using fluorescent tracer carboxyfluorescein (CF) and epiflourescent microscopy. Phloem transit will be assessed in healthy and CLas infected greenhouse trees following several tree delivery techniques. The first is a (i) novel petiole/branch feeding technique that we are developing in this proposal for use in citrus; (ii) direction injection (Arborjet); (iii) continual release injection; (iv) a soil drench and (v) a foliar spray application.Silver (Ag) NP synthesis: We will synthesize a range of AgNPs with different sizes using developed methods. In the first method, AgNPs are produced using a "green" synthesis methodology whereby Ag(NH3)2+ ions are reduced to Ag0 and subsequently form AgNPs by reacting the ions with a carbohydrate in a high pH environment. In the second method, silver acetate is dissolved in an organic solvent and refluxed with oleylamine. Regardless of the synthesis method, the AgNPs can be functionalized (made hydrophilic and, thus, more phloem mobile) by mixing them with a thiol-containing molecule. Sulfur (S) NP synthesis: Synthesis of SNPs will be based on the method developed by Chen et al. (2013). Copper (Cu) and Zinc (Zn) NPs: Several versions of CuNPs and ZnNPs are being developed for use against the bacterium Xanthomonas axonopodis pv. citri and CLas by other research groups. Thus, we will use CuNPs and ZnNPs as an important comparison in our studies. In vitro inhibition assays using NPs. L. crescens BT-1 is a culturable close relative of CLas and is identified as a suitable surrogate for the uncultivable CLas. We will use this system to establish the minimal inhbitory concentrations (MIC) and minimal lethal concentrations (MLC) for each nanoformulation.Objective 2: Harnessing the power of bactericides produced by citrus-inhabiting microbes. Citrus samples will be collected in at least 5 FL groves. Tissues will be lyophilized prior to shipping to UCR. Similar samples from healthy trees (grown under ACP exclusion structures) will be used as negative controls. MS spectra from these controls will be used to subtract background plant metabolites and pull forward meaningful differences from the experimental sample set. To partner community level metabolic activity with microbes, it is necessary to conduct community level microbial analyses based on rDNA (bacterial) and ITS (fungal) sequencing. rDNA Libraries will be run on an Illumina MiSeq to obtain 300bp reads. Low quality and chimeric sequences will be removed using Quantitative Insights Into Microbial Ecology (QIIME). We will identify target compounds along with the microbes that map to the locations of those target compounds. Purifying the natural products in quantities large enough for testing in vitro and in vivo will require obtaining pure cultures of the citrus-associated microbes. Inhibition bioassays. The L. crescens inhibition assay will be used to guide isolation ofthe active compound from the crude extract using flash column chromatographyand high-performance liquid chromatography.Objective 3: Testing bactericides for phloem mobility and as HLB therapies. Ag, S or Cu NPs will be delivered to both healthy and diseased greenhouse trees using the delivery techniques that achieved the best systemic phloem penetration in the CF experiments in Obj. 1A. NPs will be applied to trees at a single high dose above the MLC determined in Objs. 1B. Trees that receive NP carrier buffer only will serve as the negative controls for the experiment. Application methods will include those described in Obj 1. All experiments will be repeated 3 times. We will determine the fate and distribution of NPs at the plant compartment level (roots, budwood and leaf petioles). The bark peels and the xylem sap will be weighed and then incinerated in a furnace at 550° C in air, filtered and injected into an ICP-MS for metal analysis. To obtain higher resolution on the distribution of the Ag, S or Cu between the phloem and the xylem, we will use scanning electron microscopy (SEM) coupled to energy-dispersive X-ray spectroscopy (EDX) to map the NPs inside the root, budwood or leaf tissue.The natural products that were inhibitory in our L. crescens inhibition assay in Obj. 2 will be assessed for their phloem mobility in greenhouse trees in FL using the same experimental design described for the NPs. Natural product presence in the vasculature will be monitored in budwood bark peels and xylem sap and whole root and leaf samples using MS.We will begin assessing efficacy of NPs and natural products against CLas in greenhouse grown citrus trees. Curative treatments will be performed on infected trees obtained as described in Obj. 1 and will be delivered to trees using the delivery methods and concentrations that performed best in terms of delivery to the phloem. We will two criteria to assess bactericide efficacy over the course of 12 months: HLB disease severity and CLas titer. Disease severity will be assessed on a greenhouse tree scale of 0-3, 0: no symptoms/growth, 1: mild, 2: moderate, & 3: severe chlorosis/growth inhibition/leaf deformity. CLas titer will be determined using a qPCR method.Field trials will begin in year 3, focusing on the application method and formulations that performed well in the greenhouse studies. Field blocks will be setup as follows: Test blocks of 12 trees will be setup using a randomized block design. Blocks will consist of 12 trees/block, 6 of the trees will be graft-inoculated with CLas (in year 2) and the other 6 will not (uninfected). Three of the graft -inoculated trees will receive the bactericide being evaluated, 3 will not. Three of the uninfected trees will receive the bactericide being evaluated, 3 will not. The blocks will be replicated 5 times. Two different application methods (determined from Objs. 1 and 3) will be tested. The 3 year-old trees would receive bactericide applications beginning in year 3 and evaluated over a 2-year period with repeated applications of bactericide. This experimental design will allow for simultaneous evaluation of curative and prophylactic effects of each bactericide under evaluation. Trees will be separated into 4 quadrants and each quadrant will be rated on a 0-5 scale indicative of how many limbs are showing symptoms (0=no limbs-5= all limbs).Objective 4: Integration of Research and Extension. We will develop a robust extension and outreach plan with the following end-users as our target audiences. For extension activities, our target end-users are county extension agents, grower stakeholders and academic/industry researchers. For outreach activities, our target end-users are non-commercial citrus growers and undergraduate students in agricultural sciences.Economic analysis: We will perform empirical analyses of the costs and benefits from adoption of identified bactericides that will cure or prophylactically treat CLas infection so we may best inform growers about the potential gains from these practices, reduce uncertainty about them and, when cost effective, increase adoption, contributing to long-term profitability and sustainability. The perennial nature of citrus orchards, the incidence of HLB and presence of ACP, and their relative life-expectancy suggest decisions regarding planting and care can have effects that linger long into the future. In these situations a dynamic economic model is appropriate as it captures these temporal interactions. The model simulates investment decisions under alternative control practices, accounting for differences across growing regions. The simulated results on costs and benefits under current cultural practices and alternative control practice scenarios can be compared to estimate the costs and benefits to citrus growers from adoption not just today, but into the future.

Progress 01/15/17 to 01/14/23

Outputs
Target Audience:Our target audiences are grower stakeholders, academic and industry scientists, undergraduate and graduate students and the general public. To reach these audiences, we have presented our work at national and international professional science meetings that are attended by scientists, citrus growers and agricultural industry representatives. We have input the specific locations and titles of these meetings in the Other Products (Activities and Events) section in the report. Our two Extension Specialists have disseminated information to target stakeholder audiences through our project website, presentations at stakeholder meetings and through semi-technical stakeholder publications that are listed in the Other Products (Events and Publications) section of this report. In addition, our Economist has conducted grower surveys. We have also disseminated information about Huanglongbing (HLB), its implications to the citrus industry and specifics of this project through the Citrus Clonal Protection Program (CCPP) budwood distribution system (over 9,000 users), our project website, educational aids and curricula developed by the PDs and PIs of this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PDs and PIs of this project have trained one senior scientist, six graduate students and three postdoctoral scholars in nanoparticle design and synthesis, microbial isolations from plants and soils, natural product isolations and microbial inhibition assays. We have also trained three graduate students and six undergraduates in liquid chromatography-mass spectrometry, natural product identfication and global metabolite network analyses. We have also trained three graduate students and one postdoctoral scholar in field sampling protocols and they all participated in the field expedition portion of the project. We have also trained six additional undergraduates in bacterial culturing, organic extraction and chromatographic isolation, and in mining bacterial genomes for antimicrobial natural product production using antiSMASH. Several members of the CDRE project have been trained on how to construct Illumina libraries and perform the subsequent bioinformatic analysis. Several Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, Marine Natural Products Gordon Conference, Tiny Earth Symposia, Citrus Research Board Antimicrobial Summit as well as grower stakeholder meetings and stakeholder meetings. How have the results been disseminated to communities of interest?The Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society Plant Health online as well as grower stakeholder meetings, such as UCR Citrus Day, International Citrus Congress, Citrus Research Board Antimicrobial Summit and Citrus Research Board meetings. PI Maloney gave a guest lecture in a local high school biology class. 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: Optimizing formulations of chemical bactericides to facilitate phloem mobility. Phloem studies. The Turgeon lab began transformation of Carrizo citrange, a common rootstock, with three separate versions of anchored, phloem-restricted antibodies against CLas. These trees, used as rootstocks or whole trees, are being tested for resistance by the Mandadi lab in Texas. CLas will be introduced in these studies by the Asian Citrus Psyllid, Diaphorina citri. Our structural analysis of young phloem tissue is nearing completion and will be published. Objective 2. Harnessing the power of bactericides produced by citrus-inhabiting microbes. Targeted metabolite analysis. We have optimized production of a subset of CLas-inhbiting natural products by altering growth conditions of the microbes in liquid culture. We have prepared organic extracts, and tested these in the anti-L. crescens bioassay. The cultures were subjected to solid-phase extraction and the extracts fractionated by flash-column chromatography on silica gel. Through reiterative processes of separating the active fractions from these strains by HPLC, and following up with LC-MS, we have linked the inhibitory activity to a class of compounds. We are currently performing in situ anti-CLas ihnbition assays in collaboration with the Mandadi lab at Texas A&M using the citrus hairy root assay. We are also continuing to grow and extract additional bacterial strains isolated from HLB-infected citrus trees. Objective 3. Testing bactericides for phloem mobility and as HLB therapies. Nanoparticle studies. We have conducted greenhouse assays to test prophylactic use of the Gum arabic-AgNPs in citrus trees that will be infected with CLas. These AgNPs had an unexpected effect on killing psyllids but this needs to be repeated to draw meaningful conclusions. Natural product bactericide studies. We have been testing natural products that are inhibitory in our anti-Liberibacter crescens assay in prophylactic and curative efficacy against CLas in citrus trees in the UCR Biosafety level 3 plant facility. We have also initiated a collaboration with the Mandadi lab (Texas A&M) and the Niedz lab (USDA, ARS Fort Pierce, FL) to test our inhibitory compounds or mixtures of compounds in their higher throughout anti-CLas bioassays. Objective 4. Integration of Research and Extension. In the proposal, we indicated that our economic analysis would begin in year 4 of the project. We have just completed year 6 of this project and although the public health restrictions imposed by the COVID-19 pandemic limited our economic outreach in 2020 and 2021, we were able to engage growers and other industry stakeholders through in-person meetings in 2022, which allowed us to gather data to use in our economic assessment of management strategies to combat the Asian Citrus Psyllid and HLB.

Publications

Type: Journal Articles Status: Published Year Published: 2022 Citation: Ginnan, N. A., De Anda, N. I., Campos Freitas Vieira, F., Rolshausen, P. E., & Roper, M. C. (2022). Microbial Turnover and Dispersal Events Occur in Synchrony with Plant Phenology in the Perennial Evergreen Tree Crop Citrus sinensis. MBio, 13(3). https://doi.org/10.1128/mbio.00343-22
Type: Journal Articles Status: Published Year Published: 2022 Citation: Xi, M., Deyett, E., Ginnan, N., Ashworth, V. E. T. M., Dang, T., Bodaghi, S., Vidalakis, G., Roper, M. C., Glassman, S. I., & Rolshausen, P. E. (2022). Geographic Location, Management Strategy, and Huanglongbing Disease Affect Arbuscular Mycorrhizal Fungal Communities Across U.S. Citrus Orchards. Phytobiomes Journal, 6(4), 342353. https://doi.org/10.1094/pbiomes-03-22-0014-r
Type: Journal Articles Status: Published Year Published: 2022 Citation: Su, Y., Zhou, X., Meng, H., Xia, T., Liu, H., Rolshausen, P., Roper, C., McLean, J. E., Zhang, Y., Keller, A. A., & Jassby, D. (2022). Costbenefit analysis of nanofertilizers and nanopesticides emphasizes the need to improve the efficiency of nanoformulations for widescale adoption. Nature Food, 3(12), 10201030. https://doi.org/10.1038/s43016-022-00647-z
Type: Journal Articles Status: Published Year Published: 2023 Citation: Xi, M., Deyett, E., Stajich, J. E., El-Kereamy, A., Roper, M. C., & Rolshausen, P. E. (2023). Microbiome diversity, composition and assembly in a California citrus orchard. Frontiers in Microbiology, 14. https://doi.org/10.3389/fmicb.2023.1100590
Type: Journal Articles Status: Published Year Published: 2023 Citation: Kurbessoian, T., Heimlich-Villalta, G., Ginnan, N., Vieira, F. C., Rolshausen, P. E., Roper, M. C., & Stajich, J. E. (2023). Genome Sequence and Assembly of 18 Fusarium Isolates from Florida Citrus under High Huanglongbing Disease Pressure and California Citrus under Low Huanglongbing Disease Pressure. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00101-23 (Refereed, Electronic) https://doi.org/10.1128/mra.00101-23
Type: Other Status: Published Year Published: 2022 Citation: Haynes, Samuel, Ajay Singh, and Jonathan D. Kaplan. "HLB Grower Web ToolTechnical Note." California State University, Department of Economics, Technical Note, February 2022. https://www.csus.edu/faculty/k/kaplanj/economic_tools/hlb_grower_web_tool_tech_note.pdf.
Type: Other Status: Published Year Published: 2022 Citation: Haynes, S., Kaplan, J., and A. Singh, How grower beliefs influence the efficacy of area-wide coordinated management of Asian citrus psyllids and Huanglongbing in California Topics in Subtropics, Volume 23, Spring 2022. https://ceventura.ucanr.edu/news/Topics_in_Subtropics/?newsletteritem=93524.
Type: Journal Articles Status: Published Year Published: 2022 Citation: Petras, D.; Phelan, V. V.; Acharya, D.; Allen, A. E.; Aron, A. T.; Bandeira, N.; Bowen, B. P. Belle-Oudry, D.; Boecker, S.; Cummings Jr., D. A.; Deutsch, J. M.; Fahy, E.; Garg, N.; Gregor, R.; Handelsman, J.; Navarro-Hoyos, M.; Jarmusch, A. K.; Jarmusch, S. A.; Louie, K.; Maloney, K. N.; Marty, M. T.; Meijler, M. M.; Mizrahi, I.; Neve, R. L.; Northen, T. R.; Molina-Santiago, C.; Panitchpakdi, M.; Pullman, B.; Puri, A. W.; Schmid, R.; Subramaniam, S.; Thukral, M.; Vasquez-Castro, F.; Dorrestein, P. C.; Wang, M. GNPS Dashboard: Collaborative exploration of mass spectrometry data in the web browser. Nat. Methods. 2022, 19, 134-136.
Type: Journal Articles Status: Published Year Published: 2022 Citation: Osman, F., Dang, T., Bodaghi, S., Haq, R., Lavagi-Craddock, I., Rawstern, A., Rodriguez, E., Polek, M., Wulff, N. A., Roberts, R., Pietersen, G., Englezou, A., Donovan, N., Folimonova, S. Y., & Vidalakis, G. (2022). Update and validation of the 16S rDNA qPCR assay for the detection of three Candidatus Liberibacter species following current MIQE guidelines and workflow. PhytoFrontiers". https://doi.org/10.1094/phytofr-04-22-0046-fi
Type: Journal Articles Status: Published Year Published: 2022 Citation: Rolshausen, P. Mycorrhizae: An Underground Support Network for Trees. 2022. Topics in Subtropics. Vol22.

Progress 01/15/21 to 01/14/22

Outputs
Target Audience:Our target audiences are grower stakeholders, academic and industry scientists, undergraduate and graduate students and the general public. We have presented our work at national and international professional science meetings that are attended by scientists, citrus growers and agricultural industry representatives. We have input the specific locations and titles of these meetings in the Other Products (Educational Aids and Curricula) section in the report. Our two Extension Specialists have disseminated information to target stakeholder audiences through our project website, presentations at stakeholder meetings and through semi-technical stakeholder publications that are listed in the Other Products (Educational Aids and Curricula) section of this report. In addition, our Economist has conducted grower surveys. We have also disseminated information about Huanglongbing (HLB), its implications to the citrus industry and specifics of this project through the Citrus Clonal Protection Program (CCPP) budwood distribution system (over 9,000 users), our project website, educational aids and curricula developed by the PDs and PIs of this project. Our public in-person outreach has been hampered this year due to COVID-19 restrictions regarding in-person events. Changes/Problems:The laboratories of the PDs and PIs were in a restricted research ramp up phase that includes limits on the number of personnel in the lab at a given time. Thus, research is slower than originally planned.We are still working under the new normal with somewhat loosened COVID restrictions. What opportunities for training and professional development has the project provided?The PDs and PIs of this project have trained one senior scientist, six graduate students and three postdoctoral scholars in nanoparticle design and synthesis, microbial isolations from plants and soils, natural product isolations, microbial inhibition assays. We have also trained three graduate students and one postdoctoral scholar in field sampling protocols and they all participated in the field expedition portion of the project. We have also trained four additional undergraduates in bacterial culturing, organic extraction and chromatographic isolation, and inmining bacterial genomes for antimicrobial natural product production using antiSMASH. Several members of the CDRE project have been trained on how to construct Illumina libraries and perform the subsequent bioinformatic analysis. Members of the project are also being trained in liquid chromatography-mass spectrometry and global metabolite network analyses. Several Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, Marine Natural Products Gordon Conference, Tiny Earth Symposia as well as grower stakeholder meetings and stakeholder meetings (listed in Other Products). How have the results been disseminated to communities of interest?The Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society Plant Health online as well as grower stakeholder meetings, such as UCR Citrus Day, Citrus Research Board meetings. PI Maloney gave a guest lecture in a local high school biology class. Due to COVID-19 restrictions that started during this reporting period, much of our interactions with the communities of interest have been virtual. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. The key compounds identified in the analysis of spatial distributions in the field and 3D mapping experiments will be tested in planta to determine their effect in prophylactic (administration of the compounds to uninfected plants followed by the infection with hot psyllids) or curative (administration of the compounds to infected plants) applications and will be evaluated in this next year in the Biosafety level 3 plant high containment facility. We will be testing a citrus flavonoid mixture and tangeritin, a citrus flavonoid involved in plant defense. We have also identified ferulic acid, an intermediate metabolite in flavonoid biosynthesis, is putatively toxic to CLas. This has been demonstrated in an in silico model and inhibits the surrogate, L. crescens in vitro. We have grown 9 citrus-derived bacterial strains in liquid culture at medium scale (1L), prepared organic extracts, and tested these in the L. crescens bioassay. Of these, 6 strains, including three Bacillus sp., two Pantoea sp., and one Curtobacterium sp., were selected for large scale fermentation (6L). The cultures were subjected to solid-phase extraction and the extracts fractionated by flash-column chromatography on silica gel. We are in the process of further separating the active fractions from these strains by HPLC, and will also continue growing and extracting additional bacterial strains isolated from HLB-infected citrus trees. Objetive 3. Nanoparticle studies. We have initiated greenhouse assays to test curative and prophylactic use of the Gum Arabic-AgNPs in citrus trees that will be graft-inoculated with CLas. We will take this experiment to completion and also repeat this experiment in the next reporting period. Natural product bactericide studies. We will have prophylactic studies underway for ferulic acid in the BSL3, putrescine and feruloylputrescine and a citris flavanoid mixture in the BLS3. We will take this experiment to completion and also repeat this experiment in the next reporting period. Objective 4. In the proposal, we indicated that our economic analysis would begin in year 4 of the project. We have just completed year 4 of this project and although the public health restrictions imposed by the COVID-19 pandemic limited our economic outreach in 2020, we anticipate new opportunities in 2021 to further engage growers and other industry stakeholders through in-person meetings so we may gather data to use in an economic assessment of management strategies to combat the Asian Citrus Psyllid and HLB. We will continue our other robust extension and outreach programs we have described above.

Impacts
What was accomplished under these goals? Objective 1. Our results demonstrate that trunk injection can efficiently deliver silver (Ag) nanoparticles (NPs) into trees, and NPs with polyvinylpyrrolidone (PVP) or gum Arabic (GA) coatings can move systemically both acropetally and basipetally through the tree's vascular system. We have set up an experiment in the BSL3 plant faclity at UC Riverside that is testing the efficacy of NPs against CLas in planta. We applied the NPs as a foliar application and inoculated plants with infectious Asian Citrus psyllids. We are currently monitoring the plants for HLB symptom development and CLas titer. Objective 2. We have completed all three years of the field study for the spatial and temporal metabolomic field study and the data are in preparation for publication. We determined spatial co-distributions of plant host metabolites and these chemical distributions were consistent between branch-scale mapping and single leaf mapping experiments. We found that feruloylputrescine correlates with HLB symptomology and we speculate that disrupting its biosynthesis may inhibit symptom manifestation. Our data also show distinct disruptions in the biosynthetic pathways of specific flavonoids. We now have over 1,600 bacterial and fungal isolates from CA and FL. We screened the supernatants of these isolates for activity against Liberibacter crescens using a disc diffusion assay. We grew cultures of these microbes and sent these to PI Maloney's laboratory. We fractionated extracts from culture supernatants of these isolates. We are currently working towards isolating more compounds using flash column chromatography HPLC and assaying the fractions against L. crescens in the Roper and Maloney labs. Objective. 3. During the last reporting period we detailed how we tracked nanoparticle mobility in planta using AgNPs because there is little to no background silver content in plant tissue so we began our phloem mobility studies using silver nanoparticles (AgNPs) delivered to 2 year-old citrus trees via trunk injection. Polyvinylpyrrolidone (PVP) or gum Arabic (GA) coated AgNPs transport within the plants, but the GA-modified NPs had the best transport performance with the highest content of Ag in leaves and roots distal from the point of injection. As mentioned above, we are now testing the efficacy of NPs for suppression of CLas titer and HLB symptom development in the BSL3 plant facility. We are also esting if application of the metabolites (ferulic acid) in the disrupted flavonoid synthesis can prevent manifestation of disease symptoms and suppress CLas titer in the BSL3 plant facility. Objective 4. The goal of this objective is to communicate our research to the stakeholder community, the greater academic community and the general public. During this report period, we have accomplished this by participating in the Extension events listed in the Other Products (Activities and Events). In addition, we included HLB brochures in 1,407 budwood shipments to 1,024 Citrus Clonal Protection Program Budwood users.The economic analysis for this project began in year 4 with PI Kaplan and collaborator Ajay Singh conducting interviews among citrus industry stakeholders. In the past year, the information gathered during these interviews guided the design of survey questionnaires on managing ACP/HLB, which was distributed among California citrus growers and other industry stakeholders. We have posted an online research note that discusses the survey and the results from an analysis of the responses. In addition, the Economics team completed a computer-based simulation model of citrus production, citrus flush cycles, and ACP/HLB population dynamics in California to assess management strategies that will be used to provide growers with information they can use to best manage their groves from the threat of HLB. We have also posted an online research note discussing the model and some of the key insights from an analysis of initial simulation results. We have also begun assessing alternative ACP/HLB management strategies including microbial biocontrols.

Publications

Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Y. Su, X. Zhou, H. Meng, T. Xia, A.A. Keller, H. Liu, P. Rolshausen, C. Roper, J.E. McLean, Y. Zhang, D. Jassby 2022. Nanotechnology is Key to the Sustainable Agricultural Practices, Nature Food, Under Review.
Type: Journal Articles Status: Published Year Published: 2020 Citation: Pagliaccia D., Bodaghi S., Chen X., Stevenson D., Deyett E., De Francesco A., Borneman J., Ruegger P., Peacock B., Ellstrand N., Rolshausen P.E., Popa R., Ying S. and Vidalakis G. 2020. Two food waste by-products selectively stimulate beneficial resident citrus host-associated microbes in a zero-runoff indoor plant production system. Frontiers in Sustainable Food Systems. 4. (Refereed, Electronic) https://doi.org/10.3389/fsufs.2020.593568
Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Rolshausen P.E. 2021. Healthy Roots, Healthy Trees. Topics in Subtropics 19 (Winter issue Issue):8-9.
Type: Journal Articles Status: Published Year Published: 2021 Citation: Haynes, SIngh, Kaplan, "An Agent Based Model of ACP/HLB in California Citrus - Preliminary Results on the Effects of Insecticide and Coordination on the Spread of HLB," California State University, Department of Economics, Research Note 2021-2, December 2021. https://www.csus.edu/faculty/k/kaplanj/researchnotes/researchnote2021-2.pdf
Type: Journal Articles Status: Published Year Published: 2021 Citation: Haynes, Singh, Kaplan, "California Citrus Greening Survey - Preliminary Results on ACP/HLB Risk Perception and Information Confidence," California State University, Department of Economics, Research Note 2021-1, September 2021. https://www.csus.edu/faculty/k/kaplanj/researchnotes/research_note-2021_1_survey.pdf
Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Ginnan, N., Dang, deAnda, N. I., Campos Vieira, F., Rolshausen, P.E., and Roper, M.C. 2022. Microbial turnover and dispersal events occur in sync with plant phenology in the perennial evergreen tree crop, Citrus sinensis, Under Revision at mBio

Progress 01/15/20 to 01/14/21

Outputs
Target Audience:Our target audiences are grower stakeholders, academic and industry scientists, undergraduate and graduate students and the general public. To reach these audiences, we have presented our work at national and international professional science meetings that are attended by scientists, citrus growers and agricultural industry representatives. We have input the specific locations and titles of these meetings in the Other Products (Activities and Events) section in the report. Our two Extension Specialists have disseminated information to target stakeholder audiences through our project website, presentations at stakeholder meetings and through semi-technical stakeholder publications that are listed in the Other Products (Events and Publications) section of this report. In addition, our Economist has conducted grower surveys. We have also disseminated information about Huanglongbing (HLB), its implications to the citrus industry and specifics of this project through the Citrus Clonal Protection Program (CCPP) budwood distribution system (over 6,000 users), our project website, educational aids and curricula developed by the PDs and PIs of this project. Our public in-person outreach has been hampered this year due to COVID-19 restrictions regarding in-person events. Changes/Problems:The laboratories of the PDs and PIs were closed for a period of time due to COVID-19 mandates from our institutions and research ceased for a period of time. We are all now in a restricted research ramp up phase that includes limits on the number of personnel in the lab at a given time. Thus, research is slower than originally planned. As public health measures due to COVID-19 restricted travel and cancelled in-person events it became necessary to change the plans for the economic outreach component of this project as it originally relied on in-person meetings to gather data for the economic analysis and subsequent outreach and extension efforts. In order to continue to move forward with this part of the project, the economic outreach team chose to implement an online questionnaire to gather data from growers and other industry stakeholders even though this method of data collection is less productive than in-person meetings. Once travel restrictions and in-person events resume, the economics team will work to conduct in-person clicker-oriented survey presentations to gather additional data from growers and others to inform the economic assessment of alternative strategies to combat and manage Asian Citrus Psyllid and HLB. What opportunities for training and professional development has the project provided?Training activities: The PDs and PIs of this project have trained one senior scientist, six graduate students and three postdoctoral scholars in microbial isolations from plants and soils, natural product isolations, microbial inhibition assays. We have also trained three graduate students and one postdoctoral scholar in field sampling protocols and they all participated in the field expedition portion of the project. We have also trained two undergraduates in fungal fermentation, organic extraction and chromatographic isolation. Two undergraduates were trained in mining bacterial genomes for antimicrobial natural product production using antiSMASH. Two more senior undergraduates continued their training and helped in the mentoring of the junior students. Several members of the CDRE project have been trained on how to construct Illumina libraries and perform the subsequent bioinformatic analysis. Members of the project are also being trained in liquid chromatography-mass spectrometry and global metabolite network analyses. Professional Development: Several Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, Marine Natural Products Gordon Conference, Tiny Earth Symposia as well as grower stakeholder meetings and stakeholder meetings (listed in Other Products). How have the results been disseminated to communities of interest?The Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society Plant Health online as well as grower stakeholder meetings, such as UCR Citrus Day, Citrus Research Board meetings. PI Maloney gave a guest lecture in a local high school biology class. Due to COVID-19 restrictions that started during this reporting period, much of our interactions with the communities of interest have been virtual. What do you plan to do during the next reporting period to accomplish the goals?What do you plan to do during the next reporting period to accomplish the goals? Objective 1: Optimizing formulations of chemical bactericides to facilitate phloem mobility. Phloem studies. We have begun transformation of Carrizo citrange, a common rootstock, with three separate versions of anchored, phloem-restricted antibodies against CLas. These trees, used as rootstocks or whole trees, will be tested for resistance by the Mandadi lab in Texas. CLas will be introduced in these studies by the Asian Citrus Psyllid, Diaphorina citri. Our structural analysis of young phloem tissue is nearing completion and will be published. We will conduct a RNA-seq analysis of young citrus shoot issue at early time points to identify early response genes in our callose synthase project. Promoters of these genes will be used to trigger callose synthesis in infected tissue. Optimizing nanoparticles chemistry for transit in phloem. Because the gum arabic coated NPs performed the best in planta in terms of translocation into above ground and below ground plant tissues, we will initiate greenhouse assays to test curative and prophylactic use of the Gum arabic-AgNPs in citrus trees that will be infected with CLas. Objective 2. Harnessing the power of bactericides produced by citrus-inhabiting microbes. Untargeted metabolite analysis. The key compounds identified in the analysis of spatial distributions in the field and 3D mapping experiments will be tested in planta to determine their effect in prophylactic (administration of the compounds to uninfected plants followed by the infection with hot psyllids) or curative (administration of the compounds to infected plants) applications and will be evaluated in this next year in Fort Pierce, FL as well as Riverside, CA in the Biosafety level 3 plant high containment facility. We will be testing a citrus flavonoid mixture and tangeritin, a citrus flavonoid involved in plant defense. We have also identified ferulic acid, an intermediate metabolite in flavonoid biosynthesis, is putatively toxic to CLas. This has been demonstrated in an in silico model and inhibits the surrogate, L. crescens in vitro. Targeted metabolite analysis. In the next reporting period, we will continue growing and extracting bacterial and fungal strains isolated from HLB-infected citrus trees, and will begin the purification and chemical identification of antibacterial molecules from these extracts. Objective 3. Testing bactericides for phloem mobility and as HLB therapies. Nanoparticle studies. We will initiate greenhouse assays to test curative and prophylactic use of the Gum Arabic-AgNPs in citrus trees that will be graft-inoculated with CLas. Natural product bactericide studies. This work encompasses discoveries both from the untargeted metabolite studies and targeted metabolite studies. For the targeted metabolite studies, any microbially-derived purified anti-CLas molecules for which we obtain initial high yields, will go immediately into our pipeline to test both prophylactic and curative efficacy against CLas in citrus trees in Fort Pierce, FL or the UCR Biosafety level 3 plant facility. If the yields are low, we will need to optimize growth conditions to increase yields of these compounds. We will initially test the natural products, radicinin, which is produced in high yield from several fungi in our collection and is strongly inhibitory to L. crescens. For the untargeted studies, we have identified several steps of specific flavonoid biosynthesis that appear to be blocked in HLB-infected trees and are currently assessing if we can alleviate this blockage through interference with this disruption of flavonoid biosynthesis in planta. We will also test prophylactic and curative applications of ferulic acid, tangeritin, a citrus flavonoid involved in plant defense and a mixture of commercially available flavonoids. Objective 4. Integration of Research and Extension. In the proposal, we indicated that our economic analysis would begin in year 4 of the project. We have just completed year 4 of this project and although the public health restrictions imposed by the COVID-19 pandemic limited our economic outreach in 2020, we anticipate new opportunities in 2021 to further engage growers and other industry stakeholders through in-person meetings so we may gather data to use in an economic assessment of management strategies to combat the Asian Citrus Psyllid and HLB. We will continue our other robust extension and outreach programs we have described above.

Impacts
What was accomplished under these goals? Objective 1. Optimizing formulations of chemical bactericides to facilitate phloem mobility. Phloem studies. Since the detrimental effects of CLas infection are due to bacterial migration, we are pursuing a strategy to arrest CLas movement, thus protecting either rootstocks or the entire tree. The principle is to express single-chain (scFv) antibodies against surface proteins of CLas as extensions of an abundant, native (SEOR) protein, in sieve elements. The SEOR proteins serve as anchors. Natural immune mechanisms kill the immobilized bacteria. Because sieve tubes are highly redundant there is no reduction in food transport capacity. ScFv antibody sequences were provided by the Hartung lab (USDA). In collaboration with Dr. Kranthi Mandadi at Texas A&M University, using a novel "hairy root" system, we have now shown that these anchored, nontoxic antibodies provide 100% protection against movement of CLas (PCR analysis), thus rendering the tissue fully immune from the disease. In principle this strategy can be used to protect rootstocks or whole trees. Rootstock protection is important since root damage is largely responsible for tree decline, and the fruit from such grafted trees will be nontransgenic. We are currently producing transgenic Citrus X Poncirus trifoliata (Carrizo citrange) a common rootstock variety, which the Mandadi lab will test by infection with Diaphorina citri. In related tests in tobacco we have shown that another form of anchored single-chain antibodies (camelid nanobodies) are functional in sieve tubes. As a second strategy we have shown that sieve element pores in the end walls can be closed by overexpression of callose synthase. The concept is to use the promoter of an early response gene to selectively and rapidly close the pores in response to infection, thus arresting the movement of CLas. We have also conducted electron microscopy studies on developing and mature sieve elements of Citrus sinensis. This work is in preparation for publication. The purpose of these studies is to better understand the ultrastructure of citrus phloem at the point of infection in the shoot apex and young leaves. Optimizing nanoparticles chemistry for transit in phloem. Our results demonstrate that trunk injection can efficiently deliver silver (Ag) nanoparticles (NPs) into trees, and NPs with polyvinylpyrrolidone (PVP) or gum Arabic (GA) coatings can move systemically both acropetally and basipetally through the tree's vascular system. We are now set up to test if these NP formulations can suppress CLas infection in planta in the UCR BSL3 plant facility. Objective 2. Harnessing the power of bactericides produced by citrus-inhabiting microbes. Untargeted metabolite analysis: We have collected, processed and analyzed the data for the third year of field samples collected from the orchards trees. The full dataset for all three years of the field study has been reprocessed to include the latest samples to align it with our publication (In preparation) on the spatial and temporal microbiome field study. We determined spatial co-distributions of plant host metabolites and these chemical distributions were consistent between branch-scale mapping and single leaf mapping experiments. We found that feruloylputrescine correlates with HLB symptomology and we speculate that disrupting its biosynthesis may inhibit symptom manifestation. Our data also show distinct disruptions in the biosynthetic pathways of specific flavonoids. We are testing if application of the metabolites (ferulic acid) in the disrupted flavonoid synthesis can prevent manifestation of disease symptoms. Targeted metabolite analysis: We isolated bacteria and fungi from the same field collected citrus trees described for the untargeted metabolite analyses. During the last reporting period we had 200 bacterial and fungal isolates. We now have over 1,600 bacterial and fungal isolates from CA and FL. We screened the supernatants of these isolates for activity against Liberibacter crescens using a disc diffusion assay. During the last reporting period, we had seven bacterial isolates fungal isolates that produced compounds inhibitory to L. crescens. We now have twenty-three isolates that are inhibitory to CLas. We grew cultures of these microbes and sent these to PI Maloney's laboratory. We fractionated extracts from culture supernatants of these isolates and several of these were published in Applied and Environmental Microbiology. We are currently working towards isolating more compounds using flash column chromatography HPLC and assaying the fractions against L. crescens. We have also synthesized a derivative of one of those compounds (deoxyradicinin) that is a strong inhibitor of CLas and this work has been published in the Journal of Natural Products. Objective 3. Testing bactericides for phloem mobility and as HLB therapies. During the last reporting period we detailed how we tracked nanoparticle mobility in planta using AgNPs because there is little to no background silver content in plant tissue so we began our phloem mobility studies using silver nanoparticles (AgNPs) delivered to 2 year-old citrus trees via trunk injection. Polyvinylpyrrolidone (PVP) or gum Arabic (GA) coated AgNPs transport within the plants, but the GA-modified NPs had the best transport performance with the highest content of Ag in leaves and roots distal from the point of injection. We have propagated the plants and the Asian Citrus Psyllid colony in the UCR Biosafety level 3 facility and will soon test GA-AgNPs for their ability to suppress CLas in citrus. Objective 4. Integration of Research and Extension. The goal of this objective is to communicate our research to the stakeholder community, the greater academic community and the general public. During this report period, we have accomplished this by participating in the Extension events listed in the Other Products (Activities and Events). In addition, we included HLB brochures in 1,404 budwood shipments to 1,050 Citrus Clonal Protection Program Budwood users. The Economic analysis for this project began in year 4 with PI Kaplan conducting semi-structured interviews and developing a survey questionnaire on managing ACP/HLB and distributing it among California growers and other industry stakeholders. Due to COVID-19 restrictions our original plan to conduct in-person clicker survey presentations at various citrus events was altered, leading to distribution of the questionnaire through online/web-based outlets such as the Citrus Research Board, UCANR Tropics in Subtropics, CPDPC's Citrus Insider, and several online citrus industry websites. In addition, the Economics team developed a proof-of-concept mathematical programming model of citrus production and ACP/HLB dynamics in California to assess management strategies that will be used to provide growers with information they can use to best manage their groves from the threat of HLB.

Publications

Type: Journal Articles Status: Published Year Published: 2020 Citation: Blacutt, A., Ginnan, N., Dang, T., Bodaghi, S., Vidalakis, G., Ruegger, P., Peacock, B., Viravathana, P., Campos Vieira, F., Drozd, C., Jablonska, B., Borneman, J., McCollum, G., Cordoza, J., Meloch, J., Berry,V., Lozano Salazar, L., Maloney, K.N., Rolshausen, P.E. and M. Caroline Roper. 2020. An in vitro pipeline to screen and select citrus associated microbiota with potential anti-Candidatus Liberibacter asiaticus properties, Applied and Environmental Microbiology, doi:10.1128/AEM.02883-19
Type: Journal Articles Status: Published Year Published: 2020 Citation: Ginnan, N.A, Dang, T., Bodaghi,S., Ruegger,P.M., McCollum, G., England, G., Vidalakis, G., Borneman, J., Rolshausen, P.E., and M. Caroline Roper*. 2020. Disease-induced microbial shifts in citrus indicate microbiome-derived responses to Huanglongbing across the disease severity spectrum, Phytobiomes Journal, 4(4), 375387. doi:10.1094/pbiomes-04-20-0027-r
Type: Journal Articles Status: Published Year Published: 2020 Citation: Brandenburg, C.A., Castro, C.A, Blacutt, A.A., Costa, E.A., Brinton, K.C., Corral, D.W., Drozd, C.L., Roper, M.C., Rolshausen, P.E., Maloney, K.N. and Jonathan W. Lockner. 2020. Synthesis of Deoxyradicinin, an inhibitor of Xylella fastidiosa and Liberibacter crescens, a culturable surrogate for Candidatus Liberibacter asiaticus, Journal of Natural Products, 83 (6), 1810-1816
Type: Journal Articles Status: Published Year Published: 2021 Citation: Michelle A. Schorn, Stefan Verhoeven, Lars Ridder, Florian Huber, Deepa D. Acharya, Alexander A. Aksenov, Gajender Aleti, Jamshid Amiri Moghaddam, Allegra T. Aron, Saefuddin Aziz, Anelize Bauermeister, Katherine D. Bauman, Martin Baunach, Christine Beemelmanns, J. Michael Beman, Mar�a Victoria Berlanga-Clavero, Alex A. Blacutt, Helge B. Bode, Anne Boullie, Asker Brejnrod, Tim S. Bugni, Alexandra Calteau, Liu Cao, Victor J. Carrion, Raquel Castelo-Branco, Shaurya Chanana, Alexander B. Chase, Marc G. Chevrette, Leticia V. Costa-Lotufo, Jason M. Crawford, Cameron R. Currie, Bart Cuypers, Tam Dang, Tristan de Rond, Alyssa M. Demko, Elke Dittmann, Chao Du, Christopher Drozd, Jean-Claude Dujardin, Rachel J Dutton, Anna Edlund, David P. Fewer, Neha Garg, Julia M. Gauglitz, Emily C. Gentry, Lena Gerwick, Evgenia Glukhov, Harald Gross, Muriel Gugger, Dulce G. Guill�n Matus, Eric J. N. Helfrich, Benjamin-Florian Hempel, Jae-Seoun Hur, Marianna Iorio, Paul R. Jensen, Kyo Bin Kang, Leonard Kaysser, Neil L. Kelleher, Chung Sub Kim, Ki Hyun Kim, Irina Koester, Gabriele M. K�nig, Tiago Leao, Seoung Rak Lee, Yi-Yuan Lee, Xuanji Li, Jessica C. Little, Willam W. Metcalf, Katherine N. Maloney, Daniel M�nnle, Christian Martin H., Andrew C. McAvoy, Hosein Mohimani, Carlos Molina Santiago, Bradley S. Moore, Michael W. Mullowney, Mitchell Muskat, Louis Felix Nothias, Ellis C. O'Neill, Elizabeth I. Parkinson, Daniel Petras, J�rn Piel, Emily C. Pierce, Karine Pires, Raphael Reher, Diego Romero, M. Caroline Roper, Michael Rust, Hamada Saad, Carmen Saenz, Laura M. Sanchez, S�ren Johannes S�rensen , Margherita Sosio, Roderich D. Suessmuth, Doug Sweeney, Kapil Tahlan, Regan J. Thomson, Nicholas J. Tobias Amaro E. Trindade-Silva, Gilles P. van Wezel, Mingxun Wang, Kelly C. Weldon, Fan Zhang, Nadine Ziemert, Katherine R. Duncan, Max Cr�semann, Simon Rogers, Pieter C. Dorrestein, Marnix H. Medema, Justin J. J. van der Hooft. 2021. A community resource for paired genomic and metabolomic data mining. Nature Chemical Biology, https://doi.org/10.1038/s41589-020-00724-z.

Progress 01/15/19 to 01/14/20

Outputs
Target Audience:Our target audiences are grower stakeholders, academic and industry scientists, undergraduate and graduate students and the general public. To reach these audiences, we have presented our work at national and international professional science meetings that are attended by scientists, citrus growers and agricultural industry representatives. We have input the specific locations and titles of these meetings in the Other Products section in the report. Our two Extension Specialists have disseminated information to target stakeholder audiences through our project website, presentations at stakeholder meetings and through semi-technical stakeholder publications that are listed in the Other Products section of this report. We have also disseminated information about Huanglongbing (HLB), its implications to the citrus industry and specifics of this project through our project website, educational aids and curricula developed by the PDs and PIs of this project. In addition, we have engaged in public outreach at local citrus events held at the Riverside, CA Citrus State Historic Park where we inform the general public of HLB, HLB resources available to them and general information about our project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The Principal Directors and Investigators of this project have trained two senior scientists, six graduate students and three postdoctoral scholars in microbial isolations from plants and soils, natural product isolations and microbial inhibition assays. We have also trained three graduate students and one postdoctoral scholar in field sampling protocols and they all participated in the field expedition portion of the project. We have also trained two undergraduates in fungal fermentation, organic extraction and chromatographic isolation. Two more senior undergraduates continued their training and helped in the mentoring of the junior students. Several members of this CDRE project have been trained on how to construct Illumina libraries and perform the subsequent bioinformatic analysis. Members of the project are also being trained in liquid chromatography-mass spectrometry and global metabolite network analyses. Professional Development: Several Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society and American Chemical Society as well as grower stakeholder meetings and stakeholder meetings (listed in Other Products). How have the results been disseminated to communities of interest?The Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society, the International Society of Plant Microbe Interactions and the American Chemical Society as well as grower stakeholder meetings and stakeholder meetings, such as International HLB conference (co-organized by PI Vidalakis), UCR Citrus Day, Citrus Research Board meetings California Citrus conference, California Citrus Nursery Society Annual Conference and the Lindcove Fruit Display. We have also had press coverage of our project in several news outlets. 1. Co-PI Maloney and Advisory Board member Ben McLean III were featured in the cover story of the June 10 issue of Chemical Engineering News (widely read chemistry news magazine published by the American Chemical Society). 2. Zhang, C. "Citrus greening is killing the world's orange trees. Scientists are racing to help." 2019 Chemical & Engineering News, 97 (23): 30. https://cen.acs.org/biological-chemistry/biochemistry/Citrus-greening-killing-worlds-orange/97/i23. 3. Student Connor Brandenburg's poster was selected by the American Chemical Society's press office for featuring in press release that was picked up by a few platforms, including EurekAlert! ScienceDaily and (e) Science News: "Juice plant pathogen could be treated with newly identified antibacterial agent" EurekAlert!, published online April 1, 2019 https://www.eurekalert.org/pub_releases/2019-04/acs-jpp022019.php. 4. Co-PI Maloney and undergraduate student Connor Brandenburg spoke about the work in a press conference at the American Chemical Society National Meeting in Orlando, April 2, 2019: https://www.youtube.com/watch?v=tUSH1DC7aeU&list=PLLG7h7fPoH8LNYjJHshZ3TPkijXaSsvLg&index=8 What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Optimizing formulations of chemical bactericides to facilitate phloem mobility. Phloem studies. We will continue our "seize and capture" studies with emphasis on the nanobody method of arresting movement of CLas. Our structural analysis of young phloem tissue is nearing completion and will be published. We will conduct an RNAseq analysis of young citrus shoot issue at early time points to identify early response genes in our callose synthase project. Promoters of these genes will be used to trigger callose synthesis in infected tissue. Optimizing nanoparticles chemistry for transit in phloem. Because the gum arabic coated NPs performed the best in planta in terms of translocation into above ground and below ground plant tissues, we will initiate greenhouse assays to test curative and prophylactic use of the GA-AgNPs in citrus trees that will be infected with CLas. Objective 2. Harnessing the power of bactericides produced by citrus-inhabiting microbes. Untargeted metabolite analysis. The key compounds identified in the analysis of spatial distributions in the field and 3D mapping experiments will be now tested in planta to determine their effect in prophylactic (administration of the compounds to uninfected plants followed by the infection with hot psyllids) or curative (administration of the compounds to infected plants) applications and will be evaluated in this next year in Fort Pierce, FL as well as Riverside, CA in the BSL3p high containment facility. We will be testing a citrus flavonoid mixture and tangeritin, a citrus flavonoid involved in plant defense. Targeted metabolite analysis. In the next reporting period, we will continue growing and extracting bacterial and fungal strains isolated from HLB-infected citrus trees, and will begin the purification and chemical identification of antibacterial molecules from these extracts. Objective 3. Testing bactericides for phloem mobility and as HLB therapies. Nanoparticle studies. We will initiate greenhouse assays to test curative and prophylactic use of the GA-AgNPs in citrus trees that will be graft-inoculated with CLas. Natural product bactericide studies. This work encompasses discoveries both from the untargeted metabolite studies and targeted metabolite studies. For the targeted metabolite studies, any microbially-derived purified anti-CLas molecules for which we obtain initial high yields, will go immediately into our pipeline to test both prophylactic and curative efficacy against CLas in citrus trees. If the yields are low, we will need to optimize growth conditions to increase yields of these compounds. We will initially test the natural products, radicinin, which is produced in high yield from several fungi in our collection and is strongly inhibitory to L. crescens. For the untargeted studies, we have identified several steps of specific flavonoid biosynthesis that appear to be blocked in HLB-infected trees and are currently assessing if we can alleviate this blockage through interference with this disruption of flavonoid biosynthesis in planta. We will also test prophylactic and curative applications of tangeritin, a citrus flavonoid involved in plant defense. Objective 4. Integration of Research and Extension. We have just completed year 3 of this project and we have begun the economic outreach. We will continue our other robust extension and outreach programs we have described above.

Impacts
What was accomplished under these goals? Objective 1. Optimizing formulations of chemical bactericides to facilitate phloem mobility. Phloem studies. We conducted electron microscopy studies on developing and mature sieve elements of Citrus sinensis. This work is in preparation for publication. The purpose of these studies is to better understand the ultrastructure of citrus phloem at the point of infection in the shoot apex and young leaves. We are also pursuing a "seize and capture" strategy to arrest the movement of CLas in sieve tubes at the point of infection. We have produced transgenic tobacco plants that express single-chain (scFv) antibodies against surface antigens of CLas. The antibody sequences were provided by the Hartung lab (USDA, ARS). These antibodies are expressed as extensions of the immobile sieve element occlusion related (SEOR) proteins in sieve elements, which serve as anchors. In collaboration with Dr. Kranthi Mandadi at Texas A&M University at Westlaco we are testing the ability of these antibodies to arrest the movement of CLas in citrus using a novel "hairy root" system that allows for rapid screening of therapies. As a second strategy we have shown that sieve element pores in the end walls can be closed by overexpression of callose synthase. The concept is to use the promoter of an early response gene to selectively and rapidly close the pores in response to infection, thus arresting the movement of CLas. Optimizing nanoparticles chemistry for transit in phloem. We demonstrate that trunk injection can efficiently deliver silver (Ag) nanoparticles (NPs) into trees, and NPs with polyvinylpyrrolidone (PVP) or gum Arabic (GA) coatings can move systemically both acropetally and basipetally through the tree's vascular system. We also demonstrate PVP-, GA-, are negatively charged, resulting in high attractive forces between the NPs and xylem/phloem surface. It is important to take steric interaction into consideration for explaining mobility of NP in plants. Our data suggest that repulsive steric interactions are the main reason for the mobility of NPs in plants. Objective 2. Harnessing the power of bactericides produced by citrus-inhabiting microbes. Untargeted metabolite analysis: We have collected, processed and analyzed the data for the third year of field samples collected from the orchards trees. The full dataset for all three years of the field study has been reprocessed to include the latest samples to align it with our publication (In preparation) on the spatial and temporal microbiome field study. We determined spatial co-distributions of plant host metabolites and these chemical distributions were consistent between branch-scale mapping and single leaf mapping experiments. We found that feruloylputrescine correlates with HLB symptomology and disrupting its biosynthesis may inhibit symptom manifestation. Our data also show distinct disruptions in the biosynthetic pathways of specific flavonoids that we also observed in a separate greenhouse experiment. We are testing if application of the metabolites in the disrupted flavonoid synthesis alleviates subsequent disease symptoms. Targeted metabolite analysis: We isolated bacteria and fungi from the same field collected citrus trees described for the untargeted metabolite analyses. We have over 1300 fungal and bacterial isolates and continue to isolate additional microbes. We screened the supernatants of these isolates for activity against Liberibacter crescens using a disc diffusion assay. Four bacterial isolates and three fungal isolates produce compounds inhibitory to L. crescens. The bacteria include three different Bacillus spp., and one Pantoea sp., The fungi include two Cladosporium spp, and one Epicoccum sp. We grew cultures of Epicoccum sp. and Cladosporium sp. and sent these to PI Maloney's laboratory. We fractionated extracts from large-scale cultures of Epicoccum nigrum and Cladosporium sp. using flash column chromatography and assayed the fractions against L. crescens. We identified the major compounds in the active fractions as epicoccamide A (E. nigrum) and cladosporols A, C, and D (Cladosporium sp.). We purified these compounds by HPLC and tested them in the L. crescens bioassay and found that epicoccamide A did not inhibit L. crescens, suggesting that a minor component in the active extract must be responsible for activity of E. nigrum. Cladosporols A, C, and D were found to inhibit L. crescens in a dose-dependent fashion. This work has been published. We also grew 1L cultures of several bacterial strains isolated from asymptomatic citrus trees in orchards under high HLB pressure and prepared organic extracts for bioassay against L. crescens. We have also synthesized a derivative of one of those compounds (deoxyradicinin) that is a strong inhibitor of CLas and this work has been submitted for publication. Objective 3. Testing bactericides for phloem mobility and as HLB therapies. There is little to no background silver content in plant tissue so we began our phloem mobility studies using silver nanoparticles (AgNPs) delivered to 2 year-old citrus trees via trunk injection. 1000 ppm PVP-, GA-, and Ct-AgNPs suspension were injected into citrus trees at the base of trunk (above the graft union), and after 1, 3, and 7 days leaf samples were collected for Ag content analysis. All three types of AgNPs transport within the plants, but the GA-modified NPs had the best transport performance with the highest content of Ag in leaves and roots distal from the point of injection. Our results showed that 20-50% of total Ag injected was recovered among these trees regardless of the coating type and of that 20-50% of the AgNPs we recovered, over 99% of the Ct-AgNPs, 68% of the PVP-AgNPs, and 66% of the GA-AgNPs remained in the trunk. This suggests that different size and surface modification impact the transport performance of NPs in plants and that the smaller GA-coated particles transport more readily in 2 year-old citrus trees. Because GA-AgNPs showed higher transport capability in plants than Ct- and PVP-AgNPs at a high concentration (1000 ppm), we assessed transport of lower GA-AgNPs concentrations on NP transport in plants. Increasing AgNPs concentration from 10 ppm to 100 ppm did not lead to a significant increase of Ag content in leaves. 10 ppm AgNPs injection application had better NP transport performance than the 100 ppm AgNP injection. This implies that at high injecting concentrations, NPs tend to aggregate which further retards their transport. Our initial observations indicate the xylem is the main mode of transport for NPs. We confirmed the mobility of NPs via measuring total Ag mass in plant tissues, and identifying NPs in tissues with transmission electron microscopy (TEM) and hyperspectral imaging technology. However, the dissolution rate of the NPs in trees is still unclear. UV-Vis spectroscopy plus ICP-MS enabled us to estimate the change of NP concentration. The ICP-MS analysis found that after 7 days in synthetic sap ionic Ag (dissolved) accounted for less than 1% of total Ag. Because the anti-microbial properties of AgNPs are partly associated with their dissolution (as silver ions are responsible for the anti-microbial activity), the slow dissolution of AgNPs in sap may reduce their antimicrobial performance, although it is unknown what concentration of silver ions is actually needed to induce a satisfactory anti-microbial response. Objective 4. Integration of Research and Extension. The goal of this objective is to communicate our research to the stakeholder community, the greater academic community and the general public. During this report period, we have accomplished this by participating in the Extension events listed in the Other Products. In addition, we included HLB brochures in over 3,500 budwood shipments to Citrus Clonal Protection Program Budwood users. The Economic analysis (PI Kaplan) for this project will begin in year 4.

Publications

Type: Journal Articles Status: Published Year Published: 2019 Citation: Su, Yiming; Ashworth, Vanessa; Geitner, Nicholas; Wiesner, Mark; Ginnan, Nichole; Rolshausen Philippe; Roper, Caroline ; Jassby, David. Delivery, Fate, and Transport of Silver Nanoparticles in Citrus Trees. 2019. Environ. Sci.: Nano,6, 2311-2331
Type: Journal Articles Status: Published Year Published: 2019 Citation: Su, Y., Ashworth, V., Kim, C., Adeleye, A. S., Rolshausen, P., Roper, C., White, J. and Jassby, D. (2019). Delivery, uptake, fate, and transport of engineered nanoparticles in plants: a critical review and data analysis. Environmental Science: Nano, 6(8), 23112331. doi:10.1039/c9en00461k
Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Brandenburg, C.; Castro, C.; Blacutt, A.; Costa, E.; Brinton, .; Corral, D.; Roper, C.; Rolshausen, P.; Maloney, K.; Lockner, J. Synthesis of deoxyradicinin, an inhibitor of Xylella fastidiosa and Liberibacter crescens, a culturable surrogate for Candidatus Liberibacter asiaticus. Under revision for J. Nat. Prod.
Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Ginnan, N., Dang, T., Bodaghi, S., Ruegger, P.M., MCollum, G., England, G., Vidalakis, G., Borneman, J., Rolshausen, P.E., and Roper, M.C. 2020. Disease-induced microbial shifts in citrus indicate microbiome-derived responses to Huanglongbing across the disease severity spectrum, In preparation
Type: Journal Articles Status: Published Year Published: 2019 Citation: Rolshausen P.E. 2019. Harnessing the plant microbiome for commercial applications. Open Access Government. April 362-363.
Type: Journal Articles Status: Published Year Published: 2020 Citation: Blacutt, A., Ginnan, N., Dang, T., Bodaghi, S., Vidalakis, G., Ruegger, P., Peacock, B., Viravathana, P., Campos Vieira, F., Drozd, C., Jablonska, B., Borneman, J., McCollum, G., Cordoza, J., Meloch, J., Berry,V., Lozano Salazar, L., Maloney, K.N., Rolshausen, P.E. and M. Caroline Roper*. 2020. An in vitro pipeline to screen and select citrus associated microbiota with potential anti-Candidatus Liberibacter asiaticus properties, Applied and Environmental Microbiology, 86 (8), doi:10.1128/AEM.02883-19

Progress 01/15/18 to 01/14/19

Outputs
Target Audience:Our target audiences are grower stakeholders, academic and industry scientists, undergraduate and graduate students and the general public. To reach these audiences, we have presented our work at national and international professional science meetings that are attended by scientists, citrus growers and agricultural industry representatives. We have input the specific locations and titles of these meetings in the Other Products (Activities and Events) section in the report. Our two Extension Specialists have disseminated information to target stakeholder audiences through our project website, presentations at stakeholder meetings and through semi-technical stakeholder publications that are listed in the Other Products (Events and Publications) section of this report. We have also disseminated information about Huanglongbing (HLB), its implications to the citrus industry and specifics of this project through our project website, educational aids and curricula developed by the PDs and PIs of this project. In addition, we have engaged in public outreach at local citrus events held at the Riverside, CA Citrus State Historic Park where we inform the general public of HLB, HLB resources available to them and general information about our project. Changes/Problems:We have not been able to initiate work in the BSL3 greenhouse in Riverside, CA yet because the facility is not yet commissioned. We anticipate that the facility will be commissioned during the next reporting period. What opportunities for training and professional development has the project provided?Training activities: The PDs and PIs of this project have trained six graduate students and three postdoctoral scholars in microbial isolations from plants and soils, natural product isolations and microbial inhibition assays. We have also trained three graduate students and one postdoctoral scholar in field sampling protocols and they all participated field expedition portion of the project. Several members of the CDRE project have been trained on how to construct Illumina libraries and perform the subsequent bioinformatic analysis. Members of the project are also being trained in liquid chromatography-mass spectrometry and global metabolite network analyses. Professional Development: Several Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society as well as grower stakeholder meetings and stakeholder meetings (listed in Other Products). How have the results been disseminated to communities of interest?The Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society as well as grower stakeholder meetings and stakeholder meetings, such as UCR Citrus Day, Citrus Research Board meetings, International HLB conference, California Citrus conference, California Citrus Nursery Society Annual Conference and the Lindcove Fruit Display. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Optimizing formulations of chemical bactericides to facilitate phloem mobility. Phloem studies. We will continue to develop the CLas "seize and capture" method using phloem specific proteins, Sieve Element Occlusion Related (SEOR proteins). To do this, we will continue to optimize our methods for visualization of SEOR proteins in phloem using liquid N2 slush to fix phloem structures in place before sectioning and microscopy. As the project progresses, we will attempt, in collaboration with the Kranthi Mandadi lab at Texas A&M to inhibit disease progression in transgenic citrus roots using the seize and capture technique described above in the Accomplishments section. Optimizing nanoparticles chemistry for transit in phloem. Because the gum arabic coated NPs performed the best in planta in terms of translocation into above ground and below ground plant tissues, we will move forward with the NP transit studies and focusing only on the gum arabic-coating only. We will initiate greenhouse assays to test curative and prophylactic use of the GA-AgNPs in citrus trees that will be graft-inoculated with CLas. In addition, we will begin engineering NPs made from other materials, such as manganese. Objective 2. Harnessing the power of bactericides produced by citrus-inhabiting microbes. Untargeted metabolite analysis. When conducting the field level metabolite study in Florida, we also collected microbiome (bacteria and fungi) data from the same root, stem, leaf and soil samples that we analyzed for the untargeted metabolite analysis study. We have completed the microbiome analyses (publication is in preparation) and are now poised to overlay the microbiome data over the metabolite data to mine the data for tissue specific metabolites of microbial that correlate with trees that have lower rates of disease progression. Targeted metabolite analysis. In the next reporting period, we are focusing our efforts on fractionation of the inhibitory supernatants. We plan to further purify the active molecules using reverse-phase high performance liquid chromatography (HPLC) to isolate and identify the antibacterial molecule(s). From this, we will identify single, purified molecules that retain L. crescens inhibition. We will also continue our culturing and screening efforts using the L. crescens inhibition bioassay to assess if there are additional microbes that are promising candidates for incorporation into our natural product discovery pipeline. Objective 3. Testing bactericides for phloem mobility and as HLB therapies. Nanoparticle studies. We will initiate greenhouse assays to test curative and prophylactic use of the GA-AgNPs in citrus trees that will be graft-inoculated with CLas. In addition, we will begin engineering NPs made from other materials, such as manganese. We will then perform transport studies using these newly engineered NPs. Natural product bactericide studies. This work encompasses discoveries both from the untargeted metabolite studies and targeted metabolite studies. For the targeted metabolite studies, any microbially-derived purified anti-CLas molecules for which we obtain initial high yields, will go immediately into our pipeline to test both prophylactic and curative efficacy against CLas in citrus trees. If the yields are low, we will need to optimize growth conditions to increase yields of these compounds. For the untargeted studies, we have identified several steps of specific flavonoid biosynthesis that appear to be blocked in HLB-infected trees and are currently assessing if we can alleviate this blockage through interference with this disruption of flavonoid biosynthesis in planta. Objective 4. Integration of Research and Extension. In this original proposal, we indicated that our economic analysis would begin in year 3 of the project. Thus, during this reporting period, we will initiate these studies. We will continue cultivating our robust extension and outreach programs we have described above.

Impacts
What was accomplished under these goals? Objective 1. Optimizing formulations of chemical bactericides to facilitate phloem mobility. Phloem studies. We conducted electron microscopy studies on developing and mature sieve elements of Citrus sinensis. Of particular interest are the SEOR (Sieve Element Occlusion Related) protein bodies which disperse in mature sieve cells and are potential impediments to CLas as movement in genetically modified plants. We are developing a "seize and capture" technology to prevent CLas transport in sieve tubes. To do this we made DNA constructs which attach single-chain antibodies to the SEOR proteins. As proof of concept we used a published sequence for a single-chain nanobody raised against GFP and expressed this in tobacco. We transformed tobacco with free GFP expressed in the phloem. When we grafted the two plant types together, the nanobodies trapped free-flowing GFP. Several attempts to generate nanobodies to CLas were unsuccessful, but the Hartung lab (USDA, ARS) published on scFv antibodies (another single-chain type) against CLas and may be as effective as nanobodies. We obtained 8 unique sequences against 3 CLas surface proteins and made SEOR-scFv constructs to be expressed in citrus phloem. Optimizing nanoparticles chemistry for transit in phloem. Food-grade polymers (polyvinylpyrrolidone (PVP), gum Arabic (GA), and citrate (Ct) were used to stabilize Ag nanoparticles (AgNPs). The size of the metallic core of the synthesized PVP-, GA-, and Ct-AgNPs, measured via transmission electron microscopy (TEM), were 17.87±7.45, 9.15±4.21, and 28.67±10.95 nm, while the hydrodynamic (HD) diameter (which accounts for attached organic coatings) of these NPs measured in nanopure water/synthetic phloem sap were 85.1/141.2, 40.5/162.1, and 94.3/170.1 nm, respectively. The HD size successfully stabilized the NPs. The pore size of the porous membrane structures in plants ranges between 160 and 850 nm and thus the AgNPs are small enough to be transported through the vascular tissue. Because surface charge is critical for transport of NPs in environment, we also analyzed surface charge of PVP-, GA-, Ct-AgNPs in synthetic phloem sap. PVP-, GA-, and Ct-AgNPs had charges of -57.1, -51.4, and -67.4 mV, respectively. A Derjaguin-Landau-Verwey-Overbeek model that predicts a constant repulsive force between AgNPs and the xylem or phloem walls indicating deposition of NPs on these surfaces is not energetically favorable which would facilitate transport. Objective 2. Harnessing the power of bactericides produced by citrus-inhabiting microbes. Untargeted metabolite analysis: We performed two controlled greenhouse studies to compare the metabolites in HLB-infected trees to healthy citrus using high resolution spatial metabolite mapping both at the individual leaf level and at individual branch level (6 leaves per branch). We determined spatial co-distributions of plant host metabolites and these chemical distributions were consistent between branch-scale mapping and single leaf mapping experiments. We found that feruloylputrescine correlates with HLB symptomology and we disrupting its biosynthesis may inhibit symptom manifestation. We also conducted a grove level study in Florida. We sampled root, stem, leaf and soil samples from 4 groves from several varieties of citrus. These data show distinct disruptions in the biosynthetic pathways of specific flavonoids that we also observed in the greenhouse experiment. We will test if application of the metabolites in the disrupted flavonoid synthesis alleviates subsequent disease symptoms. Targeted metabolite analysis: We isolated bacteria and fungi from the same field collected citrus trees described for the untargeted metabolite analyses. We have 120 bacterial isolates and 80 fungal isolates. We screened the supernatants of these isolates for activity against Liberibacter crescens using a disc diffusion assay. Four bacterial isolates and three fungal isolates produce compounds inhibitory to L. crescens. The bacteria include three different Bacillus spp., and one Pantoea sp., The fungi include two Cladosporium spp, and one Epicoccum sp. We grew cultures of Epicoccum sp. and Cladosporium sp. and sent these to PI Maloney's laboratory. We fractionated the active extracts by liquid-liquid partitioning, and normal-phase flash column chromatography, testing each round of fractions in our bioassay. The current fractions still contain mixtures of compounds, but initial data indicate that most of the active fractions contain phenolic moieties typical of polyketide natural products. Polyketides are known to have antimicrobial activity. Objective 3. Testing bactericides for phloem mobility and as HLB therapies. There is little to no background silver content in plant tissue so we began our phloem mobility studies using silver nanoparticles (AgNPs) delivered to 2 year-old citrus trees via trunk injection. 1000 ppm PVP-, GA-, and Ct-AgNPs suspension were injected into citrus trees at the base of trunk (above the graft union), and after 1, 3, and 7 days leaf samples were collected for Ag content analysis. All three types of AgNPs transport within the plants, but the GA-modified NPs had the best transport performance with the highest content of Ag in leaves and roots distal from the point of injection. After 6 weeks, all trees were destructively sampled and separated into leaf, branch, trunk, and root samples to determine the relative abundance of AgNPs in all the parts of the tree and if the amount of total AgNPs that were input into the tree remained in the tree after 6 weeks. Plant tissues were combusted, digested with acid and Ag concentration was analyzed using inductively coupled plasma mass spectrometry (ICP-MS). Our results showed that 20-50% of total Ag injected was recovered among these trees regardless of the coating type and of that 20-50% of the AgNPs we recovered, over 99% of the Ct-AgNPs, 68% of the PVP-AgNPs, and 66% of the GA-AgNPs remained in the trunk. This suggests that different size and surface modification impact the transport performance of NPs in plants and that the smaller GA-coated particles transport more readily in 2 year-old citrus trees. Because GA-AgNPs showed higher transport capability in plants than Ct- and PVP-AgNPs at a high concentration (1000 ppm), we assessed transport of lower GA-AgNPs concentrations on NP transport in plants. Accordingly, 10 ml of 10 ppm or 100 ppm AgNPs suspension were injected into citrus trees, and after 1, 3, and 7 days, trees were destructively sampled and separated into leaf, branch, trunk, and root samples. The average Ag recovery rate in plants with 10 ppm and 100 ppm AgNPs injection were 35.35±21.61% and 46.15±20.29%, respectively. Increasing AgNPs concentration from 10 ppm to 100 ppm did not lead to a significant increase of Ag content in leaves. 10 ppm AgNPs injection application had better NP transport performance than the 100 ppm AgNP injection. This implies that at high injecting concentrations, NPs tend to aggregate which further retards their transport. We are currently determining the route these NPs take in planta, but initial observations indicate the xylem is the main mode of transport for NPs. Objective 4. Integration of Research and Extension. The goal of this objective is to communicate our research to the stakeholder community, the greater academic community and the general public. During this report period, we have accomplished this by participating in the Extension events listed in the Other Products (Activities and Events). In addition, we included HLB brochures in 706 budwood shipments to Citrus Clonal Protection Program Budwood users. The Economic analysis (PI Kaplan) for this project will begin in year 3.

Publications

Type: Journal Articles Status: Published Year Published: 2018 Citation: Rolshausen, P.E., Dang, T., Bodaghi, S., Ginnan, N., Ruegger, P., Peacock, B., Roper, C., Borneman, J., McCollum, G., Vidalakis, G., and England, G.K. 2018. Correlating citrus tree health with microbes. Citrograph 9(4):52-56.
Type: Journal Articles Status: Published Year Published: 2018 Citation: Rolshausen P.E. 2018. Biological Pesticides and the Future of Sustainable Agriculture. Open Access Government May:332-333.
Type: Journal Articles Status: Published Year Published: 2018 Citation: Rolshausen P.E. 2018. Utilizing the plant microbiome in agriculture. Scientia 117:80-83
Type: Journal Articles Status: Published Year Published: 2018 Citation: Ginnan, N. A., Dang, T., Bodaghi, S., Ruegger, P.M., Peacock, B. B., McCollum, G., England, G., Vidalakis, G., Roper, M.C*., Rolshausen. P. and Borneman, J. 2018. Bacterial and Fungal Next Generation Sequencing Datasets and Metadata from Citrus Infected with Candidatus Liberibacter asiaticus, Phytobiomes, https://doi.org/10.1094/PBIOMES-08-17-0032-A
Type: Journal Articles Status: Published Year Published: 2018 Citation: Yin, X., Kelly, K., Maharaj, N., Rolshausen, P.E., and Leveau, J. 2018. A microbiota-based approach to citrus tree health. Citrograph 9(4):58-63.

Progress 01/15/17 to 01/14/18

Outputs
Target Audience:The target audience for this work are scientists, grower stakeholders, students and the general public. To reach these audiences, we have presented our work at national and international professional science meetings that are attended by scientists, citrus growers and agricultural industry representatives. These meetings are specifically described in the appropriate sections throughout the report. We have also disseminated information to target audiences through peer-reviewed scientific publications, and semi-technical stakeholders publications that are listed in the appropriate sections of this report. We have also disseminated information about Huanglongbing, its implications to the citrus industry and specifics of this project through a website, educational aids and curricula developed by the PDs and PIs of this project. Changes/Problems:One problem that we have encountered is that since the initiation of this project in the UC-Riverside campus are now under HLB quarantine regulations. This means that nurseries shipping to the quarantine must comply with federal and state regulations prior to shipment of citrus plant material to UCR. Because of this, we have experienced a slight delay in the receipt of the latest order of citrus trees. This next shipment is expected in the second week of January 2018. What opportunities for training and professional development has the project provided?Training activities: This project has trained several members of the CDRE project in microbial isolations from plants and soils, natural product isolations and microbial inhibition assays. In addition, PD Rolshausen has trained several members of the project in field sampling protocols and led the field expedition portion of the project. Several members of the CDRE project have been trained on how to construct Illumina libraries and perform the subsequent bioinformatic analysis. Members of the project are also being trained in liquid chromatography-mass spectrometry and global metabolite network analyses. Professional Development: Several Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society as well as grower stakeholder meetings and stakeholder meetings (listed in Other Products). How have the results been disseminated to communities of interest?The Principal Directors and Investigators, students and postdoctoral scholars have attended and presented at professional meetings, such as the American Phytopathological Society as well as grower stakeholder meetings and stakeholder meetings, such as UCR Citrus Day, Citrus Research Board meetings, International HLB conference, California Citrus conference, California Citrus Nursery Society Annual Conference and the Lindcove Fruit Display. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Optimizing formulations of chemical bactericides to facilitate phloem mobility: Our initial results indicate that the method works well; we can detect 14C radiolabel in the stem and roots. We are beginning more comprehensive studies on transport patterns. These experiments also provide baselines for assessing the deleterious effects of bacterial infection on transport. We will begin transport studies on CLas infected trees during the next reporting period. Objective 2: Harnessing the power of bactericides produced by citrus-inhabiting microbes: Microbial community analyses: We plan to continue examining survivor trees in Florida. The key element in this research is to examine these trees over time. By following trees over time, we will be able to determine which trees are able to maintain a survivor status over several years. By characterizing the bacterial and fungal communities in survivor trees, we anticipate that we will be able to identify individual microbes and/or consortia of microbes that inhibit the progress of HLB. These organisms will then be isolated in axenic culture, enabling them to be put into the bactericide discovery pipeline. These organisms also represent putative biological control organisms. Untargeted metabolite analysis: Untargeted mass spectrometric analysis data are currently being analyzed with molecular networking to propose chemical annotations of the compounds of interest that discriminate the survivor status. Introduction of the data from cultures of microbes into molecular networks will be conducted to establish whether any of the metabolites of interest are of microbial origin. Correlations of metabolites with microbes that are found to be discriminant of the survivor status will also be conducted. We are also establishing spatial locations of microbes throughout parts of the plant (roots, budwood or leaves) and their associated metabolic chemistries. The work on the mapping of chemical and microbial features will take place in the next reporting period. The detailed plan for the experimental design, including all of the necessary logistics have been outlined during our USDA/NIFA project group meeting on November 13-14, 2017. Targeted metabolite analyses: We will continue screening our citrus isolates in our Citrus Isolation Repository for inhibition of L. crescens. We will also continue isolating microbes from citrus to add to our repository. Using the microbes that display inhibitory activity, we will continue identifying purified molecules that retain the inhibitory activity. Objective 3: Testing bactericides for phloem mobility and as HLB therapies: We will develop a more reliable NP injection method that will enable the effective introduction of NPs into the tree using the pneumatic tree injector. We will track the movement of NPs throughout the tree by measuring NP (initially AgNPs) concentrations in different parts of the tree as well as with microscopy. We will also determine transport of NPs and whether they transport primarily in the xylem and/or phloem. We will begin assessing NPs that transit well in citrus for their efficacy against CLas infection and HLB development in Florida greenhouse experiments. Objective 4: Integration of Research and Extension: We will continue transferring new knowledge learned from our project to stakeholders through the mechanisms described under Accomplishments for Obj. 4. We will use this communication to receive feedback from stakeholders and incorporate this feedback into our experimental designs as the project evolves.

Impacts
What was accomplished under these goals? Objective 1: The initial studies focus on mapping phloem transport in healthy trees. Of particular importance is determining how effectively bactericides introduced to one branch travel transversely to other sectors of the tree. Analysis of photoassimilate transport patterns provides the foundation for understanding bactericide movement in uninfected and infected trees. To defeat the bacteria, either by application of bactericides or by other means, we need to understand the transport patterns the bacteria take. One possibility is that bactericide transport is limited to single branches and the connected phloem in the stem and roots (sectoring). Another possibility is that bactericides are able to move from one branch to another. If this is true it will greatly simplify treatment strategies. We have followed the pattern of photoassimilate transport in healthy citrus trees using fluorescent dyes and 14C-tracer methodologies. The fluorescent dye approach has been disappointing due to an unexplained inefficiency in delivering the dye to the phloem. Rather than waste time trying to understand the basis of this problem we have switched to the 14C method in which single leaves are exposed to 14CO2 and radiolabel is extracted from tissues on the same and different branches at different time intervals. Initial results indicate that the method works well; we can detect radiolabel in the stem and roots. We are beginning more comprehensive studies on transport patterns. Fabricating and characterizing coated and uncoated nanoparticles (NPs): We have synthesized and/or purchased silver (AgNPs), copper (CuNPs), and sulfur (SNPs) NPs, coated them with three different food-grade polymers (polyvinylpyrrolidone (PVP), gum Arabic (GA), and citrate) and characterized the particles in terms of size, stability in water and artificial phloem sap, and surface charge. We determined that the NP coatings are effective at controlling the stability (i.e., prevent NP aggregation) in both water and artificial phloem sap. This is critically important considering that the transport of the NPs throughout the tree is dependent on the size of the particles, with larger particles more likely to get trapped in the intricate vascular structures of the tree. PVP and GA were particularly effective at maintaining NP stability with the smallest hydrodynamic diameter. The dissolution rate of AgNPs and CuNPs was evaluated in the synthetic phloem sap. AgNPs exhibited a slow dissolution rate. In contrast, CuNPs showed a faster dissolution rate. Since AgNPs dissolve slowly, it may be necessary to introduce more NPs to achieve the minimum inhibitory NP loading in a tree. However, once loading is achieved, the dissolved ion concentrations can be maintained over long periods of time. Using Liberibacter crescens, a culturable relative of CLas, we have set up two inhibition bioassays: 1) a solid plate bioassay that tests for bactericidal activity by quantifying zones of inhibition of growth around discs containing putative antimicrobials being tested 2) a 96-well plate assay that tests for bactericidal activity in liquid medium. Using the solid plate bioassay, we have demonstrated both SNPs and AgNPs inhibit the growth of the L. crescens. SNPs with GA, PVP and citrate coatings all inhibited L. crescens. AgNPs with a GA coating inhibited, whereas AgNPs with PVP or citrate coatings did not. Objective 2: Harnessing the power of bactericides produced by citrus-inhabiting microbes: We have identified bacterial and fungal sequences that are associated with a minimal to no change in HLB severity ratings over a one-year period. These organisms include Actinoplanes sp., Bacillus sp., Corynebacterium sp., Exiguobacterium sp., Kocuria sp., Methylobacterium sp., Spirosoma sp., Streptomyces sp., Cladosporium sp., Sporobolomyces sp., Sordariales sp., Epicoccum sp., Leotiomycetes sp., and various mycorrhizal fungi. To study the microbial natural products produced by microbes associated with citrus, we have taken two approaches. 1. Untargeted metabolite analysis: We analyzed leaves, budwood, roots and microbial extracts. Of the 470 field samples provided, we found that different tissue types have disparate metabolic profiles and thus need to be considered separately. Most prominent chemical differences according to the survivor status have been found in the budwood tissue. 2. Targeted metabolite analysis: We focused on analyzing the metabolite profiles of two bacteria, a Bacillus and Paenibacillus sp., because they exhibited antagonism against L. crescens. We have initiated systematic screening of all the microbes housed in our Isolate Repository. To fractionate and isolate antimicrobial metabolites, we constructed a reverse-phase prep column. This protocol fractionates metabolites based on interactions with an organosilane stationary phase and a polarity-gradient mobile phase. Through continued fractionation, we aim to arrive at purified molecules that could be developed as anti-CLas treatments. Objective 3: Coated NPs were introduced to the trees using four methods: soil drenching, trunk slashing, foliar spreading, and petiole tube feeding. During the soil drenching, the NP suspension was poured onto exposed roots; during the trunk slashing, the NP suspension was carefully applied to a small trunk wound; in the foliar spreading, the suspension was deposited onto an abraded leaf; in petiole feeding, a petiole tube was used to introduce the suspension to a branch. Metal concentrations in the tree were evaluated at 24 hours and 7 days after exposure. It was determined that soil drenching was not an effective method of introducing NPs into trees. The volume of NP suspension that was introduced into the tree was highly variable - in some occasions the suspension dried up before it was fully introduced. Thus, we determined that a more effective method is needed to introduce a prescribed volume of NP suspension into the tree. Advisory Panelists, S. Slinski (Citrus Research and Development Foundation) suggested we try a pneumatic tree injector pump designed to inject materials into citrus trees. We have purchased this machine and are preparing to use it to inject NPs into trees. To assess the ability of the NPs to be taken up via foliar applications, we conducted a detailed mass-balance analysis on all parts of the tree. Leaves from Mexican lime trees were gently abraded, AgNP suspensions were placed on the leaf (NPs were coated with PVP, GA, or citrate). After 7 days, the entire tree was dissected, and all parts (roots, trunk, leaves, branches) were separated, combusted, digested with acid, and analyzed using ICP-MS. In the best case (when citrate was used as the NP coating), only 3% of the NPs added to the leaf were actually found in the tree, with a majority in the stem (2.5%), some in the leaves (0.4%), and a small amount in the roots (0.1%). However, this experiment demonstrated that foliar application is not a viable method to introduce NPs into a tree, as the vast majority of the NPs (97%) remained on the leaf and were "wasted". Objective 4: We have linked our research to a robust extension and outreach program through establishing a network of farm advisors in Florida and California. This has enabled us to contact growers to perform sampling in their groves. We have given presentations at a series of venues that are listed in the "Other Products" section. We have also held an initial PD/PI Kick-off cyber meeting (August 2017) and an in-person PD/PI meeting (November 2017). Both of these project meetings were attended by 2 growers (Ben McLean, III (FL) and Richard Bennett (CA)), a CRDF Bactericide trial manager (Stephanie Slinski), a Bayer Crop Science Biologics Scientist (Jean Broadhvest) and a PI on another CDRE project (Bryce Falk), all participating as advisory panelists. PD Rolshausen has developed a project website at http://ucanr.edu/sites/Citrus@UCR/ that will be updated regularly as the project evolves.

Publications

Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Nichole A. Ginnan, Tyler Dang, Sohrab Bodaghi, Paul M. Ruegger, Beth B. Peacock, Greg McCollum, Gary England, Georgios Vidalakis, Caroline Roper, Philippe Rolshausen and James Borneman. 2018. Bacterial and Fungal Next Generation Sequencing Datasets and Metadata from Citrus with Huanglongbing. Phytobiomes (Under Consideration).
Type: Journal Articles Status: Other Year Published: 2018 Citation: David Jassby and Yiming Su. Review article on the use of nanoparticles as anti-microbials in agricultural applications. In preparation.
Type: Journal Articles Status: Published Year Published: 2017 Citation: Philippe E. Rolshausen. 2017. Utilising the Plant Microbiome in Agriculture. Science Share
Type: Websites Status: Published Year Published: 2017 Citation: http://ucanr.edu/sites/Citrus@UCR/