Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1160 - Pathology | 100% |
Citrus greening or Huanglongbing (HLB) disease is devastating the citrus industry in Florida and starting to spread in Texas and California. There is an urgent need for tools that can speed screening and identification of antimicrobials by overcoming the culturing bottlenecks associated with CLas. The overall goal of the project is to implement and validate an innovative screening strategy to identify potent antimicrobials as deliverable products to manage HLB. Specifically, we will conduct in silico and in vitro screening to identify novel inhibitors targeting conserved CLas virulence proteins, as well as broad-spectrum antimicrobial peptides, utilizing state-of-the-art molecular modeling algorithms and an innovative bioassay for culturing CLas. Promising antimicrobials will be further evaluated in small-scale greenhouse and field experiments, aimed to determine the effect of the antimicrobials on CLas titers. Furthermore, we will engage and educate the stakeholders and the end-users (e.g., citrus industry, commercial and residential citrus growers, and scientific community) in Florida, Texas and California, as part of the project extension and outreach activities. The near- to intermediate-term goal is to identify an assortment of potent antimicrobials suitable for commercial deployment in the field and to provide a sustainable alternative to antibiotic/bactericide therapies currently being used to manage HLB.
The major goal of this project is to implement and validate an innovative, pilot-scale CLas culturing and screening biossayto identify potent antimicrobials that can be used to manage HLB. The specific objectives are:
The project director (PD) is Kranthi Mandadi (Texas A&M AgriLife Research) and will oversee project execution with the collaboration of co-PDs, Sandun Fernando (Texas A&M University), Veronica Ancona (Texas A&M University-Kingsville, Citrus Center), Gitta Coaker (University of California, Davis), Elizabeth Grafton-Cardwell (University of California, Riverside), William O. Dawson (University of Florida, IFAS), Stephen Futch (University of Florida, IFAS), and Mike Irey (Southern Gardens Citrus).With regards to the methodology, Obj. 1 is the responsibility of Coaker. Her team will lead the selection of conserved CLas virulence proteins for molecular dynamic simulations. Obj. 2 is the responsibility of Fernando. His team will use the ELIXIR to perform the in silico ligand-screening to identify pharmacophores and potential inhibitors of CLas virulence and effector proteins. Obj. 3 is the responsibility of Mandadi. His team will lead the production of CLas cultures and conduct bioassays using the pharmacophores and antimicrobial peptides (AMPs). For Obj. 4, Ancona will help in the efforts to validate selected pharmacophores in small-scale greenhouse and field trials at TAMUK-Citrus Center in Texas, while Dawson and Irey will pursue the greenhouse and field trials with selected AMPs in Florida using Citrus tristeza virus (CTV) viral vectors. The extension and outreach efforts to engage stakeholders and end-users, as described in Obj. 5, is a collaborative effort among Ancona as lead in Texas, while Futch and Grafton-Cardwell will be the leads in Florida and California, respectively.
Target Audience
The proposed activities in this reporting period are targeted to impact plant pathology and microbiology scientific community by advancing tools and knowledge required for studying fastidious plant pathogens.
Changes / Problems
Nothing Reported
Training & Professional Development
The project provided research training and mentoring opportunities to several undergraduate students, graduate students, as well as early-career postdocs, and research scientists. All the staff were routinely mentored by the PIs in laboratory techniques, and scientific inquiry. The staff was also given opportunities to attend and present their research at professional conferences. Additionally, the senior staff was provided opportunities to mentor junior staff, thus enhancing their teaching and mentoring skills.
Dissemination Streams
The results were mainly disseminated through research and extensionpublications. Periodic updates were also provided by the project team members to multiple stakeholdersin-person/board meetings and online media.
Next Reporting Steps
Nothing Reported
Target Audience
The proposed activities in this reporting period are targeted to impact plant pathology and microbiology scientific community by advancing tools and knowledge required for studying fastidious plant pathogens.
Changes / Problems
Nothing Reported
Training & Professional Development
The project provided research training and mentoring opportunities to several undergraduate students, graduate students, research technicians, as well as early career postdocs, and research scientists. All the staff were routinely mentored by the PIs in laboratory techniques, and scientific inquiry. The staff were also given opportunities to attend and present their research in professional conferences. Additionally, the senior staff were provided opportunities to mentor junior staff, thus enhancing their teaching and mentoring skills.
Dissemination Streams
The results were mainly disseminated through research and extension publications. Periodic updates were alsoprovided by the project team members to multiple stakeholders in TX, FL and CA at growers board meetingsand online media.
Next Reporting Steps
This is the final year and we will prepare and submit the final report. <br><br>
<br>What was accomplished under these goals? The overall goal of this project was to implement and validate an innovative, pilot-scale CLas culturing and screening bioassay to identify potent antimicrobials that can be used to manage HLB. We have completed most of the experiments related to the identification and testing of new antimicrobials in hairy root bioassays, thus demonstrating the versatility and efficiency of the hairy root-based pipeline.Due to unforeseenpandemic which resulted in nationwide shutdowns and stay-at-home orders, we extended the project to this year. This gave enough time to completepending activities, and for preparation and submission ofpublications/reports. <br><br><b>Publications</b><br>
Target Audience
The activities in this reporting period impacted plant pathology and microbiology scientific community by advancing tools and knowledge required for studying fastidious plant pathogens/diseases such as citrus greening.
Changes / Problems
Nothing Reported
Training & Professional Development
The project has provided research training and mentoring opportunities to several Hispanic-minority undergraduate students, graduate students, research technicians, as well as early career postdocs, and research scientists. All the staff were routinely mentored by the PIs in laboratory techniques, and scientific inquiry. The staff were also given opportunities to attend and present their research in professional conferences. Additionally, the senior staff were provided opportunities to mentor junior staff, thus enhancing their teaching and mentoring skills.
Dissemination Streams
The results were disseminated by publications, trade magazines and conference presentations. Additionally, project results were periodically disseminated by the project team members to stakeholders in TX, FL and CA, and to the broader scientific community through a variety of avenues including in person field and site visits, and online media.
Next Reporting Steps
We will continue following up onexperiments related CTVfield trials (Obj. 4). The CTVtrial can take several years to completion, andare beyond the proposed project timeframe. However, we will continue monitoring them and resultswill beupdated in any future reports. Additionally, we will continue our outreach and extension activities to transfer knowledge and recommendations to stakeholders and end-users (Obj. 5) in TX, FL and CA, and to the broader scientific community through a variety of avenues including field and site visits/meetings, conferences, publications and online/social media. <br><br>
<br>What was accomplished under these goals? Obj. 1. Completed. We identified two potential CLas virulence proteins and six potential anti-CLas antimicrobial peptides (AMPs). Obj. 2: Completed. We identified sevenpotential CLas pharmacophoreinhibitors by molecular modeling approaches of the two CLas virulence proteins. Obj. 3. Completed. CLas-citrus hairy root bioassays were successfully employed to prescreen the seven potential CLas inhibitors and six AMPs. The screening identified two inhibitors and four AMPs that inhibited CLas. Obj. 4. In planta trials are ongoing with the candidate inhibitors and AMPs. Pharmacophoreinhibitor plant trials: We evaluated efficacy of two potential inhibitorsidentified from the hairy root bioassaysin HLB-infected citrus trees in the field.Briefly, multiple dosages of the inhibitors, along with reference bactericides, penicillin and oxytetracycline, were applied by foliar-sprayingonto leaves and branches of HLB-infected citrus treesperiodically (once a week for two months). Subsequent to the treatments, leafsamples were collected and molecular diagnostics was performed to quantify CLas titers among controls and treatments. Unfortunately, these results were inconclusive. First, we saw large variations in CLas titersamong the untreated controls and reference treatments alike. This could be due to the dynamics of the CLas/HLB system. The CLas accumulation is highly random and non-uniform in the trees. Second, it is not clear ifthe standarddelivery approach by foliar spraying ofthe citrus trees is thateffectivetoget the therapies and active ingredients (AIs) into the phloem tissues where the bacteria resides. This scenariois more in linewith a recent reportthat showed that foliar spraying was not effective to deliver a bactericide oxytetracycline when compared to trunk injections ( Li et a., 2019). There are mixed reports on the utility of the trunk injections and still not clear whether it can deliver AIs effectively into phloem. Nevertheless, these results suggest that there isa dire need for alternative and effective approaches to deliver chemicals/AIs into citrus tree phloem. As aproxi to CLas-citrus trials, we evaluated the efficacy of the two inhibitors against a closely related pathogen, Candidatus Liberibacter solanacearum (CLso) in potatoes. In silico analysis revealed that the target STP protein iswell-conserved between CLas and CLso, and importantly the two inhibitors demonstrated good binding affinities to both CLas and CLso STPs. We then pursued foliar spray application of the two inhibitors at multiple dosages onto CLso-infected potatoes (twice a week for four weeks).Potatoleaf tissues are much softer and moreamenable to foliar spraying-based application than citrus.Subsequent to the treatments, leafsamples were collected and molecular diagnostics was performed to quantify CLso titers among controls and treatments.Theresultsshowed that the two inhibitors did inhibitCLso accumulaton and alleviated CLso-induced disease symptoms. The results are promising and suggest that the two inhibitors could bepotentially useful to control also HLB/CLas, if they can be delivered effectivelyinto citrus tree vasculature. AMP-CTV plant trials:We completed cloning ofselected AMPs, identified fromthe hairy root bioassays, intoCTV transient vector constructs and inoculated them into citrus seedlings. The stability of theconstructs was also verified by RT-PCR and Sanger sequencing, followed by grafting the inoculuminto HLB-infected citrus trees in the field. Currently, we are monitoring the trees for HLB phenotype and trait improvements. References:Li J, Pang Z, Duan S, Lee D, Kolbasov V, Wang N. 2019. The in planta effective concentration of oxytetracycline against Candidatus Liberibacter asiaticus for suppression of citrus Huanglongbing. Phytopathology 109:2046-2054. Obj. 5. Outreach and extension to transfer knowledge and recommendations to stakeholders and end-users. Project updates and results were periodically disseminated by the project team members to stakeholders in TX, FL and CA, and to the broader scientific community through a variety of avenues including publications, online meetings and trade magazine articles. Meetings: February 21, 2020. TAMUK-Citrus Center Winter Festival cutreach event,Weslaco, TX(Ancona). June 16, 2020. Stakeholder meeting, Texas Citrus Pest and Disease Management Corporation, Mission, TX (Mandadi, Ancona). October 18, 2020. Stakeholder meeting, Texas Citrus Pest and Disease Management Corporation, Mission, TX (Mandadi, Ancona). Magazines/social media: An education article was developed for a citrus grower/industry magazine, Citrograph, that is published by the Citrus Research Board (California, USA). The primary goal was to educate and extend the project findings to the grower and industry stakeholders. Mandadi, K., Irigoyen, S., Ancona, V., Sétamou, M., Coaker, G., Borneman, J., and Irey, M. (2020) Hairy roots to the rescue: Speeding up Discovery for HLB Management. Citrograph 11 (2) 20-22. http://www.citrusresearch.org/wp-content/uploads/CRB-Citrograph-Mag-Q2-Spring-2020-Web-1.pdf <br><br><b>Publications</b><br>
Target Audience
The proposed activities in this reporting period are targeted to impact plant pathology and microbiology scientific community by advancing tools and knowledge required for studying fastidious plant pathogens.
Changes / Problems
Nothing Reported
Training & Professional Development
The project has provided research training and mentoring opportunities to several Hispanic-minority undergraduate students, graduate students, research technicians, as well as early career postdocs, and research scientists. All the staff were routinely mentored by the PIs in laboratory techniques, and scientific inquiry. The staff were also given opportunities to attend and present their research in professional conferences. Additionally, the senior staff were provided opportunities to mentor junior staff, thus enhancing their teaching and mentoring skills.
Dissemination Streams
The results were disseminated by publications andconference presentationsat international conferences. Additionally, projectresults were periodically disseminated by the project team members to stakeholders in TX, FL and CA, and to the broader scientific community through a variety of avenues including in person field and site visits, and online media.
Next Reporting Steps
We will continue our experiments in greenhouse and field trials (Obj. 4). Selected inhibitors and AMPs that demonstrated positive activity in the hairy root assays will be further tested in small-scale greenhouse and field trials. Additionally, we will continue our outreach and extension activities to transfer knowledge and recommendations to stakeholders and end-users (Obj. 5) in TX, FL and CA, and to the broader scientific community through a variety of avenues including in person field and site visits, conferences, publications and online/social media. <br><br>
<br>What was accomplished under these goals? Obj. 1. Identification and selection of conserved CLas virulence proteins. In order to identify and select conserved CLas virulence proteins, we have taken a comparative genomics approach of diverse C. Liberibacter species, including those that can infect citrus. We have also sequenced and/or annotated an additional 24 strains of the HLB-associated pathogen, CLas, including strains from multiple locations in California, Texas, Florida and Mexico. A manuscript describing the genomic characterization of each strain in the context of all citrus-infecting Liberibacters is currently under review. Furthermore, using a comparative approach, we identified several proteins that are specific and conserved in CLas, as well as additional Liberibacter species (C. Liberibacter americanus, C. Liberibacter africanus, C. Liberibacter solanacearum). These proteins represent promising targets for disease control and are likely required for Liberibacter survival in citrus. After investigating the predicted biological function of each protein and consulting with the PD/Co-PDs, we selected multiple proteins (a serine/tyrosine phosphatase (STP), a Serralysin and an ABC transporter) for molecular modeling and in silico screening of small molecule inhibitors (Obj. 2). Some of these proteins are conserved in all CLas genomes sequenced to date, and are predicted to be required for virulence, and have surface-localized features enabling inhibition by pharmacological inhibitors. Additionally, in consultation with Co-PD Irey (Southern Gardens Citrus), we shortlisted another set of eight broad spectrum antimicrobial peptides (AMPs) that could be evaluated for their potential to inhibit CLas. Obj. 2. Molecular modeling and in silico screening of CLas pharmacological inhibitors. Target-based inhibitor screening was conducted on the three conserved CLas proteins. Inhibitor screening comprised of several steps including the development of the homology model, identification of active sites and pharmacophores, inhibitor screening, and in silico confirmation before experimental verification. Simulation platforms including Autodock Vina, Visual Molecular Dynamics (VMD), Drugui, Enhanced Ligand Exploration and Interaction Recognition Algorithm (ELIXIR-A), Nanoscale Molecular Dynamics (NAMD) were used during in silico analysis. In this manner we identified seven potential inhibitors. The inhibitors were also isolated based on Lipinski's rule of 5 and bioactivity scores and had higher binding affinities to the target protein than the native substrates. For example, two compounds (C1 and C2) that bind to the CLas STP had greater affinities, -5.30 ± 0.283 kcal/mol and -5.27± 0.320 kcal/mol, respectively vs. native G6P substrate with -4.38 ± 0.379 kcal/mol binding affinity for to the active site on CLas STP. This would suggest that the small molecules can competitively bind to the active site and interfere with the native function of the target protein, thus would inhibit CLas in plant cells. The seven potential inhibitors targeting the three different CLas proteins were further evaluated using the CLas hairy root cultures per Obj. 3. Obj. 3. Production of CLas cultures and high-throughput screening of inhibitors and AMPs (Mandadi). The efficacy of the seven in silico predicted CLas inhibitors, and eight selected broad-spectrum antimicrobial peptides (AMPs) identified from spinach were tested using the hairy root cultures. For the inhibitor screening, CLas hairy root cultures were generated and validated by i) Fluorescence microscopy-based visualization of the co-transformed GFP marker in the hairy root cultures, and ii) presence of CLas via. diagnostic PCR analysis with RNR primers for CLas. Next, the CLas hairy root cultures were treated with the seven small molecules at various concentrations in a multi-titer plate for three days, along with a negative control (0.1% DMSO) and a reference bactericide oxyteracycline (500 ppm) that appears to have moderate CLas inhibitory activity in planta. All samples were subjected to qPCR molecular diagnostics to estimate the relative CLas titers. Results for the screening identified two out of seven inhibitors targeting the STP and the ABC transporter inhibited CLas, in a manner similar to oxytetracycline. For evaluating the eight antimicrobial peptides (AMPs), briefly, the coding regions of the AMPs were cloned into binary expression vectors and transformed into CLas-hairy root cultures. Tissue samples were collected for molecular diagnostics in order to confirm the expression of defensins and CLasaccumulation/titers using RT-qPCR and qPCR techniques, respectively. Each experiment was performed with three to five biological replicates. The results of the antimicrobial assays identified four out of eight AMPs reduced CLas titers in the hairy root cultures. Together, the hairy root bioassays allowed us to rapidly short-list potentially effective inhibitors and eliminating those that are ineffective. The small molecules and AMPs are being evaluated in planta using foliar sprays and Citrus tristeza virus viral vector (CTVvv)-based delivery, respectively, per Obj. 4. Obj. 4. Validation of antimicrobials in greenhouse and field. Selected inhibitors and AMPs that demonstrated positive activity in the CLas hairy root assay are being further tested in small-scale greenhouse and field trials. These experiments are still ongoing, and results will be reported in the coming period. Obj. 5. Outreach and extension to transfer knowledge and recommendations to stakeholders and end-users. Project updates and results were periodically disseminated by the project team members to stakeholders in TX, FL and CA, and to the broader scientific community through a variety of avenues including in person field and site visits, conferences, and online media. Field/site visits: August 2018: Stakeholder meeting and visit to Wonderful Citrus (TX) and US Citrus (TX) (Mandadi, Futch, Ancona). November 2018: Stakeholder meeting and visit to Southern Gardens Citrus (FL) (Mandadi, Irey). 19 February 2019: Stakeholder meeting, Texas Citrus Pest and Disease Management Corporation, Mission, TX (Mandadi, Ancona). 19 June 2019: Stakeholder meeting, Texas Citrus Mutual, Mission, Texas (Mandadi, Ancona). August 2019: Stakeholder meeting and visit to Southern Gardens Citrus (FL) (Futch). Conferences/workshops: December 2018: Texas Plant Protections Association Conference (TX) (Mandadi). March 2019: IRCHLB meeting (CA) (Mandadi, Ancona, Futch, Grafton-Cardwell). May 2019: Texas Citrus Mutual Annual Meeting (TX) (Mandadi, Ancona). Online/social-media activities: May 2019: A research snapshot article was developed and published on the UCANR Science for Citrus Health website. "Hairy root matrix used to culture CLas to rapidly screen antimicrobials for improved HLB management. 2019. Kranthi Mandadi, Veronica Ancona, Steve Futch, Elizabeth Grafton-Cardwell, Peggy G. Lemaux, Lukasz Stelinski, Monique Rivera & Sara Garcia-Figuera. Webdistribution:https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/Mandadi/ Thedocument was also sent to all the citrus extension agents to be distributed in citrus newsletter as well as to many of the citrus researchers around FL, CA and TX at various REC. <br><br><b>Publications</b><br>