Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1100 - Bacteriology | 100% |
The devastating effects of the bacterial disease known as huanglongbing or HLB have severely crippled the citrus industry in the US by both increasing the production costs and dramatically decreasing revenue. Florida citrus production dropped from over 240 million boxes in 2003/04 to below 100 million boxes in 2014/15 (USDA-NASS) while per-acre spray cost increased fourfold during that period. Not only are 80% of trees in citrus operations infected by HLB in Florida but HLB is impacting citrus production in other states as well. The development and full deployment of any cost-effective strategies that can control the Las bacterium or mitigate the effects of HLB in the United States are vital to the survival of the citrus industry. This makes the topic of chemotherapy an important one amongst shareholders in the citrus industry because unlike other plant bacterial diseases, no resistant citrus cultivars are currently available for the control of HLB.The project presented here will identify and assess the most cost-effective treatment for HLB control by 1) deploying an inexpensive and accelerated screening procedure that greatly enhances our ability to find novel, active compounds through the incorporation of chemical libraries often involved with medicinal drug discovery; 2) evaluating pre-selected effective chemicals to determine the optimum application times and effective dosages for a field setting; 3) determining if the fruit quality and quantity in treated HLB-affected trees can recover to healthy unaffected tree levels; and 4) assessing the practicality of the treatment options from the growers perspective by providing an economic analysis of chemotherapy methods; all while 5) continuously communicating project advancements with the stakeholders to facilitate the acceptance and integration of new chemistries into growers' management programs.Upon completion of the objects presented, we will have ascertained the most successfulantimicrobial for the control of HLB based on the ability of the chemical to 1) eliminate/suppress Las and restore an infected tree to ahealthy, productive state;2) be excluded from the fruit and juice upon harvest; and 3) be economically viable in regards to large-scale field application. In addition, we will also have identified several new potentialantimicrobials warranting further investigations in field trials. Overall, this will allow us to reach our ultimate goal of providing actionable recommendations to the growers regarding advancements made in chemotherapy by providing the critical research necessary before products can be made commercially available for the treatment of HLB.
Our overarching goal is to revitalize the citrus industry across the United States by finding a sustainable solution that eliminates the spread of huanglongbing and restores HLB-affected trees back to a healthy, productive state through the discovery and application of antimicrobial compounds that either kill or suppress Candidatus Liberibacter asiaticus (Las). The objectives listed below aim to define chemotherapy treatment methods that will reduce Las titer in areas such as Florida, where the disease is endemic, as well as protect new plantings in groves not yet infected in Florida, Texas and California.
High throughput screening of small moleculesTwo small molecule libraries, one from the Torrey Pines Institute for Molecular Studies (TPIMS) and the other from Chembridge Research Laboratories, will be screened for chemicals effective against Las. In order to facilitate the screening of these large chemical libraries (totaling over 30 million compounds) on HLB-affected plants and overcome the time/space limitations of previous approaches, we have developed a high throughput screening method that utilizes a two-prong approach for assessing the chemicals. For the first part of this approach, detached periwinkle leaves from infected plants (a HLB model system) are placed in 96-well blocks containing the chemicals to be tested for a total of 14 days. A sample consisting of ½ of the leaf tissue is taken prior to being immersed in the chemicals and the remaining ½ is taken at the end of the immersion period in order to test for the Las titers using real-time PCR. Each chemical treatment is performed in triplicate in order to help eliminate any false positives/negatives associated with sampling. The effectiveness of each chemical can then be deduced from the change in level of Las at these two time points, with an increased Ct value indicating a lower level of bacteria in the sample. The second part of the system involves the testing of the chemical on the growth of Liberibacter crescens, the closest cultivable relative to Las currently known. Optical density measurements taken at the initiation of the experiment and 10 days post-chemical exposure will be usedto determine the level of growth inhibition for each chemical assayed.The Chembridge library was screened using Xylella fastidiosa, as a surrogate bacterium to conduct a functional analysis of the key virulence genes in Las. This screen yielded three compounds that affected the pathogenicity of the Las complimented pilG mutant. These three compounds, along with any chemicals identified from the TPIMS library mentioned above will be evaluated in the greenhouse for efficacy against Las and phytotoxicity using the traditional graft analysis previously published.Field analysis of a chemicals effectivenessIn addition, a field analysis of Validoxyamine A, Aliette 80WG, and Carvacrol will be conducted using a randomized block design to investigate the effects of chemotherapy on HLB infected citrus trees. Trials will be conducted on two citrus cultivars (grapefruit and sweet orange) at locations in both Florida and Texas with Las titers being measured at set time points along the treatment schedule using real-time PCR. Treatments will then be compared via statistical analysis to determine their effectiveness. The recovery of the tree's health will also be assessed using tree appearance, trunk diameter, and canopy volume/density.Juice and fruit analysisBecause antimicrobial compounds may have unwanted side effects for the consumers, their presence in the fruits and juices will also be analyzed using a macro matrix solid-phase dispersion method to extract the antimicrobial chemicals from the citrus juice samples in conjunction with gas chromatography-mass selective detection to define the chemicals and quantify the residues. The flavor and nutritional quality of the fruits and juices will also be analyzed using an electronic tongue and high performance liquid chromatography along with HPLC-mass spectrometry, respectively.Economic analysisThe economic viability and performance of proposed treatments are the ultimate factors determining whether the treatment will be adopted by growers so we will assess the costs and benefits of the different control methods at the grower level as well as the economic impact of the optimal control strategies at the industry level, accounting for both increased yield and enhanced quality of the fruits.
Target Audience
The target audience of our outreachare citrus agricultural stakeholders which are citrus growers, crop consultants, county extension agents, extension and research faculty, agroindustry, regulators, and state residents. Working with growers and industry representatives, we have demonstrated to stakeholders that chemotherapy of citrus HLB is a challenging task, and the deliverable solutions may be reachablewhen we integrated it with other management tools. In Fort Pierce, Florida, PIs communicated with growers on weekly base, and updated the results with the Florida Citrus Research and Development Foundation production committee members. Every two months, Co-PI Dr. Ancona meets with growers at the Texas Citrus Pest and Disease Management Corporation to discuss HLB issues and management strategies. The evaluated treatments were discussed, and they were informed on the progress of the research. The research results and hurdles were also presented and discussed with growers and scientists in the International Research Conference on Huanglongbing.
Changes / Problems
Nothing Reported
Training & Professional Development
Chemotherapy of citrus HLB is the most challenging project in management of plant diseases because it requires an effective chemical that can kill the intracellular and systemic HLB bacteria and easily diffuse throughout all citrus tissues, and is environment-friendly. This project has provided the researchers an unprecedented opportunity to conduct this challenging research on multiple aspects: from chemical synthesis and high throughput screening to delivery into citrus; from evaluation of chemical effectiveness and side effects to potential economic impact; from Chemistry and Pathology to Economics. All PIs, postdoc research associates, graduate students, technicians and student aids learned a great deal from this tough project, which includes lab and field trial experience. The experience and knowledge obtained in this project will facilitate future research more efficiently.
Dissemination Streams
Working withindustry representatives and extension agents, we have demonstrated to stakeholders that how chemotherapyworks for mitigatingcitrus HLB in greenhouse andfields.Research progress and advancements were disseminated by the project personnel at meetings targeted to grove owners, grove managers, and other stakeholders atgrower's meetingsat the annual Citrus Shows in Ft. Pierce,and Ft. Myers; at local/regional/national/international conferences particularly for other researchers and extension professions;Data were alsodisseminated in academic journals. The PD and its team members worked with a coordinated group of growers, citrus extension agents, and agricultural engineersfor practical application based on the research findingsthat showedto alleviate the detrimental effects of HLB. While working with growers, we also worked closely with Florida Citrus Research and Development Foundation, the California Citrus Research Board, and Texas Citrus Mutual to communicate our research progress with other colleagues.
Next Reporting Steps
Nothing Reported
Target Audience
Citrus growers, crop consultants, county extension agents, extension and research faculty, agroindustry, regulators, and state residents.
Changes / Problems
One-year no cost extension was granted.
Training & Professional Development
Chemotherapy of citrus HLB is the most challenging project in management of plant diseases because it requires an effective chemical that can kill the intracellular and systemic HLB bacteria, and easily diffuse to every pierce of citrus tissues, and is environment-friendly. This project has provided the researchers an unprecedented opportunity toconduct this challenging research on multiple aspects: from chemical synthesis, high throughput screening to delivery into citrus; from evaluation of chemical effectives, side effects to potential economic impact; from Chemistry, Pathology to Economics. All PIs, postdoc research associates, graduate students, technicians and student aids learned a lot from this tough project, which include lab and field trial experience. The experience and knowledge obtained in the first and second years will facilitate the research in third year and beyond.
Dissemination Streams
Working with growers and industry representatives, we have demonstrated to stakeholders that chemotherapy of citrus HLB is a challenging task, and the deliverable solution is reachable when we integrate it with other management tools. In Fort Pierce, Florida, PIs communicated with growers on weekly base, and updated the results with the Florida Citrus Research and Development Foundation production committee members. Every two months, Co-PI Dr. Ancona meets with growers at the Texas Citrus Pest and Disease Management Corporation to discuss HLB issues and management strategies. The evaluated treatments are discussed, and they are informed on the progress of the research.
Next Reporting Steps
Because of the accomplishments in the first two years, our goals for the next reporting period will primarily focus on the following three aspects: 1) continue the field trial evaluations with the selected chemicals, and we will plan a field day to allow those interested a first-hand look at the outcome of the trials performed. A special training session will also be held for citrus extension agents to inform them of the applicability of the chemotherapy agents under study; 2) start new field trial with the newly selected CRL small molecules that functions as preventive or suppressing the HLB bacterial movement, and greenhouse trials on HLB-affected citrus trees with newly selected TIPMs molecules; and file patent applications on several new molecules and work with company to initiate commercial-oriented development of these new molecules. We expect to increase the number of antimicrobials that may be used commercially by identifying new antimicrobial agents not currently used in human medicine that aid in the recovery of HLB affected trees by suppressing or eliminating the growth of Las. Effective protocols and actionable recommendations for HLB therapy will be made available to growers and stakeholders after the completion of this project regarding the effectiveness of the three chemicals in the field trials. A better understanding of how the chemotherapeutic agents affect the fruit will also be gained. Insight into the sustainability of the treatments will be acquired using a grower level economic analysis so that stakeholders can define the true costs of each treatment. We will dissimilate our research accomplishments with citrus growers, crop consultants, county extension agents, extension and research faculty, agroindustry, regulators, and state residents. <br><br>
<br>What was accomplished under these goals? Objective 1. High throughput screening of small molecules effective against HLB We have screened two small molecule libraries, one from the Torrey Pines Institute for Molecular Studies (TPIMS) and the other from Chembridge Research Laboratories (CRL), and have identified a number of effective compounds that may be useful for the control of citrus HLB. By utilizing the two-prong approach for assessing the chemicals, we have identified 8 scaffold compounds (some of them were narrowed down to individual compounds) of TPIMS. Interestingly, these compounds either inhibited or enhanced the growth of Liberibacter. The identified individual compounds were advanced to graft-based assay in citrus. Using Xylella fastidiosa (Xf), as surrogate bacterium for functional analysis and interference of Ca. Liberibacter asiaticus (Las) virulence genes, we have successfully substituted Las genes including pilG, popP and rpfA and mopB into Xf genome through a cross-over chromosome recombination technique. All these genes have been found to be important in attributing toward pathogenicity. This system allows us real-time evaluation of the effect of anti-virulence compounds on twitching motility, or evaluation of in vitro suppression of the PopP kinase and RpfA aconitate hydratase activities and the interruption of MopB surface attachment ability. Several virulence parameters including cell growth, colony formation, cell-cell aggregation, and biofilm formation were used to measure the effects. Altogether, we have identified 22 molecules that were most effective. It is worth noting that the results from cell attachment experiment further identified two molecules, P02G02 and P01B02 that were found to be the even better in terms of virulence suppression as compared to other candidates and antibiotic compounds. Thus far, we have successfully screened and functionally validated several new anti-virulence molecules under in vitro assays. While screening these small molecular libraries, we have developed a method to culture Ca. Liberibacter asiaticus in liquid culture. Although this method remains to be improved, it allow us to screen the chemicals in an 96-well plate using qPCR evaluation, and the results showed that it has a better correlation with the graft-based bioassay in citrus than the use of Liberibacter crescens. After identification of individual compounds effective against Liberibacter in vitro, we further evaluated 14 (9 from TPIMS, and 3 from CRL) newly identified compounds in citrus and periwinkle plants using spraying, injection and graft-based assay in greenhouse. Three TPIMS compounds showed more effectives than the others in eliminating or suppressing Las in both assay while two CRL compounds displayed their effectives by slowing the bacterial movement, and therefore suppressing the HLB symptom development. These two compounds may be used for prevention or the treatment on early infection of HLB. Objective 2. Field analysis of a chemicals effectiveness We have initiated field evaluations of Validoxyamine A, Aliette 80WG, and Carvacrol as planned in three locations in Florida and two locations in Texas using a randomized block design to investigate the effects of chemotherapy on HLB infected citrus trees. In addition, the combination treatment of Aliette and Mycoshield was added upon grower's request since these two pesticides are legally available for commercial grove application. Trial set-up, baseline measurements (tree height, canopy spread, disease index ratings), and leaf collections for Las real-time PCR detection were conducted before application of the antimicrobials. Except the location in southwest Florida, near Immokalee, which was slightly delayed due to hurricane Irma, all the other locations yielded preliminarily positive results in the reduction of bacterial titers with different treatments. It is worth noting that our continuation of the three-year MAC-funded project revealed the accumulation effect of these selected antimicrobials on reduction of bacterial titers, disease index and revitalization of tree growth. Objective 3. Juice and fruit analysis Because antimicrobial compounds may have unwanted side effects for the consumers, their presence in the fruits and juices will also be analyzed using a macro matrix solid-phase dispersion method to extract the antimicrobial chemicals from the citrus juice samples in conjunction with gas chromatography-mass selective detection to define the chemicals and quantify the residues. The flavor and nutritional quality of the fruits and juices will also be analyzed using an electronic tongue and high performance liquid chromatography along with HPLC-mass spectrometry, respectively. Juice quality was maintained well in HLB-affected trees though yield in Navel oranges was not improved after the 1st year of treatment in Texas trial, most likely because flowering and fruit was already set when we applied the treatments. However, In Fort Pierce, Florida, fruit quality tests were carried out on all previous MAC trials. Fruit qualities including size, peel color, puncture resistance, fruit weight, juice weight, brix and acid were tested. The preliminary results showed no significant difference among these treatments. In addition, we collected grapefruit samples in middle September for residue analysis. These samples included 5 lb x 3 bags of fruit from each treatment of four treatments. Juice was extracted with 1 gallon juice per bag (replicate) of fruit and stored at -20°C. We will be run GCMS analysis on juice samples from year 2 (2018) as proposed. Objective 4. Economic analysis The economic viability and performance of proposed treatments are the ultimate factors determining whether the treatment will be adopted by growers so we will assess the costs and benefits of the different control methods at the grower level as well as the economic impact of the optimal control strategies at the industry level, accounting for both increased yield and enhanced quality of the fruits. We have conducted an extensive literature research on HLB treatments and the economics, summarized in a review paper "A review of Huanglongbing Management and the impact on citrus production", covering major HLB treatments and their effect on citrus yield. We have also collected production cost data in the literature. This work has been summarized in a working paper titled "The downstream impact of agricultural disease: The case of U.S. orange juice market" and was presented in the International Research Conference on HLB (March 13-15, Riverside, CA). <br><br><b>Publications</b><br>
Target Audience
Citrus growers, crop consultants, county extension agents, extension and research faculty, agroindustry, regulators, and state residents.
Changes / Problems
Nothing Reported
Training & Professional Development
Chemotherapy of citrus HLB is the most challenging project in management of plant diseases because it requires an effective chemical that can kill the intracellular and systemic HLB bacteria and easily diffuse throughout all citrus tissues, and is environment-friendly. This project has provided the researchers an unprecedented opportunity toconduct this challenging research on multiple aspects: from chemical synthesis and high throughput screening to delivery into citrus; from evaluation of chemical effectiveness and side effects to potential economic impact; from Chemistry and Pathology to Economics. All PIs, postdoc research associates, graduate students, technicians and student aids learned a great deal from this tough project, which includes lab and field trial experience. The experience and knowledge obtained in the first year will facilitate the research in second year and beyond.
Dissemination Streams
Working with growers and industry representatives, we have demonstrated to stakeholders that chemotherapy of citrus HLB is a challenging task, and the deliverable solution is reachable when we integrate it with other management tools. In Fort Pierce, Florida, PIs communicated with growers on a weekly basis, and updated the Florida Citrus Research and Development Foundation production committee members with the results. Every two months, Co-PI Dr. Ancona meets with growers at the Texas Citrus Pest and Disease Management Corporation to discuss HLB issues and management strategies. The evaluated treatments are discussed, and they are informed on the progress of the research. Two manuscripts related to this project: "Antimicrobial compounds effective against Candidatus Liberibacter asiaticus discovered via graft-based assay in citrus" and "Molecular mechanisms underlying heat or tetracycline treatments for citrus HLB control" were submitted for peer-reviewed publication.
Next Reporting Steps
Because of the accomplishments in the first year, our goals for the next reporting period will primarily focus on the following three aspects: 1) continue the field trial evaluations with the selected chemicals and plan a field day to allow those interested a first-hand look at the outcome of the trials performed. A special training session will also be held for citrus extension agents to inform them of the applicability of the chemotherapy agents under study; 2) start new field trial with the newly selected CRL small molecules that function as preventive or suppress the HLB bacterial movement, and greenhouse trials on HLB-affected citrus trees with newly selected TIPMs molecules; and file patent applications on several new molecules and work with the company to initiate commercial-oriented development of these new molecules. We expect to increase the number of antimicrobials that may be used commercially by identifying new antimicrobial agents not currently used in human medicine that aid in the recovery of HLB affected trees by suppressing or eliminating the growth of Las. Effective protocols and actionable recommendations for HLB therapy will be made available to growers and stakeholders after the completion of this project regarding the effectiveness of the three chemicals in the field trials. A better understanding of how the chemotherapeutic agents affect the fruit will also be gained. Insight into the sustainability of the treatments will be acquired using a grower level economic analysis so that stakeholders can define the true costs of each treatment. We will disseminate our research accomplishments with citrus growers, crop consultants, county extension agents, extension and research faculty, agroindustry, regulators, and state residents. <br><br>
<br>What was accomplished under these goals? Objective 1. High throughput screening of small molecules effective against HLB We have screened two small molecule libraries, one from the Torrey Pines Institute for Molecular Studies (TPIMS) and the other from Chembridge Research Laboratories (CRL), and have identified a number of effective compounds that may be useful for the control of citrus HLB. By utilizing the two-prong approach for assessing the chemicals, we have identified 8 scaffold compounds (some of them were narrowed down to individual compounds) of TPIMS. Interestingly, these compounds either inhibited or enhanced the growth of Liberibacter. The identified individual compounds were advanced to graft-based assay in citrus. Using Xylella fastidiosa (Xf), as surrogate bacterium for functional analysis and interference of Ca. Liberibacter asiaticus (Las) virulence genes, we have successfully substituted Las genes including pilG, popP and rpfA and mopB into Xf genome through a cross-over chromosome recombination technique. All these genes have been found to be important in contributing to pathogenicity. This system allows us real-time evaluation of the effect of anti-virulence compounds on twitching motility, or evaluation of in vitro suppression of the PopP kinase and RpfA aconitate hydratase activities and the interruption of MopB surface attachment ability. Several virulence parameters including cell growth, colony formation, cell-cell aggregation, and biofilm formation were used to measure the effects. Altogether, we have identified 22 molecules that were most effective. It is worth noting that the results from cell attachment experiment further identified two molecules, P02G02 and P01B02 that were found to be the even better in terms of virulence suppression as compared to other candidates and antibiotic compounds. Thus far, we have successfully screened and functionally validated several new anti-virulence molecules under in vitro assays. While screening these small molecular libraries, we have developed a method to culture Ca. Liberibacter asiaticus in liquid culture. Although this method remains to be improved, it allow us to screen the chemicals in an 96-well plate using qPCR evaluation, and the results showed that it has a better correlation with the graft-based bioassay in citrus than the use of Liberibacter crescens. After identification of individual compounds effective against Liberibacter in vitro, we further evaluated 14 (9 from TIMPS, and 3 from CRL) newly identified compounds in citrus and periwinkle plants using spraying, injection and graft-based assay in greenhouse. Three TMIP compounds showed greater efficacy than the others in eliminating or suppressing Las in both assays, while two CRL compounds effectively slowed the bacterial movement, and therefore suppressed HLB symptom development. These two compounds may be used for prevention or treatment of early HLB infection. Objective 2. Field analysis of chemicals' effectiveness We have initiated field evaluations of Validoxyamine A, Aliette 80WG, and Carvacrol as planned in three locations in Florida and one location in Texas using a randomized block design to investigate the effects of chemotherapy on HLB-affected citrus trees. In addition, the combination treatment of Aliette and Mycoshield was added upon grower's request since these two pesticides are legally available for commercial grove application. Trial set-up, baseline measurements (tree height, canopy spread, disease index ratings), and leaf collections for Las real-time PCR detection were conducted before application of the antimicrobials. Except the location in southwest Florida, near Immokalee, which was slightly delayed due to Hurricane Irma, all the other locations yielded preliminarily positive results in the reduction of bacterial titers with different treatments. It is worth noting that our continuation of the three-year MAC-funded project revealed the accumulation effect of these selected antimicrobials on reduction of bacterial titers, disease index and revitalization of tree growth. Objective 3. Juice and fruit analysis Because antimicrobial compounds may have unwanted side effects for consumers, their presence in the fruits and juices will also be analyzed using a macro matrix solid-phase dispersion method to extract the antimicrobial chemicals from the citrus juice samples in conjunction with gas chromatography-mass selective detection to define the chemicals and quantify the residues. The flavor and nutritional quality of the fruits and juices will also be analyzed using an electronic tongue and high performance liquid chromatography along with HPLCmass spectrometry, respectively. Juice quality was maintained well in HLB-affected trees though yield in Navel oranges was not improved after the 1st year of treatment in the Texas trial, most likely because flowering and fruit was already set when we applied the treatments. However, In Fort Pierce, Florida, fruit quality tests were carried out on all previous MAC trials. Fruit qualities including size, peel color, puncture resistance, fruit weight, juice weight, brix and acid were tested. The preliminary results showed no significant difference among these treatments. In addition, we collected grapefruit samples in mid-September for residue analysis. These samples included 5 lb x 3 bags of fruit from each of four treatments. Juice was extracted with 1 gallon of juice per bag (replicate) of fruit and stored at -20°C. We will run GCMS analysis on juice samples from year 2 (2018) as proposed. Objective 4. Economic analysis The economic viability and performance of proposed treatments are the ultimate factors determining whether the treatment will be adopted by growers. Therefore, weare assessing the costs and benefits of the different control methods at the grower level as well as the economic impact of the optimal control strategies at the industry level, accounting for both increased yield and enhanced quality of the fruits. We have conducted extensive literature research on HLB treatments and the economics, summarized in a review paper "A review of Huanglongbing Management and the impact on citrus production", covering major HLB treatments and their effect on citrus yield. We have also collected production cost data in the literature. <br><br><b>Publications</b><br>