Development of alternative or complementary approaches for effective management of citrus greening is highly desirable and will greatly help the citrus industry due to the difficulty to control the HLB disease. Considering the highly destructive nature of HLB disease and the lack of control measures, there is a huge potential to develop antimicrobial small molecules against the causal agent thus to suppress the population of Ca. L. asiaticus in planta and to reduce the innoculum for psyllid transmission. Treatment of Ca. L. asiaticus infected citrus could be pursued by applying antimicrobials to infected trees. The most common targets for antimicrobial agents development include receptors, proteins and enzymes, DNA, RNA and ribosomal targets. Among them, proteins have become the major target due to their druggable characteristics. In this study, we presented our research on screening small molecule inhibitors against SecA. SecA is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane. The Sec pathway was also shown to be required for virulence of Ca. L. asiaticus in our study. SecA is the protein translocase ATPase subunit, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. In our recent study, we further expanded our previous study in identifying lead antimicrobial compounds with higher activities by targeting SecA using various computational techniques like homology modeling, virtual screening, molecular docking & minimization. Due to the uncultivable nature of Ca. L. asiaticus, we tested the potential inhibitory effect of the selected compounds against Agrobacterium tumefaciens, which is phylogenetically related to Ca. L. asiaticus. Twenty compounds were selected for biological activity study against SecA of Ca. L. asiaticus and A. tumefaciens. Five compounds were found to inhibit the ATPase activity of SecA of Ca. L. asiaticus in nano molar concentrations and showed antimicrobial activities against A. tumefaciens with MBC ranging from 128 ug/ml to 256 ug/ml. These compounds appear to be suitable as lead compounds for further development of antimicrobial compounds against Ca. L. asiaticus. Those findings were published in the article entitled: Identification of small molecule inhibitors against SecA of Candidatus Liberibacter asiaticus by structure based design. on Eur J Med Chem (http://dx.doi.org/10.1016/j.ejmech.2012.05.035). To test the application potential of those compounds on plants, the phytotoxicity studies were performed on the five compounds against citrus. At higher concentrations (0.25mg/ml), all five compounds showed phytotoxicity. When the concentrations were diluted to 0.025mg/ml, the toxicity went down to mild to low. Currently, we are testing the inhibitory effects of the five compounds again Ca. L. asiaticus in planta. Currently, we are optimizing these five antimicrobial compounds to identify compounds higher antimicrobial activity. Utilizing similarity search methods on these five active structures, fourteen compounds were selected from commercially available compound database for antimicrobial activity study. The selected fourteen compounds are being tested for their inhibition against SecA ATPase activity.
Pathogenic microorganisms affecting plant health are major and chronic threat to food production and ecosystem stability worldwide. As agricultural production intensified over past few decades, producers became more and more dependent on agrochemicals as a relatively reliable method of crop production helping with economic stability of their operations. However, increasing use of chemical inputs causes several negative effects, e.g. development of pathogen resistance to the applied agents and their non target environmental impacts. Furthermore the growing cost of pesticides and consumer demand for pesticide free food has led to the search of substitute of these products. In addition, there are a number of fastidious diseases for which chemical solutions are few, ineffective or nonexistence. Biological control is thus being considered as an alternative or a supplemental way of reducing the use of chemicals for agriculture and to manage fastidious diseases. Citrus Huanglongbing (HLB, greening) is one of the most devastating diseases and presents an unprecedented threat to Florida citrus industry. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las) (Duan et al. 2009). Thus, alternative management approaches to manage HLB are necessary. The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. We have developed a culture collection of approximately 400 bacteria initially isolated from the root and rhizosphere of citrus. These bacterial isolates have been screened for various beneficial traits related to P solubilization, siderophore production, nitrogen fixation, indole acetic acid (IAA) synthesis, production of antibiotic and lytic enzymes, induction of systemic resistance, production of ACC deaminase1-amino-cyclopropane-1-carboxylate deaminase and production of quorum sensing [N-acyl homoserine lactones] signals. We are also evaluating the antagonistic activity of these bacterial strains against some well-known plant pathogenic fungi. Especially we have screened a bacterial isolate designated as 43A which possess multiple plant growth promoting activity and is also able to antagonize different fungi. We are also testing the plant growth promoting activity of 24 isolates using seed germination pouch in greenhouse. We have also selected several Bacillus spp. possessing multiple beneficial traits to develop bacterial consortium which can be further developed as carrier based bioformulation. Assay for compatibility between isolates using antagonistic survival tests showed that all the selected beneficial bacteria are compatible with each other. Plant growth promoting activity of six selected isolates was evaluated using the model plant Arabidopsis grown in vitro. The results suggested that three isolates could promote plant growth. The plant growth promoting activity of these six isolates is being tested using citrus (grapefruit) seedlings in greenhouse.
Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. As requested by CRDF and SAB, we have revised project and will focus on the following two objectives: to characterize how Las causes HLB disease symptoms and how Las manipulates plant defense response by investigating the roles of putative virulence factors; to test different compounds in controlling HLB and characterize their mechanisms in controlling HLB. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed two times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB. Currently, We are checking the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We will continue to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove.
The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. The hypotheses of the proposed research are (1) Biofilm formation and QS play important roles in X. citri ssp. citri infection of citrus fruit; (2) Control of citrus canker could be improved by interfering with biofilm formation and QS of X. citri ssp. citri. The hypotheses are based on previous studies and our preliminary studies. The specific objectives of this proposed research are as follows: Objective I: To understand the role of biofilm formation and QS in X. citri ssp. citri infection of citrus fruit. Objective II: To prevent X. citri ssp. citri infection of fruit by combining copper with inhibitors of biofilm formation and QS. Recently, we compared the attachment of the QS mutants on the citrus fruit surface. Compared with wild type stain Xac 306, the quorum sensing mutant ‘rpfF showed significantly reduced attachment to the fruit surface as revealed by CLSM (confocal laser scanning microscopy) observation with the GFP-labeled bacterial strains. We also evaluated the effect of nine compounds on Xac biofilm formation on abiotic surfaces using the crystal violet staining method. The data obtained showed that three compounds were active in inhibiting Xac biofilm formation in NB liquid medium at. Plant test in greenhouse showed that treatment with the three compounds prior to infection could reduce biofilm formation of Xac on leaf surface, reduce the formation of canker lesions on spray-inoculated grapefruit leaves with the wild-type strain. Effects of the three compounds on Xac on detached immature citrus fruit were also tested using spray inoculation. Preliminary results showed that these small molecules affected the infection of Xac 306 on unwounded and wounded citrus fruits at sub-inhibitory concentrations. We are currently testing the effect of those compounds in different combinations with copper based bactericides in controlling Xac infection of grapefruit plants in the greenhouse.
Per the requirements from CRDF, we have revised our goal of this proposal to focus it on the identification of the causal agent of citrus blight. Citrus blight has imposed consistent losses and challenges to citrus industry since the causal agent of the disease remains unknown. The present study would be instrumental in knowing the mysterious pathogen causing citrus blight and pave way for devising efficient management or control methods to help citrus industry to tackle citrus blight. We will characterize the microbiomes of the blight diseased and healthy citrus roots through metagenomic approaches. Recently, we have survey three groves at Lake Alfred, Auburndale, and Haines city. Citrus blight trees at different development stages and healthy trees are being confirmed based on symptoms, water injection, and P12 antibody that have been known as the diagnosis tools for citrus blight. We finalize the blight diseased and healthy citrus trees to be used for sampling. Root samples were collected from 24 trees. The first set of DNA and RNA samples have been purified and sent for deep sequencing to identify the microbes associated with blight diseased and healthy citrus.
Since the date of submission of the first report for this project (on 8/7/12), I have learned that progress reports should be written in accordance with the calendar trimesters. For that reason, this report is describing only the period between the second week of August and the end of September. During this time, Citrus Core Transformation Facility maintained its level of productivity and continued to produce transgenic Citrus plants. Number of new orders that were placed was very high for such a short period of time. Altogether, nine orders were placed and all for production of transgenic Duncan grapefruit plants. The binary vectors with the genes of interests were named: pX7, pX11, pX16, pX19, pX20, pX28, pNah, pMED14, and pMED16. All nine vectors were successfully mobilized into appropriate strains of Agrobacterium; glycerol stocks were created, and stored at -80oC. For some of those cultures, the first co-incubation experiments were done. Transgenic plants that were produced during these 7 weeks belonged to some recent orders but also to a few older orders. Altogether, 25 Carrizo citrange plants were produced for the same ‘Y’ order: eleven Y150 plants, six Y102 plants, five Y141 plants, and three Y46 plants. Five Duncan grapefruit plants were produced carrying MYB2 gene from pBCR2 vector and additional six carrying gene from the pAZI1 vector. Mix of 10 Carrizo and five Duncan plants were produced with the BI gene from the pAtBI vector. Three Duncan plants with the gene from pDPR1 vector were made. Finally, a mix of seven additional Duncan plants were produced transformed with the genes from following vectors: three with pEDS5, one with pELP3, one with pWG24-13, and two with pWG25-13. Future work will focus on the newly placed order and on completion of old orders.
IRREC-ACPS project (Barrett Gruber, co-PI) The second objective of this project is to establish an ACPS replant trial at the UF-Indian River Research and Education Center (IRREC, Fort Pierce). The goal of this objective is to evaluate the performance of ACPS for citrus fresh-market citrus varieties and regional disease problems such as canker. After receiving funds in July 2012, RTK-DGPS mapping was used to stake the end-of-tree-rows on 8 bedded acres of research property at IRREC and was done to obtain an estimate of total tree numbers. This mapping was used to plan for two different planting densities: 8′ . 25′ (218 trees / acre, ‘conventional planting’); and, 9′.1/4(3’+19’+3’+25′) (387 trees / acre, ‘higher density planting in double tramlines’), all on standard 50-foot beds. After estimates were obtained, a total of 3,098 new grapefruit trees (1,305 Ray Ruby/US897, 1,305 Ray Ruby/Kuharske, and 488 Ray Ruby/Swingle) were ordered on contract from a certified, commercial nursery. The nursery estimated that these trees would be delivered to IRREC no later than August 2013. CREC-ACPS project at Auburndale The 14-acre ‘Hamlin’ orange project with C-35 and Swingle rootstocks continues to grow well with the in-ground open hydroponics system. Fruit yields from the highest planting density being tested (363 trees/acre) are expected to reach 400-500 boxes/acre in this, the fourth year. Corresponding nitrogen fertilizer rates for that density are expected to reach 180 to 190 lb /acre. Due to the rapidly increasing HLB infection in the block, we decided to lower the priority of high nutrient efficiency in the block because any nutrient stress could adversely exacerbate the development of HLB symptoms. So far this strategy seems to help, and a recent survey of symptom severity on all identified HLB-affected (symptomatic) trees revealed that more than 80% of the tree displayed mild to moderate symptoms, having set and developed a fairly normal crop of fruit. We expect many of the more symptomatic fruits to drop before the harvest in December, but hopefully every HLB-affected tree will still produce a partial yield in order to keep the average yield in the block economically viable. CREC-ACPS project at block 22, CREC A new 4-acre ‘Valencia’ orange experiment using in-ground drip fertigated open hydroponics was planted in August 2012 near the CREC. Rootstocks US897 and Swingle are being compared, as well as a calcium-free versus a calcium-rich hydroponics fertilizer source. The other factor being investigated is the use of alternating double rows or “tramlines” to better utilize grove space in higher planting densities. We are comparing 484 trees/acre in single rows with 538 trees per acre in tramline rows.
Selection of nano-particles (Edgardo Etxeberria, co-PI) Due to its capacity to bind both neutral and charged ions/molecules, we selected PAMAN [poly(amido amine)-tris(hydroxymethyl) amidomethane ] dendrimer as the experimental nanoparticle. This particle can be used to carry any of the ionic or neutral elements components of the foliar fertilization cocktail. Nanoparticle size was also scrutinized in terms of quantitative efficiency to carry nutrient elements and ease of isolation. Due to its size of 5 nm, we selected 4th generation PAMAM dendimers. Isolation of nanoparticles from unbound elements. Separation of element-loaded nanoparticles from unbound elements is a key step in the purification system. We examined three isolation methods using fluorescence-bound nanoparticles: 1. dialysis, 2. ultracentrifugation, 3. low speed nano-filter centrifugation. Nanoparticles were loaded with fluorescent Alexa-488 and recovery determined fluorometrically at 519 nm. We determined that low speed nano-filter centrifugation resulted in higher nanoparticle recovery without loss of volume. The same system was also used to determine the maximum amounts of nanoparticles per filtration step. Efficieny of the system is highest at xxx nanoparticles/mL. Urea as test substrate. Urea was selected as test substance due to its carbon based skeleton that can be followed by 14C scintillation spectroscopy. Binding of urea to nanoparticles. The number of urea molecules carried per nanoparticle was determined using 14C-urea. After a 30 min binding process of urea to nanoparticles consisting of agitation at 30’C and pH 7.0, unbound 14C-urea was removed by low speed filtration. The number of 14C-urea molecules bound to nanoparticles was determined by scintillation spectroscopy. From the standard curve (Fig. 1), it was determined that each nanoparticle binds to approximately 370 molecules of urea. Setup for tests of electrostatically charged spraying and conventional spraying efficacy A computer-controlled motorized turntable was constructed to serve as an infinitely controllable spray platform for potted citrus. The spray nozzles were mounted on a stationary vertical boom to spray the tree canopy on the rotating platform. An ESS single nozzle electrostatic sprayer module was purchased and is being incorporated into the same turntable spray platform. Initial tests with the experimental platform will include 1) nutrient coverage and penetration of citrus leaves with or without electrostatic charging, and 2) with or without commercially available surfactants.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently facilitates citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. Maintenance of this grove requires extra financial commitment as grove care costs tend to be higher and fruit production is lower than commercial groves due to the nature of many of the research projects, especially those involving the management of the HLB vector or other damaging pests. During this funding cycle significant research findings related to Asian citrus psyllid, citrus leafminer and citrus rust mite management were obtained in the MFCF grove and incorporated into the UF/IFAS Extension Florida Citrus Pest Management Guide. Other trials in the grove have been established or continue to evaluate antibiotics for HLB management, nutritional programs for increasing tree health, root and foliar disease management and herbicide efficacy evaluation, to name a few. Also rootstock and cultivar evaluations have continued and been initiated. The Mid Florida Citrus Foundation also serves as a forum to educate growers, scientists, governmental agencies and others on research results. It has been the venue for numerous field days and tours focusing on trials conducted at the site.
In this quarter, we applied three therapeutic strategies to infected and healthy Valencia orange on Kuharske Carrizo rootstocks at Florida. Citrus rootstock trees were HLB inoculated with bark pieces from citrus infected trees using a standard inverted ‘T’ budding technique. The trees were kept in the greenhouse under natural light conditions at 17-25’C until the HLB infection was confirmed by quantitative real-time PCR (qRT-PCR) on August 2012. Eight treatments were applied on 80 healthy and 80 HLB-infected trees, grouped in three experiments. The therapeutic compounds used for the different foliar treatments were: L- Arginine, gibberellin in combination with 6-benzyl adenine (BA), and atrazine in combination with sucrose. The surfactant Silwet, LK-phite and LDKP3XTRA were added to all treatments. All treatments were sprayed on the citrus foliage; the volume sprayed per tree was enough to wet both the upper and lower leaf surfaces just to the point of runoff. Phosphites were added to our therapeutic compound mixtures, since our data mining of HLB infected citrus suggests the presence of genes associated with phosphate deficiency, the symptoms include starch accumulation and collapse of healthy root function. Our rationale is that by eliminating these deficiencies, we will be able to better evaluate if our therapeutic treatments enhance citrus response to HLB, prolong the life of HLB-infected plants, reduce the bacterial titer and counteract the detrimental effects of the infection on citrus production. For the first experiment we used 2 different concentrations of L-arginine dissolved in water with Silwet as surfactant and LK-phite and LDKP3XTRA. We use 10 healthy and 10 HLB-infected trees (1 tree = 1 biological replicate) for each L-arginine concentration. Ten healthy and 10 HLB-infected trees were treated with water containing just the surfactant and used as control trees. Also, 10 healthy and 10 HLB-infected trees were treated with mixture of surfactant, LK-phite and LDKP3XTRA in water and used as control trees. All HLB-infected treated trees and the healthy control sets were sampled. This will provide us 4 treatments x 6 trees x 3 biological replicates X 2 sampling points = 144 samples for analysis (RNA). For the second experiment we used 2 different concentrations gibberellic acid in combination with a single 6-benzyladenine concentration, the solutions were prepared as explained above. We use 10 healthy and 10 HLB-infected trees for each combination. This experiments will provide 2 treatments x 6 trees x 3 biological replicates X 2 sampling points = 72 samples for analysis (RNA). For the third experiment we used 2 different concentrations of atrazine in combination with one concentration of sucrose, solutions were prepared as explained above. We use 10 healthy and 10 HLB-infected trees for each combination. This experiments will provide us with 2 treatments x 6 trees x 3 biological replicates X 2 sampling points = 72 samples for further analysis (RNA). Three leaves that were mid symptomatic based on observation of blotchiness, mottling and hardening of the veins were collected at 0 time and 3 days after treatment from each individual treated or control tree and processed individually. Leaves were immediately frozen in liquid nitrogen and keep at -80’C until extracted. Total RNA was extracted and stored at -80’C. Expression levels of biomarker genes for each treatment will be determined by qRT-PCR.
In the period between the May 1st and the end of July, Citrus Core Transformation Facility continued to offer its services to researchers who needed transgenic Citrus material. Interest for the transgenic Citrus plants remained high. Six new orders were placed: to produce transgenic Carrizo plants transformed with genes from pY48 and pY109 vectors; for transgenic plants of Duncan grapefruit and sweet orange transformed with the genes from pSF1 vector and for transgenic plants of Duncan grapefruit transformed with the genes from pNW3 and pNW4 vectors. Work on these orders has begun immediately upon receipt of DNA material. At the same time facility continued to service old orders. As a result, bulk of the plants produced within this quarter belong to the orders placed previously. Altogether, 106 plants were produced within this quarter: thirty seven Carrizo pY102 plants, fifteen Carrizo pY46 plants, seven Carrizo pY141 plants, thirteen Carrizo pY150 plants, twelve Duncan pBCR2 plants, eighteen Carrizo pAtBI plants, and four Duncan pAZI1 plants. There are about 30 putatively transgenic shoots grafted on rootstock at different stages of early growth before transfer to soil. Productivity of the facility remains on the high level and operation is continuing without any problems.
Quarterly report for July-September 2011 The citrus extension agents work collectively to educate growers on HLB and production practices to minimize psyllids in groves with the use of citrus health management areas (CHMAs). The agents were actively involved in developing additional CHMA areas throughout all citrus production regions and to support those already developed areas with the local team captains. CHMAs offer the greatest opportunity to manage psyllid within a larger geographical area as compared to individual management efforts. The HLB photo series is being hosted on the agents’ website (http://citrusagents.ifas.ufl.edu/hlb_photos/index.htm) whereby monthly photos are posted to allow growers to view the progression of HLB over time. The agents made 29 grove visits, conducted 7 local educational programs, in addition to assisting with the Citrus Expo that is conducted annually in Ft. Myers. All events are designed, developed, and conducted to maximize educational opportunities to enhance grower knowledge within the citrus industry to ensure all are aware of HLB and suggested management strategies. Three local field days were conducted during the period to enhance surveying tasks for HLB or psyllid monitoring. Agents are also actively involved in assisting county extension offices with Master Gardener trainings or to assist with urban citrus issues and problems.
Quarterly report Jan. – March 2011 To assist in answering growers questions and to identify grove specific problems, the citrus agents conducted 45 field visits during the period of January ‘ March 2011. The citrus agents were actively involved with the Florida Citrus Show (formerly Indian River Citrus Seminar) whereby over 500 attendees gained knowledge via the 11 presentations related to HLB, canker or black spot identification. On a local level, 9 production schools or meetings were conducted to disseminate educational information to the local growers on a wide range of topics that have been determined to local grower needs or advisory committees. All citrus agents participated in the International HLB conference hosted in Orlando to gain information and then share with growers throughout the citrus industry. To ensure that low volume sprayers are delivering the proper spray droplet size as required on some pesticide labels, a sprayer rodeo calibration event was hosted in Bartow. This event utilized resources provided by the USDA researchers and provided printed reports to each participant to document droplet size. Programs were also offered to enhance pesticide and equipment safety to area citrus workers. These programs offer printed certificates to document training as required by worker protection standards or various insurance agencies. HLB photo series is available to all growers and interested parties via the web at: http://citrusagents.ifas.ufl.edu/hlb_photos/index.htm. This site allows interested individuals to view the condition of selected citrus trees that have been confirmed to have HLB over time.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing and citrus canker. Using input from stakeholders and scientists, threatened germplasm has been identified, collected, and established at Ft. Pierce. Presently 66 accessions are being held at Ft. Pierce. These accessions have been subjected to antibiotic therapy, and are testing negative for Las by qPCR. In collaboration with the Florida Citrus Germplasm Introduction Program, the accessions are beginning thermotherapy, which will be conducted for a 16 week time period. In another 3-4 weeks, the thermotherapy treatment will be finished. Experience has indicated that laboratory testing before 12 weeks post treatment will result in false negative results. Thus in September, the accessions will be testing using laboratory methods for Las as well as other graft transmissible diseases of citrus, including viroids. Following the testing and depending on the results, the accessions will be forwarded to the USDA ARS Repository in Riverside for further clean up and testing before release from quarantine. In Riverside, the penicillin/streptomycin mixture, cyclohexamide, D4E1 (antimicrobial peptide) treatments used in Florida are being evaluated for elimination of Candidatus Liberibacter associated with tomato psyllid yellows and citrus stubborn from tomato and citrus, respectively.
The purpose of this project is to determine methods to easily eliminate Candidatus Liberibacter asiaticus (Las), the bacterium associated with huanglongbing (HLB) in Florida, from citrus with emphasis on cryotherapy, and to determine the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. In this first quarter of the project, we have begun activities while waiting for the funds. In Riverside, young plants are being evaluated for use for biological indexing of Citrus tatterleaf virus, citrus vein enation, and citrus viroids. The results obtained from using young indicator plants (less than 4 months old) are being compared to the conventional method of biological indexing for these pathogens. In Colorado, permits have been obtained to permit shipment of huanglongbing infected material from Florida so that cryotherapy protocols can be done. In Florida, elite breeding material has been identified and prioritized for testing/treatment.
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