The purpose of this project is to determine methods to effectively 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 the last quarter from Riverside, we have forwarded plant materials infected with viroids, citrus tatterleaf virus, phytoplasmas, citrus stubborn, citrus psorosis, and Citrus tristeza virus to Ft. Collins, CO for treatment by cryotherapy. From Florida, three clones infected with HLB were forwarded to Ft. Collins, CO. All of these materials have been cryotherapied using the current protocol. Surviving plants following treatment are held for 12 weeks, and then leaves are removed, nucleic acid extracted, and forwarded to riverside for laboratory testing of the target pathogens. Personnel from Ft. Collins, CO traveled to Riverside for training on shoot tip grafting and also to train Riverside personnel on recently developed protocols for cryotherapy. Jointly they conducted cooperative research on modification of the cryotherapy protocols was in order to more efficiently eliminate graft transmissible pathogens. Following the training in shoot tip grafting, a comparison of effectiveness of elimination of graft transmissible pathogens by cryotherapy and shoot tip grafting will be done. Research is continuing in Riverside on the use of very young plants as indicator plants for biological indexing, and a comparison of results will be made with the results from biological indexing by the conventional protocol.
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. We have received the sequencing result and are currently analyzing the data. The publication of Sweet orange genome significantly helps our analysis. Lately, Dr. Brlansky joined this project as a co-PI. The addition of expertise will help us in sample collection and improve our research capacity in characterizing the involvement of RNA virus as potential pathogen.
During the first year of this proposal we cloned, purified and performed small molecule screens on CLIBASIA_01180 and CLIBASIA_01510 transcription factors. The effects of these ligands were further confirmed in vitro performing thermal denaturation experiments at different concentrations. During this quarter we focused on the optimization of secondary assays (in vitro and in vivo) to prove the specificity of the chemicals identified. The choice of the secondary assay was tailored to each of the proteins and was based on its biological activity. We then focus on the identification and determination of ligand binding pockets on these proteins by building a structural model of each protein. For CLIBASIA_01510, seventeen chemicals were identified that increased its thermal denaturation in a concentration dependent manner. CLIBASIA_01510 is a homolog to proteins involved in gene regulation through direct interaction with the beta subunit of the RNA polymerase, not by direct binding to DNA. Based on these observations the effect of the chemicals identified in the screen were tested in vivo using a two hybrid system. We further determined the optimal growth media and growth phase to obtain tight protein:protein interactions. Then, the effect of the ligands was tested. It was observed that two chemicals identified on the chemical screens were able to disrupt the CLIBASIA_01510:RpoB interactions. The structural model of this protein indicated several hydrophobic regions that may be involved in protein:protein interactions. In order to determine the location and specificity of the ligand interactions we are currently performing site directed mutagenesis on the residues identified. These mutants will be tested for its capacity to interact with the RNA polymerase and the chemicals identified on the screens. A similar approach is being used for the location of the ligand binding site in CLIBASIA_01180.
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. We are evaluating different solvents and adjuvants for the five compounds. Their antimicrobial activities against Liberibacter crescens, which is closely related Ca. L. asiaticus and could be cultured, are being tested. 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 . 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 was tested using citrus (grapefruit) seedlings in greenhouse. Three isolates could promote growth of grapefruit seedlings. Those isolates are being used to evaluate their potential to prevent citrus seedlings from Las infection vectored by psyllids in a greenhouse. Several isolates also exhibit inhibitory activity again the citrus canker pathogen Xanthomonas citri ssp. citri.
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 three times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We are characterizing the two putative virulence genes sndA and stbA of Las, e.g. subcellular localization and host proteins interacting with them.
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. The sensitivity of biofilm and planktonic cells of Xac 306 to copper (copper sulfate) were evaluated by measuring the MICs. Biofilms are less susceptible to copper than planktonic cells. Effect of the selected compounds on sensitivity of Xac planktonic cells and biofilm cells to copper sulfate was also investigated. Two compounds could significantly increase the sensitivity of Xac planktonic cells to copper sulfate. In a in vitro biofilm system test, the combined use of copper sulfate and the compounds individual or both resulted in significantly increased killing compared to killing by copper sulfate alone. Currently, we are further evaluating the compounds combining with copper in control citrus canker.
Citrus canker is an economically important bacterial disease of most commercial citrus cultivars resulting in significant losses worldwide. Spread of citrus canker has been a severe problem to the citrus industry of Florida. How bacteria escape from infected plants is underexplored. Understanding the molecular determinants of lesion rupture, how Xcc survives in the intercellular spaces, and how Xcc releases from lesions of host plants will provide many fundamental and practical benefits. Despite the tremendous effort to eradicate citrus canker, the pathogen has spread to most citrus production areas in Florida and continues to spread. Understanding the genetic mechanism of release of Xanthomonas axonopodis pv. citri (Xac) from citrus canker lesions will help develop effective control and containment strategies to stop citrus canker pathogen from spreading. The goal of the proposed research is to understand the genetic mechanism of release of Xac from citrus canker lesions. The specific objectives are to: 1. characterize critical genes involved in release of X. axonopodis pv. citri from citrus canker lesions; 2. understand the release mechanism by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions. We have identified 12 EZ-Tn5 transposon mutants of Xac with reduced capacities of release from citrus canker lesions. The insertion sites of the 12 mutants have been identified with insertions in 11 different genes. Currently, complementation analysis of the mutants is underway. Bacterial growth assays of the mutants and the wild type strain in grapefruit ‘Duncan’and sweet orange ‘Valencia’ have been conducted. Three mutants were tested for affect in release from citrus canker lesions (dispersal assay) on grapefruit for 5, 7, 10 and 14 days as compared to wild type strain. The preliminary assay showed differences in growth rate (total count) and dispersal rates (surface count). The dispersal will thus be calculated according to percentage point difference to negate any effect due to growth changes. Pectate lyase assay, proteinase assay and motility tests, and EPS assay have been completed for all the mutants. Characterization of the mutants on LPS, capsule and biofilm formation are complete. Currently, we are investigating the release process by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions. The plant RNA is extracted and is being subjected to qRT-PCR and microarray analysis. Anatomical analysis of the inoculated tissues is being conducted usign transmission electron microscopy.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports 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. The management of this grove requires extra financial commitment as grove care costs tend to be higher than commercial groves due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. During the recently completed quarter (October 1 to December 31, 2012), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ The Sugar Belle’ harvest was completed with fruit utilized for fresh market packing. ‘ Plant Improvement Team: o The G-3 Eucalyptus clone was identified to be a good candidate as a windbreak plant due to plant growth habit. Per consultation of Dr. Rockwood, a harvest of one of two rows of the trial suggested for near future. o Rootstock is being grown in the ‘Arboretum’ for budding scions for HLB evaluation o Sprouting and additional horticultural care completed on Dr. Gmitter’s Hybrid evaluation o Rootstocks for Dr. Gmitter’s HLB tolerance evaluation established in ‘New Block’ o Another cultivar has been added to the New Varieties Development and Management Corporation area ‘ Dr. Futch is evaluating trifoliate rootstocks for HLB tolerance. ‘ Applications continued in the ‘commercial scale’ nutritional trial. ‘ Topping of 6 year old Hamlin orange is having good rejuvinative response ‘ Commercial Trials: o BASF has observed Hamlin size improvement in programs evaluating applications of Headline’ o Eurofins area planted o Evaluations of Agri Quest Citrus Root Health Improvement Project continue o ‘ Drs. Stelinski and Rogers have continued evaluations of Asian citrus psyllid and citrus leafminer management in their areas. ‘ Drs. Albrigo and Wong have continued to evaluate antibiotics to manage HLB o Hedging and toping improved overall tree condition in trial area
The Blood orange and the Cara cara orange derive their unique colors, flavors and health benefits from the expression of anthocyanins and lycopenes in the ripening fruit, respectively. Activation of the anthocyanin pathway has become well characterized in the past ten years. A MybA transcriptional activation gene has been shown to up-regulate the expression of anthocyanin biosynthetic genes in many species, including citrus (Butelli et al., 2012). In fact, the native citrus MybA1 gene (aka. CsRuby; GenBank JN402334) was recently isolated from the blood orange variety Moro (Butelli et al., 2012). Lycopene production requires a minimal metabolic pathway of three genes for most plants that generate and ripen fruit. Up-regulation of lycopene production in citrus has produced the flavorful Cara cara cultivar. The lycopene pathway is well understood and three genes sufficient for its biosynthesis (crtE AAA64977.1, crtI AAA64981.1 and crtB AAA64982.1) have been characterized and tested (Yoon et al., 2007). To date the MybA gene from the blood orange ‘Moro’ has been determined. The gene has been synthesized and cloned into an expression construct. Currently the gene is scheduled for testing in Tomato and Citrus tissues. Backup MybA gene’s have also been obtained from Plum, Grape and Arabidopsis. The genes for the lycopene metabolic pathway have been determined and those necessary for over-expression needed to produce a Cara-cara like fruit are currently being synthesized. Backup lycopene gene’s from Tomato and Arabidopsis have also been determined. Five promoters providing fruit specific expression have been identified within the Citrus genome. We are currently ordering primers for amplification from the genome. Backup promoters from the Tomato genome have also been determined. Finally, post-doctoral researchers are currently being interviewed to fill this position.
The objective of the proposed research is to develop genetically engineered (GE) citrus founder lines containing a platform that will allow the precise insertion of desired traits. In addition to allowing the targeted integration of transgenes, the proposed system also enables the removal of unneeded sequences such as antibiotic resistance marker genes (Wang et. al., 2010), allowing the generation of ‘clean’ (marker-free) GE citrus plants and or fruit. The targeting construct (or TAG) containing the recombinase platform has been completed and consists of the recombinase recognition sites for integration upstream of the positive/negative selection genes, CodA::kan. Kan is used for positive selection of transgenic citrus production while the codA gene will be used to detect successful recombination events. Down stream of the selection system are recognition sites for recombinase-mediated excision. Also included between the recombinase recognition sites is a minimal promoter GUSPlus gene. This gene will allow detection of the TAG construct inserting into hyperactive chromosomal expression loci. This type of loci is desirable for high expression of transgenes. Due to the placement of the minimal promoter GUSPlus gene between the recombinase recognition sites it will be removed during recombinase-mediated targeting. More recently a second version of the TAGII construct has been completed with an improved selectable expression cassette. Efforts are now underway to complete the agro-bacterium based pEXCH vector, which will be used to deliver both recombinases (for RMCE) and genes of interest (for genomic insertion). The Thomson lab has sent the CTAG constructs to the Stover lab. Transformation of citrus uses the disarmed A. tumefaciens strain EHA-105 harboring a binary vector CTAG vector. Six independent transformation events were performed for each genotype (Carrizo and Hamlin), subjected over 2,000 epicotyls and 200 cotyledons to pCTAGII vector. Putative transformed shoots, 157 from Carrizo and 149 from Hamlin, were obtained after 45 days. All putative transformed shoots have being micro-grafted. To date 19 of the initial 25 plants screened have been shown positive for the pCTAGII site by PCR. Two independent transformation events with pCTAGV-KCN3 vector. This newest CTAG vector contains DSred for non-destructive visible selection as well as an anti-apoptotic gene. The anti-apoptotic gene was included to help transformation efficiency. Both genes will be removed (along with the CodA::Kan marker) during the RMCE process. DSRed expression has been observed during co-cultivation of citrus tissues. Well-developed shoots were induced after 35 days in selection medium. This is an improvement of 10 days over the previous transformation efforts. 57 explants from Carrizo and 48 explants from Hamlin have been obtained. It is anticipated that a no-cost extension will be requested to extend the second year of funding to mid-April 2014, permitting a full year of effort on the initial funding.
We applied six therapeutic and two control treatments to infected and healthy Valencia orange on Kuharske Carrizo rootstocks at Florida. These eight treatments each contained 10 healthy and 10 HLB-infected trees growing in a greenhouse. Citrus rootstock trees used in these experiments were HLB inoculated using a standard inverted ‘T’ budding techniquewith bark with pieces from citrus infected trees. 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). The six therapeutic treatments included: 2, L- Arginine, 2, gibberellin in combination with 6-benzyl adenine (BA), and 2, 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. We are planning a second treatment in Feb 2013 on the same trees. For the first treatment we used 10 healthy and 10 HLB-infected trees (1 tree = 1 biological replicate) for 2 different concentrations of L-arginine dissolved in water with Silwet as surfactant and LK-phite and LDKP3XTRA. The controls for this treatment were 10 HLB-infected trees treated with just water containing the surfactant and used as control trees. Also, 10 healthy and 10 HLB-infected trees were treated with a mixture of surfactant, LK-phite and LDKP3XTRA in water and used as a second set of control trees. All HLB-infected treated trees and the healthy control sets were sampled. This treatment provided us 4 treatments x 6 trees x 3 biological replicates X 2 sampling points = 144 samples for analysis (RNA). For the second treatment used 2 different concentrations gibberellic acid in combination with a single 6-benzyladenine concentration, the solutions were prepared as explained above. We used 10 healthy and 10 HLB-infected trees for each combination. This experiments provided 2 treatments x 6 trees x 3 biological replicates X 2 sampling points = 72 samples for analysis (RNA). For the third treatment we used 2 different concentrations of atrazine in combination with one concentration of sucrose, solutions prepared as explained above. We use 10 healthy and 10 HLB-infected trees for each combination. This experiments provided 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 from half of the leaves the rest were stored at -80’C for extraction later. We have obtained a great deal of variability of RNA quality depending on the severity of the leaf symptoms gene expression is being determined by qRT-PCR.
The purpose of this project was to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing (HLB) and citrus canker. This project called for close cooperation among the National Clonal Germplasm Repository for Citrus and Dates (Repository), Riverside, CA, the Florida Citrus Germplasm Introduction Program (CGIP), and researchers located at the United States Horticultural Research Laboratory (USHRL), Ft. Pierce, FL. One priority of this project was to identify and prioritize valuable citrus germplasm in Florida that is being threatened and which is not already available in a protected collection. Stakeholders have met a total of three times to identify threatened citrus germplasm, and identified citrus germplasm has been propagated in a secure greenhouse located at the USDA ARS USHRL, Ft. Pierce, FL. Activity in Florida has been directed to cleaning the selected accessions from HLB and citrus canker. Currently 66 accessions are held at Ft. Pierce, FL which test negative for HLB and citrus canker. Additionally there are 25 accessions held in quarantine at the USDA ARS Repository, Riverside, CA. Eleven of these Florida quarantine accessions have been therapied, and all pathogen testing is complete except for viroid indexing from citron on sequential polyacrylamide gels, and this is anticipated to be completed within two months. There is a total of five additional accessions that have been identified and currently held by private owners which will be added to the collection at Ft. Pierce and targeted for clean up. This project has enabled the establishment of a pipeline to allow for collection of accessions in Florida, preliminary clean up in Florida, further therapy and complete testing for all known graft transmissible pathogens of citrus at the Repository, Riverside, CA. Clean accessions are then returned to Florida using the ‘Citrus Passport’ protocol which enables release of the accessions in Florida in less than a year after receipt from the Repository.
Two field experiments were established to conduct research in open hydroponics and high density advanced citrus production systems. They are located near Auburndale in the central Florida ridge region, and near Immokalee in the southwest Florida flatwoods region. The main goal of this research is to develop efficient horticultural systems which can be used for early profitable fruit production and in an IPM approach for managing / mitigating the most serious Florida citrus diseases of HLB and canker. Immokalee experiment: Results to date indicate higher NH4-N, NO3-N, P and K content in the irrigated than non-irrigated zone of drip hydroponics (DOHS). Root distribution also followed a similar pattern for DOHS, showing greater intensity (60-80%) in the irrigated than non-irrigated zone (20-40%). The nutrient concentrations and root length densities of CGP were lower compared with both OHS treatments and were uniformly distributed around the tree. Nutrient (NH4-N, NO3-N, P and K) leached was very low for all the fertigation methods. Trees grew faster under DOHS than CGP as indicated by canopy volume and trunk cross-sectional area measurements (20 to 25%). The leaf N, P and K concentration were adequate according to UF/IFAS recommendations for all treatments. However, there was greater N accumulation with DOHS (~114 kg N ha-1) than CGP (~79 kg N ha-1) supporting greater N uptake efficiency for DOHS treatments. Phosphorus and K accumulation were similar for DOHS and CGP indicating adequate applications of these two nutrients. Soil moisture was adequate in the irrigation of DOHS and grower practice falling between 7-15% on volume/volume basis suggesting that water was non-limiting in all the three irrigation methods. However, measured cumulative and daily water uptake were greater with DOHS than CGP though the crop coefficients were similar for the three irrigation practices indicating greater water use efficiency. Yield after four years was 85% greater for DOHS treatment compared with CGP. Auburndale experiment: The ACPS proved very successful for growing citrus on the Ridge, essentially reducing the time to production by 1-2 years. Those years represent real gains in early return on investment, and, more tangibly, they also represent direct savings in 1-2 years of the cost of production. The best treatment tested in this experiment consisted of the high planting density (363 trees/acre), C-35 rootstock, and the intensive drip open hydroponics (daily) fertigation system. In year 3 the yield of this treatment was an average 222 boxes/acre of ‘Hamlin’ fruit, compared to 90 boxes/acre in the conventionally grown treatment. Expected yields for year 4 in this best treatment are 500-600 boxes/acre. Unfortunately due to rapidly increasing HLB infections in this block (8.5% in year 3), the sustained yield increased seen in the first 3-4 years are unlikely to sustain in years 5 onwards because HLB infection rates are expected to exceed 50% by year 6. In November during the third year, a field day was held in the ACPS field experiment near Auburndale, attracting about 200 citrus growers, researchers and supporting industries. A comprehensive ACPS web site was developed in the first year and continues to serve visitors with timely updates on the ACPS research effort (http://128.227.177.113/ACPS/)
Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components cell-to-cell communication system. But the genome lacks the second components; luxI thar produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We confirmed the functionality of LuxR by expressing in E. coli and the acquisition of different AHLs we detect AHLs in the insect vector (psyllid) heathy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. Using different bacterial biosensor, we partly identify these AHLs (number of Carbon). Forming CLas biofilm on the surface of insect Gut confirm the presence of cell to cell communication in insect while the planctonic state of CLas in plant indicate the absence of this communication. In plant, we found molecules that bind to LuxR but inactive its function (plant defence). we try now to characterize these molecule and study their effect on biofilm formation inside insect. we use purified molecule to feed infected insect through artificial diet system. the main aim of this project is to express molecules in plant that interfere the growth of CLas in insect by feeding.
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