Three replicated experiments were undertaken to evaluate the effectiveness of reflective mulch to repel ACP, reduce incidence of HLB, and improve growth and yield of newly planted citrus compared to standard practices. The first was planted 3-4 July 2012 in a 10-acre block on 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County. Plants were provided with 2 punch-in drip emitters for irrigation and fertigation. Experimental design is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each plot is split into two subplots 5 rows wide and 13 trees long, mulch or no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film 3 mils thick and covered with a clear protective polyethylene coat. Monitoring ACP with flush inspection and sticky cards replaced weekly commenced 13 August. Of 1974 ACP found on sticky cards, more than 70% came from no-mulch plots, 11% from plots receiving insecticides and only 2% from plots with both mulch and insecticides. Of more than 59,000 flush shoots inspected, 7,368 were infested with ACP and 1565 were infested with Aphids. More than 65% of ACP and aphid infested flush were found in no-mulch plots, 477 ACP and 518 aphid infested flush in insecticide treated plots and 93 ACP and 166 aphid infested flush in plots provided with both mulch and receiving insecticide applications. Twenty positive HLB leaf samples were found from a sample collected mid-January 2014, of which none came from the mulched treatments, 12 from the no mulch + foliar nutrition but no insecticide with no other significant differences. Trunk diameter measurements made 21 January 2014 indicated better growth from trees planted in metalized mulch compared to non-mulched subplots. Greatest in growth difference among treatments between mulched and unmulched was seen without insecticides (18%) with only a 3% increase with insecticides. Foliar nutrition increased growth by 3% in all treatments when compared to no foliar nutrition. Leaf samples for nutrient analysis were collected before the March foliar nutrition spray. Normal grove care operations continued which included one gylphosate application in March for weed control, Kocide copper prayed monthly to control canker, and one application of Danitol as a dormant season spray to reduce ACP population to more realistic levels in all treatments. A 6-week insecticide drench rotation plan for designated plots was implemented in February starting with thiamethoxam and followed up mid-March with clothianidin. A second trial planted May 2013 at SWFREC consists of 24, 250 ft rows of ‘Ray Ruby’ grapefruit divided into 8 main plots, half with organic amendments of compost. Each 3-row plot is divided into 2 subplots: whiteface or metalized mulch. One of 160 leaf samples collected 28 Jan 2014 tested positive; it was located in a white mulch compost plot. Of 55 ACP found on sticky cards, 29 were from white mulch+compost, 16 white mulch no compost, 6 in metallized mulch compost and 4 in metalized mulch no compost. No infested shoots were found in trees on metalized mulch compared to 5 infested on white mulch no compost and 9 on white mulch with compost (N=7013). Trees in in compost plots had 15% more flush shoots than in plots without compost with no effect of mulch type on number of flush. The 3rd trial was planted on 17Mar14 at the Florida Sustainable Research Farm in Vero Beach to evaluate plantings in metallized reflective mulch and urban plant debris compared to a conventional cropping system. ‘Ray Ruby’ on ‘sour orange’ was planted 12′ x 25′ in a RCB with 4 reps. Irrigation and fertigation is supplied by 2 Bowsmith 2 gph drip emitters per tree. Trees were pruned after planting, trunks wrapped in 10′ aluminum foil and trees treated with imidacloprid again after planting. These last 2 trials only received funding for one year which ends 30 June 2014.
The bacterium Candidatus Liberibacter asiaticus (CLas) is closely associated with the development of HLB and is transmitted into the citrus phloem via the psyllid insect. Using the fully sequenced CLas genome, we have identified 27 proteins that are predicted to be secreted outside the bacterial cell. These bacterial proteins are called effectors. In other bacteria, effectors are required for pathogen virulence enabling nutrient acquisition, insect feeding, and suppression of defense responses. The goal of the funded research will be to investigate the expression patterns and citrus targets of four different CLas effectors that are expressed in HLB-infected citrus trees. We hypothesize that these effectors are important for bacterial survival or HLB symptom development by targeting important citrus proteins to manipulate their host. A detailed understanding of these effectors and their citrus targets will facilitate HLB detection strategies as well as provide pathogen targets that can be manipulated to enhance plant tolerance and resistance. We have made significant progress in developing tools to assess CLas effector expression. When a gene is expressed, it is first transcribed into RNA and then translated into protein. In order to assess effector expression at the protein level, the Ma lab has previously generated antibodies that can recognize each of the four CLas effectors. During the funding period, we have purified each effector and used this to affinity purify each antibody. This has resulted in significantly enhanced detection of effector proteins with no interfering background detected in navel, mandarin, or Lisbon lemon. These purified antibodies can now be used for effector detection as well as effector targets in the funded work. In the Contained Research Facility at UC Davis, we have started a time-course experiment to investigate the expression of each effector in navel and mandarin orange over time after graft inoculation with CLas. We have validated that the infected material is PCR positive for CLas, contains the four effectors, and expresses the effectors at the RNA level. Leaf tissue from plants at time zero (before inoculation) and one month after inoculation has been collected and is currently being processed to extract DNA, RNA, and proteins. We anticipate that samples will be taken for at least six months, or until the plants exhibit severe disease symptoms. In collaboration with Siddarame Gowda at the University of Florida, citrus expressing each effector is being generated using the CTV-based expression system to determine if effector expression results in any obvious morphological changes, enhances susceptibility to CLas, or promotes psyllid feeding.
The productivity of the Core Citrus Transformation Facility (CCTF) in the time between January and April of 2014 was higher than it was in the previous quarter. We have continued to produce transgenic plants for different research groups from the state of Florida and beyond. The work done within this quarter concentrated mostly on old orders. In communication with one research group, which is the biggest client at this time, the decision was made to prioritize the work on some of the orders they previously placed so CCTF had to shift the efforts towards specific orders and disregard time when the orders were placed. Also, through the communication with the CREC Director, CCTF received direct order from CRDF to produce some rootstock plants transformed with the NPR1 gene. These efforts are being coordinated with the Mature Tissue Transformation Lab (MTTL). Considering the importance of this project, CCTF started working on it immediately. Initial co-incubation experiments were done just a few days upon receipt of this order with the plant material obtained from MTTL. However, the quality of seedlings obtained from the MTTL was not good and results from that series of experiments had to be discarded. Rootstock cultivars of Carrizo, Swingle, and Citrus macrophylla are being used in this project. C. macrophylla seeds used for production of seedlings, that are the source of explants for co-incubation experiments, seem to be carrying some endophyte as most of the material obtained from these seeds ended up being contaminated. Because of this observation, the decision was made to put the emphasis on the Carrizo and Swingle cultivars. The work on this order is continuing at high pace. Plants produced by the CCTF within this three months period belong to the following orders: pNah-10 plants, pN9-seven plants, pN18-six plants, pX11- eight plants, pW14- three plants, pX20- one plant, pX28- one plant, pN7- one plant, pHGJ4- one plant, pMed16- one plant, pMed14- two plants, pELP3-G-nine plants, pELP4-G- three plants, pTMN1-five plants, and pMG105- two plants. Altogether 60 plants were produced and they were all Duncan grapefruit.
The citrus Greening Bibliographical Database http://swfrec.ifas.ufl.edu/programs/entomology/hlb_db.php was created in 2009 by the entomology group at the Southwest Florida research and Education Center (SWFREC/IFAS) in collaboration with the Florida Center for library Automation at the University of Florida. The objective of the greening database is to make available in a single location, the literature and all the information related to HLB, the vectors of Candidatus Liberibacter species, Diaphorina citri and Trioza erytrea, the effects of the disease on these insects and on plants, and also information related with the management techniques of the vectors and the disease. This information can thus serve as a useful tool for the growers, consultants, researchers and students. The database is continually updated and includes refereed and non-refereed publications, proceedings, presentations, reports, extension publications, periodicals, dissertations, book chapters and abstracts. Since October 2013, 249 new references were added to the database giving at the present 3,378 references with more than 92.5% of them linked to the original resources. The database has open information from around the world and some of this information is in different languages such as English, Spanish, Portuguese, French, Japanese and Chinese. Last year, the Citrus Greening Bibliographical Database was presented in two nationals entomological meetings: the 96th Florida Entomological Research meeting (July 14-17 2013, Naples, Florida) and the 61st Entomological society of America meeting (November 10-13 2013, Austin Texas). Our goal in this project is to continue uploading the most current HLB related information.
Obj 1A: All components of the user-friendly web versions of the single (sTCW) and multi-(mTCW) psyllid transcriptome databases have been finalized in preparation for the public release, which will allow for rapid target identification based on differences in expression, and in combination with predicted functions and tissue tropisms in the midgut and salivary glands. Twenty-two transcripts with predicted importance in nutrition, adhesion, immunity, and defense have been validated (mRNA) for ACP adults by RT-PCR. Two manuscripts reporting these efforts are ready for immediate submission. Obj 1B: Yeast-2 hybrid studies to capitalize on in vitro protein-protein interactions important in psyllid-Liberibacter interactions that support CLas infection, multiplication, and circulation in the vector that facilitate CLas infection of citrus plants. Previously, reported 15 CLas candidate genes from a list of 25 putative candidates (identified in silco using the db, obj. 1), were cloned into the Yeast 2 Hybrid (Y2H) mating experiments using the ACP gut and salivary gland libraries. To date, 17 gut library matings and 16 salivary gland library matings have been performed. Data analysis has been completed for 33 of those experiments with the remaining being in various stages (PCR, cloning, sequencing, etc.), and moving towards completion. From the 33 experiments, 50 putative ACP gene products of high interest were identified as candidates for RNA interference (RNAi) testing. Previously we reported results of the pilot test hits, optimization of RNAi feeding/transmission assays, and qPCR analysis of CLas detection in psyllid- inoculated tomato plants. Revised efforts are directed at promising candidate effectors that will be moved into the RNAi testing, many which are predicted to be important in Liberibacter adherence to host tissues. We last reported results from 9 ACP candidates mated against the CLas prey library, showing two ACP candidates with putative functions in invasion/adhesion and defense, interacting with three different CLas proteins, including a CLas pilus-associated protein. At present, 14 candidate ACP genes putatively involved in bacterial adhesion, endocytosis, nutrition, and defense were mated against the CLas prey library. Several interacting gene products (‘prey’ inserts) have been identified and additional candidates are under analysis, including a putative virulence factor identified using an ACP clathrin-like protein as bait. The identification of a potential CLas virulence factor from a mating using this ACP gene highlights the importance of our bi-directional Y2H analysis (‘ACP bait’ against ‘CLas prey’ and ‘CLas bait’ against ‘ACP prey’). This Y2H result suggests that the knockdown of this ACP candidate may inhibit CLas transmission. The identification of ACP/CLas interacting partners and subsequent knockdown of the ACP interactors is ongoing. The confirmation of Y2H interacting proteins and identification of additional interactors using immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are underway. Two CLasY2H candidates with putative involvement in adhesion and quorum sensing were selected to optimize IP and Co-IP assays among the high number of ACP ‘interactors’ identified thus far by Y2H. Outputs from this approach will be native interacting proteins for mass spectrometry identification following confirmation by western blot analysis. Obj 2: RNAi studies are underway to functionally validate candidate effectors that pass all validation steps. To this end, good quality dsRNA has been synthesized for eleven psyllid genes predicted to be involved in cytoskeleton formation, defense response, vesicle transport or transcytosis, and nutrition. Previously we reported on the knockdown of 6 of these genes on Liberibacter transmission using oral, topical, or and microinjection delivery. To date, dsRNA for 10 genes have been tested for adult PoP psyllids, and knockdowns have been assessed by qPCR analysis, with a range of 40-94% knockdown per target transcript. They also have been tested in our transmission bioassay, with five showing reduced transmission frequency. Confirmation of knockdowns and transmission experiments are ongoing for additional candidates.
In this proposal our objective is to find citrus versions for the two proteins that make up the functional components of a chimeric antimicrobial protein (CAP) previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have successfully identified a suitable replacement for the first component, the human neutrophil elastase (HNE). We obtained a close match with the tomato PR14a protein that is highly conserved in both Citrus sinensis (Cs) and Citrus clementina (Cc) genomes. We have completed the construction of two synthetic genes that encode this protein, one that just contains the coding region of CsP14a and the other is a chimeric version that contains the CsP14a coding region linked to the CecropinB (CecB) protein. We have also completed the construction constructing two CaMV35S expression cassettes to express both these synthetic genes so that the expressed proteins can be secreted. We have used CLASP to identify a citrus replacement component for CecB. Since CB has no enzymatic activity, we could not use a well-constrained motif like an active site. We chose instead the structural motif Lys10, Lys11, Lys16, and Lys29 a unique feature of CecB. We have used an approach similar to that described earlier to identify a replacement for HNE to identify a replacement component for CB. However, instead of comparing the reactive atoms as was done for the HNE matching algorithm, in this case we match for the C-alpha atoms of specific residues based on the overall shape of CB. Thus, we adopted a slightly different computational flow in this case. First, we did a keyword search, ‘plants’ in http://www.pdb.org/ that yielded about 1000 proteins. Each pdb was expanded on basis of each chain. For example, PDB X.pdb with chains A and B resulted in PDB files XA.pdb and XB.pdb. This list was filtered based on a 80% similarity redundancy. Then proteins larger than 60 aa in length were ignored. APBS (Adaptive Poisson-Boltzmann Solver) software developed to examine electrostatic properties of proteins at a nanoscale) was run on each pdb (the ones that failed electrostatic analyses, a few, were excluded). We were able to extract 200 proteins which we then analyzed further using CB motif. The CB structure is characterized by two helices. We chose specific residues from these two helices such that the residues were polar, and had stereochemical matches. For example, Lys can be replaced by either Arg or His, and thus constitutes a good candidate. We chose, Lys10, Lys11, Lys16 and Lys29 as the input motif from CB, allowing Lys to be matched by Lys, Arg or His. Since the 52 aa H+ATPase (HAT) is large so we cannot have it cheaply synthesized as a protein. We are developing additional computational tools to better evaluate individual alpha helical domains so we can identify smaller proteins that can be more cheaply synthesized for testing. We have also begun making a CAP construct where CB is replaced by HAT. This will be expressed in plants to evaluate antimicrobial properties.
Our accomplishments are: 1) We have transformed juvenile explants of succari sweet orange and carrizo citrange using transformation enhancing genes (K and I genes) we have constructed. The use of the K gene leads to 7-15 fold increases in transformation efficiency while the use of the I gene had 4-9 fold increases in transformation efficiency when compared to a conventional Ti-plasmid vector containing no K or I gene. However, many adventitious shoots produced from the I gene containing Ti-plasmid vector are abnormal. 2) We conducted one transformation experiment using explants from adult trees but the results were not satisfactory due to some unexpected problems. We are preparing more tissues from adult trees grown in greenhouse for transformation using these constructs. 3) We are constructing the other transformation enhancing gene constructs and they should be done soon for testing.
Efforts continue to obtain data for further model development and validation.
Data have been gathered from three growers, and the data were prepared for analysis. A series of statistical models were fit to assess the association between yield and the application of enhanced ground and foliar fertilization in the presence of HLB. Some promising relationships were found. However, the data are sparse, and strong recommendations should not be made from these data alone.
Data have been obtained from three additional locations. Currently the focus is on getting the data into a common format. Then the data will be cleaned and further modeling will be conducted to explore the association between yield and enhanced ground and foliar nutrient programs in the presence of HLB.
Understanding the transmission of CLas within the citrus tree remains one of the principal obstacles in the global efforts to undermine the pathogenicity of HLB (citrus greening). The movement of CLas has been assumed to follow the photoassimilate stream through the phloem. However, many observations based on our knowledge of the bacteria and general phloem anatomy have exposed inconsistencies with the accepted beliefs. The brevity of available information on the ultrastructural properties of citrus phloem sieve elements has hindered efforts to understand the spread of the disease within a tree. For example, lateral movement of CLas around an infected stem appears improbable given the size of cytoplasmic plasmodesmata connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from the roots to uninfected aerial tree parts through the phloem seems highly unlikely given the direction of phloem sap. To date we lack a thorough investigation into the ultrastructure of citrus phloem and the surrounding tissue, the potential pathways that CLas could utilize to move long distances through citrus trees, and the location of CLas habitat within different citrus tissue. Using a variety of grafting and girdling experiments, SEM, TEM, confocal, high resolution computed tomography, and PCR tissue analysis we aim to gain a better understanding of the anatomical traits that facilitate the spread of CLas through citrus. These data will allow us to develop new screening tools that breeders can use to select for resistant scion/rootstock combinations to confer resistance or tolerance to HLB. The girdling experiments have been finalized. All treatments were tested one last time and there was a high incidence of HLB transmission independent from the position of the budded material in relation to the partial girdle. Although there are still few trees that continued to be monitored, the results are clear that CLas (or an intrinsic signal) travels both in a longitudinal as well as a lateral way. The second set of grafting experiments, where graft bridges have been established between 2 independent plants, appear to be ready for the HLB-challenge. Two out of the 10 bridges appear unsuccessful, however. The remaining dead grafts were replaced with new material and are currently under observation. The initial 3 pairs of trees grafted under an initial project were tested once again. On this occasion, leaves from all branches were taken instead of one sample. The results were quite surprising in that in all trees, CLas had moved from the donor tree to the recipient tree through the xylem. This is the first data documenting xylem transmission of CLas. The anatomy of the citrus phloem has been completed and analysis of data is currently under way. A manuscript is being prepared for publication.
Understanding the proliferation and movement of Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (citrus greening), within the citrus tree remains a major obstacle in the efforts to undermine the pathogenicity and destructive nature of the disease. CLas still remains an unculturable organism, in part due to the lack of information on the phloem contents and internal environment. Once established in the phloem sieve elements, CLas movement within a tree has been assumed to follow the photoassimilate stream. Our current belief of the CLas transport mechanism is dependent on a presumed bidirectional flow of phloem sap, with basipetal movement from the site of infection, proliferation in the roots followed by systemic distribution through the tree. For CLas movement to occur under the current theory, CLas must be able to move laterally around the stems, trunks, and young roots, and then travel bidirectionally within the phloem sap. However, determinations based on general phloem physiology, our limited understanding of CLas, and on current anatomical studies have exposed serious inconsistencies with the accepted beliefs of CLas phloem transport. For example, based on general phloem anatomy and our current microscopy observations, lateral movement of CLas around a stem appears improbable given the size of cytoplasmic plasmodesmatal connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from infected roots to healthy aerial tissues through the phloem conduits is difficult to reconcile without a reversal of the phloem flow, a condition not demonstrated for any evergreen species. The objective of this study is to define the biochemical properties and transport direction of citrus phloem elements that support CLas proliferation and allow movement within a citrus tree. The data will provide the basis for developing culturing conditions for the bacteria and for mitigating strategies based on movement of the phloem sap. The phloem sap composition part of the proposal is under way as planned. Samples are being conducted monthly from HLB and healthy trees gown in shade greenhouses. Samples are being stored at -80F until all samples are collected to be analyzed under the same conditions. Preliminary results indicate significant differences on both HLB+ and healthy at different times. Data on phloem sap movement in young tissues has been completed. Is likely that an additional experiment will be carried out using a different dye to monitor phloem sap. For the determination of phloem movement in stem tissue during different seasons, the designed electronic device went through a series of tests and improvements in data collection and transfer, as well as in energy use and storage. The devices have been installed in trees grown on the field and are collecting data every 20 minutes. Devices have been placed in “healthy” as well as HLB trees. Additional devices are being built to perform tests under control conditions in the greenhouse.
Understanding the transmission of CLas within the citrus tree remains one of the principal obstacles in the global efforts to undermine the pathogenicity of HLB (citrus greening). The movement of CLas has been assumed to follow the photoassimilate stream through the phloem. However, many observations based on our knowledge of the bacteria and general phloem anatomy have exposed inconsistencies with the accepted beliefs. The brevity of available information on the ultrastructural properties of citrus phloem sieve elements has hindered efforts to understand the spread of the disease within a tree. For example, lateral movement of CLas around an infected stem appears improbable given the size of cytoplasmic plasmodesmata connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from the roots to uninfected aerial tree parts through the phloem seems highly unlikely given the direction of phloem sap. To date we lack a thorough investigation into the ultrastructure of citrus phloem and the surrounding tissue, the potential pathways that CLas could utilize to move long distances through citrus trees, and the location of CLas habitat within different citrus tissue. Using a variety of grafting and girdling experiments, SEM, TEM, confocal, high resolution computed tomography, and PCR tissue analysis we aim to gain a better understanding of the anatomical traits that facilitate the spread of CLas through citrus. These data will allow us to develop new screening tools that breeders can use to select for resistant scion/rootstock combinations to confer resistance or tolerance to HLB. The girdling experiments are well under way. Trees are frequently monitored for HLB symptoms and are routinely tested for HLB using PCR. HLB transmission has been confirmed in 40 of the 100 trees (20 trees per 4 treatments plus 20 controls). HLB transmission has been independent from the position of the budded material in reference to the girdled area. Many additional trees show classic HLB symptoms, but have resulted in PCR negative. These trees are being re-tested every 3 months. The second set of grafting experiments, where graft bridges have been established between 2 independent plants, appear to be ready for the HLB-challenge. Two out of the 10 bridges appear unsuccessful, however. These 2 pair of trees will be re-grafted while the eight remaining pairs will continue with experimentation. The anatomy of the citrus phloem has been completed and analysis of data is currently under way. A presentation on these matters was made at the Annual Meeting of the Florida State Horticultural Society.
Understanding the proliferation and movement of Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (citrus greening), within the citrus tree remains a major obstacle in the efforts to undermine the pathogenicity and destructive nature of the disease. CLas still remains an unculturable organism, in part due to the lack of information on the phloem contents and internal environment. Once established in the phloem sieve elements, CLas movement within a tree has been assumed to follow the photoassimilate stream. Our current belief of the CLas transport mechanism is dependent on a presumed bidirectional flow of phloem sap, with basipetal movement from the site of infection, proliferation in the roots followed by systemic distribution through the tree. For CLas movement to occur under the current theory, CLas must be able to move laterally around the stems, trunks, and young roots, and then travel bidirectionally within the phloem sap. However, determinations based on general phloem physiology, our limited understanding of CLas, and on current anatomical studies have exposed serious inconsistencies with the accepted beliefs of CLas phloem transport. For example, based on general phloem anatomy and our current microscopy observations, lateral movement of CLas around a stem appears improbable given the size of cytoplasmic plasmodesmatal connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from infected roots to healthy aerial tissues through the phloem conduits is difficult to reconcile without a reversal of the phloem flow, a condition not demonstrated for any evergreen species. The objective of this study is to define the biochemical properties and transport direction of citrus phloem elements that support CLas proliferation and allow movement within a citrus tree. The data will provide the basis for developing culturing conditions for the bacteria and for mitigating strategies based on movement of the phloem sap. This project is well under way, with all preliminary work completed within this period. To determine phloem movement, two separate devices have been constructed following existing literature. To follow phloem sap movement in young tissue, a device consisting of 2 arm probes has been successfully tested. In one arm, a UV light source and fluorescent sensor are mounted on the tip, whereas the second arm probe contains just fluorescent sensor. An externally applied fluorescent phloem-mobile substance is excited upon passage through the UV and its fluorescence detected by both probes as it moves down with the phloem sap. Time is recorded and velocity can be calculated. For phloem movement in mature tissue, an electronic device was constructed. The device is based on heat transfer through the plant cortex. It contains 2 heat sensors that will determine time of heat transfer from a centrally located heat supply. The instrument is being tested in greenhouse trees. Once completed, other sill be constructed to commence with field experiments.
Understanding the transmission of CLas within the citrus tree remains one of the principal obstacles in the global efforts to undermine the pathogenicity of HLB (citrus greening). The movement of CLas has been assumed to follow the photoassimilate stream through the phloem. However, many observations based on our knowledge of the bacteria and general phloem anatomy have exposed inconsistencies with the accepted beliefs. The brevity of available information on the ultrastructural properties of citrus phloem sieve elements has hindered efforts to understand the spread of the disease within a tree. For example, lateral movement of CLas around an infected stem appears improbable given the size of cytoplasmic plasmodesmata connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from the roots to uninfected aerial tree parts through the phloem seems highly unlikely given the direction of phloem sap. To date we lack a thorough investigation into the ultrastructure of citrus phloem and the surrounding tissue, the potential pathways that CLas could utilize to move long distances through citrus trees, and the location of CLas habitat within different citrus tissue. Using a variety of grafting and girdling experiments, SEM, TEM, confocal, high resolution computed tomography, and PCR tissue analysis we aim to gain a better understanding of the anatomical traits that facilitate the spread of CLas through citrus. These data will allow us to develop new screening tools that breeders can use to select for resistant scion/rootstock combinations to confer resistance or tolerance to HLB. As of this progress report the Valencia/Swingle trees have had HLB+ tissue grafted onto them. Approximately 90% of the grafted tissue has produced new flush and we are waiting for the remainder to produce new tissue. The girdling experiments have been completed and we are actively monitoring the wounds and the healing process to make sure that the redifferentiated tissue does not act as a pathway for CLas. We have hired a part-time employee to begin learning the microscopy techniques so they are well positioned to start the anatomical analysis once the trees are ready. Tissue samples have been collected and the SEM and light microscopy imaging are under way. Samples have been collected for PCR analysis. The second set of plants have been repotted and are now grafted together to form bridges between individual plants where either the phloem or the xylem is the isolated pathway. Tissue samples were collected for initial PCR analysis and samples will be collected in three month intervals.
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