Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas In order to screen a large EFR mutant population for the gain of responsiveness to elf18-CLas we have been developing reporter lines with GFP under the control of the various PAMP-inducible promoters (FRK1, WRKY30 and PER4) which could be used to screen with FACS. Of these lines PER4p:GFP produces the lowest background and clearest PAMP-induced expression. We have generated lines in both Arabidopsis cell suspension and plants. The cell suspension lines fail to respond after protoplast mediated transformation. We suspect that this is a consequence of the protoplasting procedure and are investigating alternative buffers which may enable detection of the reporter following elf18 treatment. We are currently bulking seed to test the stable transgenic plant lines to determine if protoplasts derived from plant tissue will be more reproducible. These lines have the additional advantage that some have been generated in an efr-1 background so any weak basal induction by elf18-CLas would be eliminated. In addition, we have been also investigating the possibility of targeting other PAMPs. To this end we conducted bioinformatics comparison of known PAMPs with those in C. Liberibacter asiaticus. From these search we identified CSP22 (Felix & Boller, JBC 2003, 278:6201) as a potential candidate, since it is conserved in the sequence required for recognition. After a long delay in production we have now received the CSP22-CLas peptide and are growing plants to test their activity. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. The manuscript relating to the generation of chimeric XA21:EFR receptors has now been accepted in PLOS Pathogens and will be online shortly. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Transformation experiments are ongoing; to date, a total of 10,556 ‘Duncan’ grapefruit, 2,025 sweet orange and 191 Carrizo segments have been collectively transformed with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 (empty vector control). Regenerated shoots from transformed segments are being screened for GUS expression, and GUS positive plants are transferred to soil. So far, grapefruit plantlets (110) from all 4 constructs and sweet orange plantlets (17) from the 3 constructs EFR, EFR-XA21 and EFR-XA21-EFRchim have been transferred to soil.
A manuscript has been submitted, which summarizes scion (Hamlin, Valencia, Pineapple, and Ray Ruby) introduction into the growth facility by shoot-tip grafting (STG) from Dec, 2011 to July, 2013 (20 mo). Sixty-six mother plants out of 171 STGs introduced from FDACS were determined to be disease-free after micropropagation, budding, and disease indexing. A total of 157 putative transgenics were generated, 66 survived micro-grafting, and 42 expressed the NPTII protein. NPTII immunostrips, ELISAs and Southern blots were used to characterize the transgenics. Similar to previous reports in immature and mature citrus, there were a large number of escapes using kanamycin as the selection agent. The biggest loss during this period was due to micro-grafting as only 66 out of 157 (42%) GUS or GFP positive shoots survived. We have determined that micro-grafting success is dependent on the transformation batch and shoot age; it must occur early after shoot development. Alternately, rooting mature citrus must be established. Several of these transgenics flowered after the T5 fluorescent bulbs were replaced with LED lights, night temperatures decreased, drought stress applied, or if the trees were moved to natural light. Because our facility has no natural lighting, a greenhouse with natural and supplemental lighting is necessary to obtain early flowering and fruiting of desirable events. We continue to produce transgenic mature citrus scion and rootstock using plasmids with disease resistance genes obtained from various scientists. Since most of these constructs have no GUS or GFP markers, we micro-graft all shoots and screen with PCR, which is a more rigorous process than with reporters. Transgenics are double and triple checked with PCR and NPTII immunostrips to ensure they are stable, not chimeric, and expressing the NPTII protein. We have successfully micrografted at least as many putative transgenics. There are currently 3,600 putative transgenics in the pipeline to be screened. For replicated disease screening, the number of transgenics will be increased at least threefold by budding, and expression in vegetative progeny can be determined. For one particular genetic construct, budding with transgenic immature rootstock can begin at any time to facilitate experiments to determine the contribution of each genotype in imparting tolerance. One mature Swingle rootstock tree, transgenic for a disease resistance gene, is over four feet tall and should flower soon for seed production. We continue to optimize biolistics in order to increase our productivity. Thus far, the results are promising and we have recorded 200-300 transient GUS and GFP foci per shot in mature scion and rootstock shoots. If 0.01% of these foci develop into plants, 2 to 3 transgenics might be produced after each shot. During the optimization process, we have determined optimum stage height, gold particle size, and helium pressure. The primary advantage of using biolistics is that it avoids all of the antibiotics used to suppress growth of Agrobacterium, which also suppress shoot growth in scion and rootstock. The growth facility is being certified as a nursery. This will hasten the pace of providing plants to scientists, growers, and industry. Routine disease testing in April will be conducted by FDACS.
We have concluded the remaining activities of objective 1 of our proposal which have focused around finding a native citrus protein replacement for cecropin B the C-terminal component of the chimeric antimicrobial (CAP) protein. We had identified CsHAT52 using one set of bioinformatics tools and confirmed antimicrobial activity with a portion of this protein that we designated CsHAT22. Bioassay of CsHAT22 revealed a minimum inhibitory concentration (MIC) of 50 uM with Xanthomonas, 100 uM with Xylella and 300 uM with Liberibacteria crescens (Lc). As mentioned in our last report we used two additional bioinformatics programs, PAGAL and SCAPEL and have successfully identified and tested 2 additional proteins, CsPPC20 and CsCHITI25 that were compared to CB and the N-terminal 21 amino acids of CB designated CBNT-21. Among the test strains used Xanthomonas was most susceptible to the peptides with CB and CBNT21 showing and MIC values 25 uM and the MIC values for CsPPC20 and CsCHITI25 were 50uM and 100uM respectively. Both Xylella and the BT-1 strain of Lc gave MIC values of 200 uM for CBNT21 against both Xylella and Lc BT-1. CsPPC20 was more active than BNT21 against Xylella giving an MIC value of150 uM and as active against Lc BT-1 giving an MIC value of 200uM. CsCHITI25 was as active as CsPPC20 against Xylella but not as active against Lc. The CsISS15 peptide displayed no activity and this was an expected result based on the PAGAL predictions. Based on these results we have decided to include CsPPC20 as an additional construct for testing in planta. As mentioned in earlier reports we have CsP14a as a replacement for neutrophil elastase (NE). CTV vectors for expressing CsP14a, CsP14a-CB and CsP14a-CsHAT52 have been constructed and currently being used to infect citrus plants. Binary vectors for expression of CsP14a, CsP14a-CB and CsP14a-CsHAT52 have been constructed and have been used to generate transgenic tobacco and transgenic Carizo citrus plants. The construction of both CTV and binary vectors for the expression of CsP14a-CsPPC20 are currently under way. We have also used as a positive control NE-CB to develop plants with CTV based delivery and transgenic tobacco and transgenic Charrizo citrus that can be used to validate the efficacy of the citrus derived CAP proteins against HLB.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time (~50%) to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of December 12, 2014, a total of 6,402 transgenic plants have passed through inoculation process. A total of 124,795 bacteriliferous psyllids have been used in no-choice inoculations. Additionally, since our last report we have exposed 664 plants to a total of 14,060 infected psyllids in no-choice situations to answer questions about our inoculation procedures. For example, does the presence of flush enhance transmission? In a colony of bacteriliferous psyllids, why are there sometimes large fluctuations over time in percentages of psyllids that test PCR-positive for the pathogen? Are lemon and citron equally suitable for maintaining colonies of infected psyllids? How effective is our inoculation program? Some of these questions are being answered based on transgenic material that has already been passed through the inoculation process.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time (~50%) to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of March 31, 2015, a total of 7,066 plants have passed through inoculation process. A total of 138,855 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. As reported in December 2014, we initiated a series of experiments during fall 2014 specifically to evaluate inoculation success and to investigate different parameters related to the inoculation process. For example, does the presence of flush enhance transmission? In a colony of bacteriliferous psyllids, why are there sometimes large fluctuations over time in percentages of psyllids that test PCR-positive for the pathogen? Are lemon and citron equally suitable for maintaining colonies of infected psyllids? How effective is our inoculation program? Some of these questions are being answered based on transgenic material that has already been passed through the inoculation process. Recent feedback from inoculations of rootstock material gives some insight. Eleven groups of rootstock material (3,105 plants total) were passed through the inoculation program during 2011-2014. The percentage of success was 62% for assays conducted 12 to 19 months after inoculations. There was no difference in the success rates for transformed and non-transformed seedlings.
Transgenic plants containing our stacked transgenes are being clonally propagated for disease resistance evaluation and the first trees will be challenged for HLB resistance in spring 2015. Improving Consumer Acceptance: Following the successful demonstration of the inducible cre-lox gene system, the plant transformation vector has been modified to contain our NPR1 gene and Agrobacterium mediated citrus transformation is underway to incorporate this gene. Induction of early flowering to reduce juvenility (Carrizo transformed with the FT gene): After numerous attempts, we have finally produced transgenic Carrizo citrange plants expressing the clementine-derived CFT3 gene. Several of the plants have flowered once in the greenhouse in the juvenile state. These plants have been micrografted to standard rootstock and are in the greenhouse for further evaluation and observation. The new transgenic field site at the Southwest Research and Education Center (working with Dr. Phil Stansly) was successfully established, and approximately 320 transgenic citrus plants were planted as follows: Constructs: pCIT 107O (35s-CEMA) Line/Events: 15 Constructs: pCIT 109 (35s-SABP2) Line/Events: 24 (SABP2 is a SAR-inducing gene showing great promise) Constructs: pCIT 109A (AtSUC2-SABP2) Line/Events: 57 Constructs: pCIT105 (35s-CEME) Line/Events: 34 Constructs: pLC 216 (35s-LIMA) Line/Events: 190. Note: most of these are transgenic LIMA rootstocks (Carrizo/Orange 16) with non-transgenic Valencia scion. Plants in our Indoor RES structure have not flowered this year. It is possible greenhouse temperature may have played a role in the flowering process. We will attempt to keep the greenhouse unheated this fall in hopes of initiating flowering in spring 2015. We have achieved rapidly growing transgenic sweet orange trees through thorniness. We are also planning an outdoor RES type structure for transgenics.
St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 5.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). The 2nd annual application of CRF was applied, this was the 2nd application that contained TigerSul micronutrients. The formula was modified according to our greenhouse results, and included increased concentrations of manganese and boron. Trees appear to be responding well, as even some control trees on Swingle and Volk were showing a significant improvement in health. Approximately 120 trees were reset; we added trees on rootstocks US-897 and x639 for comparison. Rootstock trial at the GREC (Balm): a 3-year old trial of Vernia on 15 rootstocks (nearly 100% infected with HLB) was treated with Compo CRF (donated) and a blend of TigerSul micronucrients and poly-coated sodium borate (Florikan) – in an effort to see if the positive results in the greenhouse study could be extended to the field. This treatment will be repeated in January. Greenhouse Experiments – Nutritional study: highly symptomatic trees on various rootstocks were treated with the 3x overdose of TigerSul manganese and polymer-coated sodium borate (Florikan); several trees are showing recovery (new healthy growth). Protecting Seed Source Trees: Transgenic Orange #4 (UFR-2)plants containing the GNA transgene have been clonally multiplied as rooted cuttings and are being sized up for evaluation. Transgenic Orange #16 and Orange #19 (UFR-4) tetrazyg plants transformed with GNA have being clonally multiplied in a mistbed to provide replicated plants for evaluation.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, we have cloned all the components necessary for this study. The plant transformation vectors containing the GNA, APA and ASAL genes driven by either the root specific RB7 promoter or the citrus derived C1867 promoter have been constructed. Vectors containing the CpTI gene driven by a SLREO promoter that targets the transgene to the mature root cortex have also been produced. In addition, plant transformation vectors containing the gus gene driven by these root specific promoters have also been produced to demonstrate proof of functionality of the root specific promoters. Transformation experiments to produce genetically modified rootstocks with each of this promoters will be initiated in the next quarter as seeds become available.
HLB’s impacts have led to grower interest in advanced production and harvesting systems with the potential for early and sustainable yield, as well as ease of harvest and other management efficiencies. The goal of this project is to identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Existing field trials previously planted with size-controlling rootstock candidates have continued to be observed, including the portion of the St. Helena project planted with dwarfing selections, a 40-acre Hamlin/Valencia cooperative rootstock trial with trees planted between 300-500/acre, and a high density planting of LB8-9 (Sugar Belle). Data are being compiled on rootstocks to identify new candidate rootstocks for larger’scale ACPS trials. Seedlings of some Flying Dragon hybrids that have come through the ‘HLB gauntlet’ screening process (grafted with CLas-infected Valencia budsticks, and then cycled through a hot psyllid house, ending with no obvious HLB symptoms) were planted in the field, under a DPI permit for further observation. Seed were collected from previously untested Flying Dragon-derived hybrids, as well as from a range of other complex interspecific hybrids with tree size control potential; these seed were characterized for polyembryony and have been planted to assess relative trueness to type and seedling vigor; observations are in progress to identify the best candidates for new trials. A large scale, ACPS trial was planned in collaboration with a major citrus outfit, and seeds collected in 2013 have been provided to the propagating nursery.
HLB’s impacts have led to grower interest in advanced production and harvesting systems with the potential for early and sustainable yield, as well as ease of harvest and other management efficiencies. The goal of this project is to identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Existing field trials previously planted with size-controlling rootstock candidates have continued to be observed, including the portion of the St. Helena project planted with dwarfing selections, a 40-acre Hamlin/Valencia cooperative rootstock trial with trees planted between 300-500/acre, and a high density planting of LB8-9 (Sugar Belle). Data are being compiled on rootstocks to identify new candidate rootstocks for larger’scale ACPS trials. Visits were made this summer to three different trials featuring tree size controlling rootstocks by team members, and assessments of tree conditions were made. Seedlings grown from previously untested Flying Dragon-derived hybrids, as well as from a range of other complex interspecific hybrids with tree size control potential, were grown to assess seedling vigor and other characteristics; selections were either discarded because of poor growth habits, excessive phenotypic variation, or poor germination. The best performers from this group were listed and substantial quantities of seed have been or are being harvested currently, to be available for new plantings and trials in the next year. Seeds are being harvested from rootstock hybrids that are bearing their first fruit, to be evaluated for potential in greenhouse tests in the coming year. Additionally, large lots of seed from candidates for ACPS planting already identified from existing field trials are being harvested to prepare for new trial opportunities in the next year. Finally, a field day has been planned to showcase some of the CRDF Matrix rootstocks in a trial near Vero Beach, several of which are showing good performance even though they are affected by HLB, and with ACPS potential by virtue of smaller tree size combined with higher yield efficiencies.
The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped from the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL, to Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, in early October, 2014. An additional seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped to Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. In summary, a total of 15 independent transgenic lines now exist for the FLT-antiNodT fusion protein expression construct. These plants will need to be grown for some time to produce larger plants, which will be used to vegetative propagation of multiple individual plants for each independent transgenic line. Interestingly, at least one of the transgenic lines at University Park is blooming, despite being less than 8 inches tall. This is an expected phenotype of the transgene, since overexpression of the FLT domain of the engineered FLT-antiNodT fusion protein is expected to trigger precocious flowering due to the “florigen” blooming promotion activity of the Flowering Locus T (FLT) protein. This bodes well for the successful expression of the FLT-antiNodT fusion protein from the transgene in these plants. Plants at University Park will be used for analysis of the transgene expression, while plants at Fort Pierce will be tested for resistance to citrus greening, in collaboration with Dr. Gottwald.
Leaf samples were collected from 895 of the original 912 plants that covered the whole field population (116 individuals in total) in Fort Pierce this May; 17 plants were no longer living from the original planting. Real-time PCR was used on these samples to quantify CLas levels, as a measure of hist responses to HLB disease. A total of 16 individuals were found to be basically PCR-negative for CLas (average Ct value >35); these included 6 Poncirus parents and 10 F1 hybrids, of which 4 F1 progenies were completely PCR-negative to for CLas (each repeat’s Ct value >35). Compared with the results from last October 2013 on the same population, the PCR-based measures of CLas were more serious; the average Ct value of the whole population in October 2013 was 32.6 but is now 31.2 for this May 2014). Last October, 6 F1 hybrids were completely PCR-negative for CLas (i.e., no CLas was detected in any of the replicates of each hybrid); however, in our May assessments, two of these F1 hybrids now had some individual replicates wherein CLas was detected although at rather low titer. Notably the other 4 F1 hybrids with all replicates remaining PCR-negative are exactly the same individuals in October 2013 and May 2014. Besides, HLB symptoms ratings, canker symptoms were ranked and stem diameters were measured for the preselected monitor population (20 individuals comprising 164 plants); this information was recorded in June 2014. The results showed that the average rating (on a scale of 0-3, 0 being none and 3 being severe) for HLB symptoms is 1.4, and for canker symptoms is 1.8. Finally, we finished the propagations of the greenhouse backup population for the field trial accessions that could be propagated; at last we obtained a 257-plant F1 population derived from 73 progenies in total. These plants were transferred to another protected greenhouse structure waiting for the inoculation Candidatus Liberibacter asiaticus. The results from the natural infection assessments in the field will be compared with the artificially inoculated plants in the greenhouse.
This July 2014, leaf samples were collected from 887 of the original 912 plants that covered the entire field population again to quantify the CLas levels by real-time PCR. At this sampling time, 6 F1 progenies remain basically PCR-negative for CLas (i.e., most replicates remain with undetectable levels of CLas), of which 3 are completely HLB free among all replicates. The average Ct value of the whole population declined further to 29.9. Combining all results of PCR monitoring over time, 4 F1 hybrids generally have been for the most part CLas-free, except for occasional low-level and inconsistent detections of CLas by PCR in a small number of the replicates. Two F1 hybrids have always tested negative for CLas, among all the individual replicates across all sample dates. In addition, we collected samples from the selected monitoring population again, and we took an additional step to have greater confidence in our results; we quantified CLas using double reference genes for both the plant and the bacterium. The results verified the accuracy of using a single reference gene for the whole population (correlation R2=0.91). Further, this step also verified the 2 constantly and completely CLas-negative F1 hybrid progenies in all replicates of each. Finally, HLB symptoms, canker symptoms, and stem diameters were assessed again for the pre-selected monitoring population. The average rating across the entire population for HLB was 1.6 and for canker was 2.1. We have begun artificial inoculation experiments in the protected greenhouse structure, by testing a recently developed inoculation method recommended to us by a colleague, using sap extracts from PCR+ plants rather than grafting the infected budsticks. We have started experiments to determine the effectiveness of this approach in our hands using 3 year-old Valencia orange trees. Finally, we have produced a sequence-based marker genetic linkage map for this population, to support subsequent QTL mapping efforts.
We have continued to monitor previously identified candidate survivor trees at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program. Most of the trees now display symptoms after 2.5 years, and the number of unaffected has decreased. Further assessments of other populations of trees has revealed new candidates among younger trees that had not previously been surveyed. Trees identified at several locations through out Florida have been sampled for both budwood and root tissues. We have propagated rootsprouts, by budding and by rooted cuttings. Groups of small trees are growing of for further propagations to test their responses to HLB. We extracted DNA from these new rootsrpouts from other survivor trees and compared their DNA fingerprints with what we produced from feeder roots collected previously, as expected, the fingerprints were identical. The use of SSR DNA fingerprinting was supported by the enhancement supplement (now ended) and we are continuing this approach so at the least we can confirm whether the rootsystems sampled are nucellar or of zygotic origin. New rootstock samples collected from trees were used to confirm nucellar embryony. Thus far, all except one of the rootstocks collected has been shown to be a nucellar seedling of the presumed rootstock. Routine fingerprinting of all scion varieties sampled has shown them all to be true to type; this does not discount the possibility of mutations for HLB tolerance/resistance. We have gathered information on several other possible survivors identified and visited some groves to assess the condition of the reported trees. In several instances, the reported survivors actually exhibited more symptoms than would qualify as a ‘healthy’ survivor. Two hybrids of unknown origin that we have been observing for several years in CREC groves, and were found to be HLB-free have begun to display symptoms; one of these is declining quickly while the other is remaining healthy overall, despite symptom development in a small sector. New reports of survivors have been decreasing, though there have been some. Materials have been collected from an unusual grove in Lake County that appears to be completely free of HLB symptoms.
We routinely monitor previously identified candidate survivor trees at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program, including those more recently identified. We continue to grow out and propagate recovered rootsprouts and scion materials collected, to produce groups of trees that can be tested for their responses to HLB. We have been informed of some additional newly suspected survivors, and have cataloged the information for visits in the autumn and winter, when HLB symptoms become more obvious. The materials collected from a grove in Lake County that appears to be completely free of HLB symptoms have been propagated and increased. PCR results have been negative; these plant materials now have placed into a growth room with CLas+ ACP populations to attempt natural inoculation to assess their responses. Materials were collected from a legacy Parson Brown tree found in Marion County, and apparently free of HLB. Plans are in place to visit several newly identified candidate trees in the coming months, as well as to revisit trees from which samples were collected previously to determine their current status and performance; scion and rootstock materials will be collected, tested, and propagated, as seen fit.
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