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. 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 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 July 7, 2014, a total of 5,824 transgenic plants have passed through inoculation process. A total of 115,175 bacteriliferous psyllids have been used in no-choice inoculations.
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. 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 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 July 7, 2014, a total of 5,824 transgenic plants have passed through inoculation process. A total of 115,175 bacteriliferous psyllids have been used in no-choice inoculations.
The Core Citrus Transformation Facility (CCTF) continued to function as a platform for testing of different genes that could potentially render Citrus plants tolerant to greening disease. Almost all of the efforts in the facility are directed towards a single goal-producing valuable transgenic plants that may survive greening bacteria-induced infection. Due to a present way of functioning, in some periods of time the CCTF becomes, to a certain degree, an extended arm of research labs. As such, we follow developments taking place in those labs. Recently, five vectors that were previously supplied to the CCTF by one lab were modified and are being used in the latest co-incubation experiments. These five vectors could be considered as new orders. There were no other new orders. Within the last three months, CCTF serviced old orders but the work was also done on the recent order placed by the CRDF. The progress was made for all the orders. For the existing and new orders, additional co-incubation experiments were performed with the appropriate plant material and Agrobacterium strains. Selection of putatively transgenic shoots based on the PCR screen continued without interruption. Production of rootstock plants carrying the NPR1 gene requested by the CRDF is continuing as planned. High number of shoots (~800) was tested in the primary PCR and about 120 of them were positive. Because of our effort to accelerate the production of this material, shoots harvested from explants were placed on medium with gibberellic acid (GA3) to promote the elongation prior to primary PCR screen. Elongated shoots are much easier to graft. The treatment with GA3 turned out to be detrimental for many of these shoots and about 30 of them that were positive in the primary PCR screen were lost as their elongated stems did not sustain grafting well. Of those shoots that were positive in the primary screen and survived the grafting, 27 were moved from in vitro environment to pots. Four of those plants were negative in the secondary PCR while 23 were positive. Those 23 plants are growing well on the light bench in the lab. For the last three months, CCTF produced plants for the following orders: pX4- 18 plants, pNah-three plants, pX11- two plants, pHGJ2- two plants, pHGJ10+ pHGJ11-five plants, pNPR1-23 plants, pNPR1-G-three plants, pELP3-G-one plant, pELP4-G- one plant, pMG105- two plants, and pTMN1-seven plants. Within this group of transgenic plants, two were C. macrophylla, three were Valencia orange, four were Swingle citrumelo, 17 were Carrizo citrange, and 41 were Duncan grapefruit.
A genetic construct from Dr. Mou has been transformed into mature scion of Valencia, Hamlin, Ray Ruby, Pineapple, and mature rootstock of Swingle and Carrizo. A number of PCR positive, putatively transgenic shoots have been micro-grafted onto immature rootstock. There are numerous PCR positive shoots in all varieties, which will be verified by additional molecular methods once the plants are larger. PCR positive Pineapple sweet orange shoots have been secondarily micro-grafted in the growth room and are in soil. We have been able to promote rooting of immature tissues in one week with IBA but not NAA. After clonal propagation of desired transgenics, budding in additional combinations will commence. The capacity of our PCR screening methods has been increased using a direct tissue PCR method, which does not involve performing DNA extractions. Putative PCR positives can also been determined by fluorescence of the PCR reaction in the tube on a UV light, without the need to run a gel, although gel electrophoresis is still mandatory. Additional constructs obtained from other UF scientist(s) have been transformed into the appropriate scion and/or rootstock. Shoots have been regenerated and micro-grafted onto immature rootstock. For some constructs, sequence and maps have still not been obtained and therefore work has not commenced with these constructs. A second-hand laminar flow bench was procured for the growth room. This will become a dedicated micro-grafting station. The numbers of transgenic shoots that survive appear to be greater if micro-grafting is performed first and the shoots are screened later. Since none of our constructs have reporter genes, earlier detection by GFP florescence or GUS staining is not possible. Southern blots of plants transformed with marker genes are underway. The nonradioactive DIG labeling kit is being used, which required a few adjustments (e.g. different membrane, different probe generation) compared to the standard protocol using radioactive labeling. Copy number information and expression data for these transgenics are being compiled for a poster presentation at the annual American Horticultural Society (AHS) meeting in Orlando at the end of July, 2014. Once copy number and gene integration has been demonstrated, it will be possible to determine the number of false positives. The gene gun is operational and is being tested on mature and immature explants of both rootstock and scion. We have been able to obtain calli suitable for biolistics by indirect embryogenesis of immature citrus explants, and plant regeneration should be relatively facile. Mature scion will also be used in biolistics experiments and it is anticipated that plant regeneration will also be improved, without the inhibitory effects of the antibiotics used to prevent Agrobacterium overgrowth after transformation.
Our focus in the last quarter was to develop a greater diversity of candidates that could serve as replacements for the CecB lytic peptide domain of our CAP previously described (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We had successfully used the structural motif Lys10, Lys11, Lys16, and Lys29 a unique feature of CecB. We were able to identify 52 aa CsHAT protein that is highly conserved among citrus. Since the protein is too large to syntheize we have begun making constructs to express these in plants. We designed 3 constructs 1) CsP14a with a secretion sequence and Flag tag, 2) CsP14a ‘ CecB (this is construct 1 expressed as a CAP with the original CecB and 3) CsP14a-CsHAT52 (here construct 1 is linked to the 52 aa CsHAT protein from Citrus). These three constructs are being incorporated into CTV vectors so that they can be used for challenging HLB, this work is in progress. We have made Construct 1 and 3 in binary vectors and incorporated these into Agrobacterium strains and these are being used to transform tobacco, construct 2 is in the process of being made. In order to understand better the functionality of the alpha helical domains of CecB we have developed two new computational tools PAGAL and SCALPEL to better predict the antimicrobial activities in portions of existing proteins. PAGAL (Properties and corresponding graphics of alpha helical structures in proteins) implements previously known and established methods of evaluating the properties of alpha helical structures, providing very useful information of the hydrophobicity and charge moments. We have successfully used PAGAL to search 4000 plant proteins in the PDB database and we now have a database that contains a listing of the properties of each and every alpha helical structure present in all of these proteins and the properties of all of the alpha helical structures present in these 4000 plant proteins. We then developed the second program SCALPEL (Search characteristic alpha helical peptides in the PDB database and locate it in the genome) to search for alpha helical structures of a particular type. Once we obtain a particular hit that has the right properties that we are interested in investigating we then use BLAST to find the corresponding citrus protein. Using these two programs we have focused on our phase 1 search which is currently underway where we are examining all small proteins and will compare them to the N-terminal 21 aa domain of CecB that we call CBNT21 (+ve charge). We determined using these two programs that this domain to be the most active of the two alpha helical domains of CecB. We would predict would be the most active in terms of lytic properties. We identified three small proteins in citrus PPC20 (+ve charge very similar to CBNT21 in its structural properties); CsHAT22 (+ve charge smaller version of the 52 aa CsHAT that is currently being tested in plants), ISS15 (-ve charge, very small protein). We also will test CATH12 smallest definsin (12 aa) to define how small a peptide we can use successfully against our pathogens of interest. All 5 proteins have been synthesized and are currently being tested for their lytic activity against E.coli, Xylella, Xanthomonas and Agrobacterium to determine their range of clearance of these pathogens.
The main accomplishments during this quarter: 1) We repeated the transformation experiments with juvenile explants of succari sweet orange and carrizo citrange and we have confirmed drastic increases in transformation efficiency using the K or I genes. 2) We have confirmed most shoots of succari sweet orange and carrizo citrange hosting the K or I genes are transgenic. 3) We grafted some transgenic shoots onto rootstock and they have survived well, and in tissue culture vessels, we have observed little morphological changes if the K gene was used while we have observed a bushy phenotype were observed if the I gene was used. 4) Some grafted transgenic shoots were transferred to soil and grown in a greenhouse. These plants also exhibited normal growth characteristics compared to the control plants. These results demonstrate that the K and I genes can drastically increase transformation efficiency of juvenile tissues of succari sweet orange and carrizo citrange, suggesting they should be useful for adult tissue transformation. Further, these results also suggest that the K gene may not need to be eliminated in transgenic citrus plants. 5) We have put much of our time and effort on adult tissue transformation but nothing is significant to report at this time.
This report is for the one-year enhancement funding provided to this project. Original goals addressed simply the identification, propagation, and testing for HLB responses of rootstock and/or scion survivors. The enhancement expanded the amount of information to be obtained, specifically by SSR marker fingerprinting to assess trueness to type, particularly of the rootstocks. As a consequence, additional work in the labs and the greenhouses resulted, and appropriate personnel worked on the project to provide the additional information promised in the enhancement request for funding. We will continue these activities as the original project moves forward after the end of the enhancement funding period, June 2014, and we will continue to report on the results. 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 have begun to display symptoms after more than 24 months of observation, though there remain a few still unaffected as of March 2014. Trees at an abandoned location in Palm Beach County, that retain reasonably good condition and freedom from obvious symptoms, were sampled for both budwood and root tissues. Budwood samples were propagated onto healthy rootstock seedlings at the CREC. In addition, root samples were provided. We were able to collect rootsprouts from some of the trees that were decapitated; we have propagated from these rootsprouts, by budding and by rooted cuttings. These small trees are growing of for further propagations to test their responses to HLB. . We extracted DNA from these sprouts and compared their DNA fingerprints with what we produced from feeder roots collected previously, as expected, the fingerprints were identical. 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 it was decided to allow more time to determine whether the these trees warranted more careful analysis because they generally represented quite a few trees in each location. In other 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 minor and scattered symptoms. Recent qPCR has also shown the presence of CLas, though at very low levels; however, these trees are exhibiting ever-increasing severity of disease. New reports of survivors have come in from Highlands, Lee, Collier, Indian River, Lake, and Marion Counties; several have been documented, and some have been visited by the PI or extension personnel; materials have not yet been collected, pending further assessment of disease in 2014.
Certain citrus cultivars, such as Cleopatra mandarin, seem to be incapable of supporting the full developmental life cycles of psyllids. Preliminary experiments with hybrids in a Cleopatra-derived family, using caged psyllid nymphs on pesticide-free, field grown trees, indicated possible genetic transmission to some of the progeny. A total of 91 trees in three families produced by crossing Cleopatra mandarin with three male parents were selected from field plantings for the project and for future evaluations. We have propagated replicates from each individual for assessments of psyllid reproduction and feeding behavior in controlled greenhouse and laboratory conditions, and transferred these to the co-PI Dr. Rogers for greenhouse and lab studies, that will be conducted once the trees have reached sufficient size. Additional trees have been propagated from substantial numbers of the hybrids to provide increased numbers of replicates for the ACP reproduction and feeding behavior studies, and these are being grown until they reach adequate size for use in experiments. A first round of caged tree limb experiments using a subset of the total trees available was completed last year. The trees were selectively pruned to provide abundant young flush. Specific numbers of nymphs were placed onto each of two branches per tree with flush, and cages were carefully placed over the branches. Data on survival, numbers of adults, egg deposition, etc. were collected. Several of the hybrids were unable to support adult psyllid development on both branches. These results were compared to results from previous preliminary experiments conducted in the field, and essentially demonstrated the consistency of the response of ACP to specific individual hybrids. In addition, new candidate plants have been identified from other studies of HLB impact on diverse citrus germplasm, which have shown either no impact or dramatically delayed infection, and new hybrid families have been produced from some of these for possible future studies. These include certain mandarins as well as other complex citrus hybrids. We have continued to observe these new plants in the field. Additional propagations have been made to expand the available supply of plant materials for the greenhouse work to be conducted later in 2014, under the direction of Dr. M. Rogers.
This project is built on the legacy of materials produced and field trials planted across the past several years. The objectives are to evaluate existing families and created germplasm in the field and in greenhouses for their responses to HLB and citrus canker, to carefully observe and document rootstock effects on severity and rates of progression of HLB symptoms, and to maintain the facilities and activities involved in the state-wide assessment of new scion and rootstock performance with a focus on HLB responses. Assessments of HLB field tolerance are continuously carried out in the vast collection of raw germplasm that we maintain, and new selections have been identified, and several previously found continue to hold up to HLB; additional evidence is accumulating supporting what may be differential sensitivity to HLB among sweet orange clones from the CREC program. We have essentially completed observations for the season on fifteen different individual rootstock trials planted throughout the citrus production regions of FL. These trials examine a diversity of rootstocks from the CREC program, as well as those introduced by the PIs from rootstock programs in California, Spain, Italy, and Argentina; scions cover the wide range of sweet orange varieties (commercially available as well as new CREC oranges), red and white grapefruit, and several fresh fruit varieties (including Minneola, LB8-9 (Sugar Belle), and N40W-6-3 (Seedless Snack). These observations were made in a quantifiable fashion, measuring tree growth, estimating severity of symptom expression and either estimating crop loads or measuring fruit yields (in 5 of 15). We continued monitoring those already identified healthy, albeit infected trees, on various rootstocks in different trials, showing high yields of normal fruit; some of these were included in the CRDF Rootstock Matrix, and it is noteworthy that they have continued to perform well. Seed fruit was harvested and seed extracted from 8 of the UF/CRDF matrix rootstocks (UF releases UFR-1, 2,3,4,5,6,15 & 16), and is being made available to the industry, through licensed nurseries. Seeds from Green #2 (now released as UFR-17) were made available. Additional materials and from the Rootstock Matrix list have been sent to the DPI PTP for STG and indexing, so certified materials can be made available to nurseries and TC companies for rapid increase of those trees for subsequent field trials and demonstrations. Seeds were harvested from more than 150 new rootstock hybrids, and they were characterized for seed production and polyembryony; of these nearly 50 groups were planted out for assessment of trueness to type, currently underway. New sour orange-like rootstocks have been found with tolerance to HLB, and seeds from 2 of these have been sent to nurseries for new trials to be planted next year. A triploid sweet orange hybrid (C4-16-12) was approved for release; this hybrid contains 8% trifoliate orange and has so far shown no sign of HLB. Two new sweet oranges, selected on the basis of earlier fruit maturity but with Valencia quality, have been entered into the DPI PTP.
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 were monitored for tree growth and disease incidence, 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). Yield data were collected, tree growth (height and trunk diameter) was measured, and either fruit load estimates were made or yields and fruit/juice were determined. Every tree in the St. Helena project (>3000) was assessed for tree size, yield and HLB status; data is being compiled per rootstock and will help to identify the best candidate rootstock for larger ‘scale ACPS trials. Of the 75+ rootstocks in the trial, many are producing small-to-medium trees with potential for use in ACPS. Seed trees for selected dwarfing rootstocks, already showing good performance, were propagated and planted, to support expanded trials in the future. Additional new rootstocks from the CRDF Rootstock Matrix selected for their potential in high density plantings through good tree size control were entered into the DPI Parent Tree Program for cleanup by shoot tip grafting followed by indexing, to provide certified budwood of these rootstocks for commercial nurseries upon their release. Seedlings of some other Flying Dragon hybrids 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); these will be planted in the field, under a DPI permit for further observation, in June 2014. 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. Seed were extracted from fruit of four new rootstocks with good potential for use in ACPS (precocious bearing small trees); all are producing abundant polyembryonic seed as needed for traditional nursery propagation. Propagation for larger-scale trials is underway. A large scale, ACPS trial will be propagated under an MTA with a major citrus outfit, containing as many as 10,000 trees on several dozen rootstocks. Distribution of seeds from UFR-2, UFR-3, and UFR-4, all good candidates for ACPS plantings, has begun to licensed nurseries, thereby moving UF CREC rootstocks into the commercial realm for growers to test and deploy.
A number of Poncirus and Citrus cultivars have been recently found to be tolerant to HLB. Microarray-based profiling of the transcriptomes of two cultivars with HLB tolerance (Poncirus hybrid US-897and rough lemon) and two cultivars without HLB tolerance have identified over 1,150 genes that are differentially expressed in HLB-tolerant cultivars. These genes constitute a valuable pool of potential candidate genes from which true HLB tolerance genes may be identified. Additional candidate genes have recently become available from an RNA-seq experiment using rough lemon and sweet orange in a comparison similar to what was done with the Affymetrix microarray work in our lab (Fan, et al., 2012). This project aims to screen these potential candidate genes using high throughput target capture, massively parallel sequencing of targeted gene regions, and genetic association and linkage analysis to find the most likely candidate gene(s) for HLB tolerance in Poncirus and rough lemon. We have continued careful analysis of the RNA-seq data, looking at changes in gene expression over time. Recent application of Pathway Studios software has provided more powerful discrimination of critical genes in particular pathways associated with disease responses of plants. Candidate lists from the RNA-seq study have been developed based on comparisons within accessions over time, but we are working now to make more complex comparisons between accessions, while still taking into consideration changes within accession over time, to potentially enable a closer targeting of the most critical genes involved with differential host responses to CLas. We are currently using two parallel, but complementary approaches, to compile lists of candidate genes from all available citrus gene expression studies of host responses to HLB (microarray and RNA-seq), in sensitive, tolerant and resistant citrus accessions. This two-pronged approach can identify commonalities, as well as highlight possible targets that one or the other approaches might miss.
A number of Poncirus and Citrus cultivars have been recently found to be tolerant to HLB. Microarray-based profiling of the transcriptomes of two cultivars with HLB tolerance (Poncirus hybrid US-897and rough lemon) and two cultivars without HLB tolerance have identified over 1,150 genes that are differentially expressed in HLB-tolerant cultivars. These genes constitute a valuable pool of potential candidate genes from which true HLB tolerance genes may be identified. Additional candidate genes have recently become available from an RNA-seq experiment using rough lemon and sweet orange in a comparison similar to what was done with the Affymetrix microarray work in our lab (Fan, et al., 2012). This project aims to screen these potential candidate genes using high throughput target capture, massively parallel sequencing of targeted gene regions, and genetic association and linkage analysis to find the most likely candidate gene(s) for HLB tolerance in Poncirus and rough lemon. We have continued careful analysis of the RNA-seq data, looking at changes in gene expression over time. Recent application of Pathway Studios software has provided more powerful discrimination of critical genes in particular pathways associated with disease responses of plants. Candidate lists from the RNA-seq study have been developed based on comparisons within accessions over time, but we are working now to make more complex comparisons between accessions, while still taking into consideration changes within accession over time, to potentially enable a closer targeting of the most critical genes involved with differential host responses to CLas. We are currently using two parallel, but complementary approaches, to compile lists of candidate genes from all available citrus gene expression studies of host responses to HLB (microarray and RNA-seq), in sensitive, tolerant and resistant citrus accessions. This two-pronged approach can identify commonalities, as well as highlight possible targets that one or the other approaches might miss.
A number of Poncirus and Citrus cultivars have been recently found to be tolerant to HLB. Microarray-based profiling of the transcriptomes of two cultivars with HLB tolerance (Poncirus hybrid US-897and rough lemon) and two cultivars without HLB tolerance have identified over 1,150 genes that are differentially expressed in HLB-tolerant cultivars. These genes constitute a valuable pool of potential candidate genes from which true HLB tolerance genes may be identified. Additional candidate genes have recently become available from an RNA-seq experiment using rough lemon and sweet orange in a comparison similar to what was done with the Affymetrix microarray work in our lab (Fan, et al., 2012). This project aims to screen these potential candidate genes using high throughput target capture, massively parallel sequencing of targeted gene regions, and genetic association and linkage analysis to find the most likely candidate gene(s) for HLB tolerance in Poncirus and rough lemon. We have continued careful analysis of the RNA-seq data, looking at changes in gene expression over time. Recent application of Pathway Studios software has provided more powerful discrimination of critical genes in particular pathways associated with disease responses of plants. Candidate lists from the RNA-seq study have been developed based on comparisons between accessions, while still taking into consideration changes within accession over time, to potentially enable a closer targeting of the most critical genes involved with differential host responses to CLas. Gene ontology (GO) analysis has been employed to reduce the complexity of the differentially expressed gene list. We are conducting KEGG PATHWAY mapping, a process to map molecular datasets (such as the large-scale transcriptomic dataset we have generated by RNA-seq), to KEGG pathway maps for biological interpretation of higher-level functions. And finally, we are using Gene Set Enrichment Analysis (GSEA), to dissect sub-networks and their involvement in host responses to HLB. These various approaches to analyze such a tremendously complex dataset have revealed the critical roles of certain aspects of host defense responses, and that rough lemon plants display an increasing response to pathogen attack, compared with a decreasing response of sweet orange plants. Critical differences are implicated in hormonal responses and signaling, signal transduction, amino acid biosynthesis and roles in plant defense mechanisms; certain epigenetic responses have also been implicated in the differential responses to CLas, as well. Differences between the results obtained from microarray and RNA-seq investigations highlight the value of multiple approaches to study global gene expression. We are currently using two parallel, but complementary approaches, to compile lists of candidate genes from all available citrus gene expression studies of host responses to HLB (microarray and RNA-seq), in sensitive, tolerant and resistant citrus accessions. This two-pronged approach can identify commonalities, as well as highlight possible targets that one or the other approaches might miss.
Field performance of several new rootstocks has been far superior to standard rootstocks in field trials over 7 years with severe HLB infection pressure. Based on good field performance and superior yield, the rootstocks US-1279, US-1281, US-1282, US-1283, US-1284, US-1293, US-1317, US-1318, US-1319, and US-1321 will be released by USDA for commercial use. Fruit yield of Hamlin trees on these rootstocks is 2-4 times the yield of trees on Swingle in the same trials, and the trees also have fruit that is larger in size and higher in sugar content. The rootstocks US-896 and US-1516 will also be released by USDA, based on superior performance as compared with standard commercial rootstocks in other trials with severe HLB infection pressure. These promising new rootstock selections have been provided to Florida DPI for establishment of certified budwood sources and are being made available to the CRDF Product Development Project to establish large scale commercial field trials. A special permit has been obtained from Florida DPI to immediately begin establishing widespread commercial field trials using clean USDA sources of these new rootstocks. Based on this permit, cooperative arrangements are being made with commercial Florida nurseries for large scale vegetative propagation of these promising new rootstocks, as needed to meet commercial demand until adequate seed sources can be developed. A material transfer agreement was signed with Agromillora Catalana, SA, to immediately allow that company to begin rapid micropropagation of these most promising rootstock selections for use in Florida. Five new rootstock field trials were planted in spring 2014. Trees were prepared for planting of three additional field trials with Supersour rootstocks later in 2014. About twelve thousand new propagations of Supersour rootstocks were completed to prepare trees for budding and planting in additional field trials in 2015. Work began to construct an additional greenhouse at USDA to propagate Supersour rootstocks for field trials. Cooperative work continued with a commercial nursery to multiply promising Supersour rootstocks to produce trees for medium-scale commercial plantings, and an agreement was being set up with a second commercial nursery to begin similar work. Greenhouse studies continued to assess Supersour tolerance of CTV, calcareous soils, and salinity. Studies continued on defense-related genes and small RNAs associated with HLB infection, in collaboration with University of Maryland and University of California research groups. A study of localized defense gene expression in shoots and roots provided evidence of striking differences that are a major advance in understanding and yield strong insights into ways to overcome the disease. A study of the interaction between rootstock tolerance and scion tolerance/susceptibility is being prepared for publication and provides additional insights into disease progression and the potential for improved management. Two hundred new transgenic US-942 and Sour orange rootstocks were produced, targeting to increase tolerance to HLB by manipulation of the citrus resistance genes CtDIR1, CtAZL1, CtERF1, CtFMO1, and CtTGA7. Two replicated tests were set up to retest fifteen transgenic rootstock selections previously showing increased disease resistance due to overexpression of CtNDR1. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with HLB. Several transgenic rootstock selections showing increased resistance to HLB have been identified from groups transformed with other resistance genes, and are also being prepared for confirmation testing.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. We have modified the CTV vector to produce higher levels of gene products to be screened. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. We are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, but are eliminating many that are not effective and are focusing on about 20 that still are under test and about half of a dozen that have some activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. A recent advance is that has greatly speeded up our screen is that we now can estimate when plants become infected with HLB and can tell whether a peptide is working more quickly.
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