Fruit quality, yield, tree health, and tree size data were collected from eight late-season rootstock field trials. All trials appear significantly affected by HLB. Some selections, including US-942, appear to perform better than other standard rootstocks in locations being affected by HLB. Rootstocks that appear to have more tolerance to HLB were identified, and selected rootstocks propagated for increase and inclusion into more and larger scale field trials. Fruit quality data was collected at multiple harvest dates for a large grapefruit rootstock trial in Indian River County. Significant differences were observed among the rootstocks for many fruit quality traits. For example, sour orange induced an intermediate level of total soluble solids early season and relatively high solids late season. US-897 induced the highest soluble solids early season, and US-812 induced the highest soluble solids late season, while X-639 and US-852 rootstocks induced the lowest soluble solids throughout the season. A field trial with new supersour rootstocks was planted at a site in Indian River County and will be used to evaluate trees for tolerance of Diaprepes, Phytophthora, and HLB. Rootstock liners were budded in preparation for planting in three new rootstock field trials later this year. Several thousand propagations of supersour rootstocks at USHRL were prepared for budding and use in field trials. Cooperative work continued with a commercial nursery to multiply promising supersour rootstocks to prepare trees for medium-scale commercial plantings. Work continues to assess supersour tolerance of CTV, salinity, and calcareous soils. The most promising new USDA rootstocks were identified for a cooperative effort with CRDF to place new rootstocks with HLB tolerance into larger scale commercial plantings. Detailed evaluation of specific defense-related citrus genes continued, including genes identified by expression studies as being associated with HLB response, such as CtCDR1 and CtPDF2. Constructs designed to alter expression of these citrus defense genes are being used to transform citrus for improvement of HLB tolerance and resistance, and derived transgenics will be tested using the pathogen. In a collaborative study with a University of California team and funded by CRB, we are comparing gene expression for trees infected with HLB to those infected with CTV. There are some common elements to the two different diseases that present good opportunities to understand citrus defense response. A study to understand the interaction of rootstock tolerance with scion tolerance/susceptibility is being completed and will be published later this year. Work continues to examine the effect of grafting height on tree response when the rootstock is an HLB-tolerant type. More than 200 new transgenic rootstock selections with potential resistance to HLB were produced this quarter, targeting increased expression of the citrus resistance genes CtNPR1, CtEDS1, CtMOD1, CtEDS5, CtPAD4, CtNDR1, and CtACD1. Eighteen new transgenic rootstocks with selected antimicrobial genes were propagated and entered into a replicated greenhouse test with ACP inoculation to assess tolerance to HLB. Evaluation and indexing of three previous groups of transgenic rootstocks under test with HLB continued, with selected transgenics showing some promise. A paper was published describing the overexpression of a citrus NDR1 ortholog and increased disease resistance. Presentations were made at Florida Citrus Show, Ft. Pierce and Florida Citrus Growers Institute, Avon Park, with exciting new information regarding rootstock effect on tree performance with HLB.
In the past few months, the most significant progress made on the FT project was the completion of the new FMVcDNA27 construct, which contains an FT3 cDNA insert in the pCAMBIA2201 vector with a constitutive FMV promoter. This construct was created as a first step towards the development of a new FT3 construct with an inducible promoter. In order to ensure that the cDNA was as effective as the genomic, this FMVcDNA27 construct will be compared to the original p27 construct which contains a genomic FT3 insert in the pCAMBIA2201 vector with the FMV promoter. Transformation of Carrizo and tobacco tissue is already underway in order to compare the action of these two constructs. Additionally, we have arranged for the materials transfer of two inducible promoter systems from the Danforth Foundation. Both of these promoters are inducible by the chemical methoxyfenozide, a widely-available pesticide, approved for field use. One system is driven by the CsMV constitutive promoter, and the other by the RTBV vascular-specific promoter. Once we have verified that the smaller and more manageable cDNA is as effective as the original genomic version of the FT3 gene, we will begin development of the inducible promoter constructs.
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 3,000 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. 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. This report marks the end of the third quarter of the project, during which we have began large-scale production of CLas positive ACP. To date on this project, a technician dedicated to the project has been hired, a second career technician has been assigned part-time, two small air-conditioned greenhouses for rearing psyllids are completed and are functioning well, and 18 individually caged CLas-infected plants are being used to rear ACP for infestations. A total of 2,124 transgenic plants have passed through the screening program. A total of 43,680 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Maintaining an open infestation of infected psyllids (phase 2 of the inoculation process) was challenging this past quarter because of surprise pest problems, notably western flower thrips which invaded the greenhouse initially attacking new flush and then first instar psyllids (facultative predation). Tamarixia radiata invaded the house during January, killing a majority of psyllid nymphs. Measures have been taken to limit these unwanted pests, but these measures are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects for the control of spider mites and thrips.
The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, has spread to citrus growing regions nearly worldwide and adults transmit phloem-limited bacteria (Candidatus Liberibacter spp.) that are putatively responsible for citrus greening disease (huanglongbing). Host plant resistance ultimately may provide the most effective, economical, environmentally safe, and sustainable method of control. In earlier experiments we identified genotypes of Poncirus trifoliata and xCitroncirus sp. (hybrids of P. trifoliata and another parent species) that were resistant to ACP. One mechanism we investigated to see whether it contributed to this resistance was plant hormones. We sprayed salicylic acid, methyl jasmonate, and abscisic acid, which are all common plant hormones, on susceptible citrus plants to test the influence on host choice, oviposition, development, and survival of ACP. Abscisic acid cut the life span of adult ACP in half compared to untreated control plants. The plant hormones had no strong effects on host choice, oviposition, or development of ACP. We are preparing the data for publication in a peer-reviewed journal. Another mechanism that may confer resistance to ACP is the structure of the scelerenchyma (a fibrous ring) surrounding the phloem. The scelerenchyma in non-citrus plant species was found to prevent feeding by herbivorous insects. We are collaborating with a Research Entomologist at our facility to test whether ACP choose different feeding positions on leaves of resistant P. trifoliate versus suceptible xCitroncirus sp. We completed two replications and found differences in feeding position. Our collaborator is currently evaluating the position of salivary sheaths from ACP and the scelerenchyma in leaves using scanning electron microscopy. Collaborators at the Fujian Academy of Agricultural Sciences conducted free-choice tests with all major groups of citrus and found differences among and within groups. Most groups of citrus were colonized by ACP, but lemons were the most preferred group and sour oranges and kumquats were the least preferred. The differences among citrus varieties within a group may be useful because volatile and phloem contents that differ between the least and most preferred species can be compared. FAAS continues their screening of germplasm including CRC accessions. To date, there is obvious resistance to ACP in Poncirus and among some distant relatives of Citrus. There is also at least a little ACP resistance within most of the Citrus groups but, in general, there is no evidence that any Citrus germplasm would offer as much genetic resistance to ACP as Poncirus. Among trifoliate hybrids, there is some material that appears as resistant as Poncirus itself, indicating that traits of interest (greatly reduced oviposition, reduced longevity) were genetically inherited.
Construction of primary structure is completed except for fabric. Fabric has been delivered.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. Early in the next quarter Dr. Grosser is removing the unsuccessful trees from the first planting and planting additional transgenics among the promising trees still under trial. Additional plantings are welcome from the research community.
Evaluation of existing standard cultivars (‘Temple’, ‘Fallglo’, ‘Sugar Belle’, ‘Tango’, ‘Hamlin’, and ‘Ruby’) for HLB tolerance/resistance is underway . Trees were planted in 2010, using a randomized complete block design, at Picos Farm, Ft. Pierce, FL. HLB symptom development and tree growth (diameter and height) are being monitored on a monthly basis. All of the cultivars in this trial exhibit symptoms of HLB and have tested positive for Candidatus Liberibacter asiaticus (CLas). Results to date support earlier observations that ‘Temple’ and ‘Fallglo’ are in the most tolerant group. Anatomical features associated with tolerant vs. susceptible closely related genotypes are being investigated. EM and visual images are being compared. FT-IR spectroscopy, which can be focused on specific tissues, displays promising differences between resistant and susceptible material. Another project involves the treatment of various resistant/tolerant citrus accessions and susceptible standards with various concentrations of antibiotics to generate a range of CLas titer levels. This has been slightly delayed and in May 2013, budwood with various concentrations of CLas, derived from the antibiotic treated plants, will be evaluated for their potential to result in HLB symptoms in disease free material. The budded plants will be evaluated for growth and HLB symptoms development over a 2-year period. Temporal progression and systemic movement of the bacteria in the inoculated plants will be determined along with HLB symptom development, and growth of the plants.. Development of periclinal chimera using resistant geneotypes and standard varieties is in progress. In vitro shoots have been established from nodal and internodal explants excised from mature, certified disease free plants of Red Carrizo, Temple, Hamlin, and Valencia. After root formation, chimeras will be generated using a procedure developed by Ohtsu (1994). After successfully generating the chimeras with HLB resistant vascular system and good fruit using the previously mentioned cultivars, additional cultivars such as ‘Sweet Orange’ and grapefruit will be added to this study. One additional study has been added to the project. Screening and evaluating new scion materials is a lengthy process and require multiple testing locations. Due to the urgency to develop tolerant/resistant material, a shorter evaluation cycle procedure is being investigated. If this screening method is successfully, it may be useful to quickly identify new sources of resistance varieties that may enhance and improve citrus production in Florida.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 30 putative transgenic plants with citGRP1 were transferred to soil. They will soon be ready for RNA isolation and RT-PCR to check gene expression. About 10 transgenic Hamlin shoots with citGRP2 are in the rooting medium for rooting. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting. In addition numerous putative transformants are present on the selective media. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is finally completed. Sequence information was confirmed and used to transform Hamlin. Some kanamycin-resistant shoots have already been obtained. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and will be use to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Other targets identified in genomic analyses are also being pursued. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and queued for testing with no-choice ACP and then free-flying ACP infection.
By collaborating with Dr. Yongping Duan’s lab, we tested whether overexpression of the CtNH1 gene confers resistance to citrus Huanglongbing (greening). Eight CtNH1 plants from the three original NPR1 overexpression lines (CtNH1-1, CtNH1-2, and CtNH1-5) were chosen. These lines have been found to be resistant to citrus canker disease, indicating that the overexpressed CtNH1 is functional. As negative controls, eight ‘Duncan’grapefruit plants were used. Half of the plants were inoculated twice with infected plant materials by grafting, while the other half was inoculated once. The overexpression of CtNH1 in the inoculated transgenic individuals was confirmed by Northern blot analyses. Disease symptoms were scored three months from the latest grafting inoculation. For the plants inoculated twice, disease developed more than six months from the first inoculation. Five of the eight ‘Duncan’grapefruit plants showed typical greening symptoms and the other three plants did not. Therefore, the success rate of our inoculation method is ~63%. Five of the eight CtNH1 positive plants showed either no disease symptoms (four plants) or mild symptoms (one plant). However, the other three CtNH1 positive plants were diseased. We carried out quantitative-PCR (qPCR) assays to detect the populations of Ca. Liberibacter in the infected plants and the results are largely consistent with observations from disease assessments. Since there is no correlation between CtNH1 overexpression and reduced or absent HLB symptoms, our results suggest that CtNH1 may not confer resistance to the disease.
Oral uptake of dsRNA targeting specific Asian citrus psyllid genes can induce psyllid mortality and reduce Liberibacter titer in infected psyllids. Research has shown that Asian citrus psyllid (ACP) mortality can be induced when adults feed on leaves from citrus that have been infected with a Citrus tristeza virus (CTV) expression vector modified to produce dsRNAs targeting specific ACP genes. The ACP mortality was shown to be directly associated with dsRNA abundance within the leaves. Also, when ACP infected Candidatus Liberibacter asiaticus (CLas) are fed on these plants for 15 days, the remaining live psyllids do not contain detectable CLas. Furthermore, none of the emergent adults that developed on these plants, from eggs laid by the CLas carrying ACP, contained detectable CLas. This is in contrast to what was observed in adults obtained from plants producing dsRNAs targeting the jellyfish green fluorescent protein (GFP) used as a control. The Clas bacterium could still be detected in adult ACP that fed on GFP-dsRNA expressing citrus and also in some adults that developed and emerged on these plants. Studies continue on comparing the effect of multiple ACP gene targeted dsRNA molecules (individually and in combination) that are fed to psyllids either in diets or produced in citrus using the CTV vector. As part of this work, a method was developed that allows complete life cycle development of the ACP on excised citrus flush thus improving efficiency of studies of dsRNA feeding on ACP nymphal stages.
In the first three months of 2013, Citrus Core Transformation Facility (CCTF) continued to operate at the steady rate without any interruptions in production of transgenic plants. Within this period, CCTF received highest number of orders ever-twelve. Five orders were placed for production of transgenic Duncan grapefruit (pW14, pHGJ1, pHGJ2, pHGJ3, and pHGJ4); four for production of transgenic Mexican lime plants (pOA1, pOA2, pOA3, and pGF1); and three for production of Valencia plants (pOA1, pOA2, and pOA3). All the binary vectors received from clients were already mobilized into appropriate Agrobacterium strains and initial co-incubation experiments were performed. Most of the work done in the CCTF revolved around the recently placed orders. Regarding the production of transgenic plants, CCTF has achieved following results. Transgenic Duncan plants carrying genes from these different vectors were produced: fifteen from the pX4, twenty six from the pX7, thirteen from the pX11, ten from the pX16, three from the pX19, five from the pX28, twelve from pNah, and five from pBI121 vector (to serve as control plants). There were also twelve Duncan plants produced carrying the gene from pMED14 vector and one carrying the gene from pMED16. Despite the high flux of workers in the facility, productivity remained high. Every effort will be made to keep production of transgenic material at satisfactory level considering high volume of incoming orders.
All signed paperwork was received on 9/17/2012.
This is a new project but 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 activities. The project’s funding came available in November 2012, but as this is based on the foundation of the long-term breeding program, activities have proceeded on a continuing basis. Individual assessments of HLB field tolerance have been carried out in the vast collection of raw germplasm that exists on UF and collaborating growers’ property, throughout the fall and early winter 2012, and this will continue through the coming months. Twelve individual rootstock trials planted in SW Florida, the Indian River region, and along the Ridge have been carefully observed for performance against HLB. In several cases these observations were made in a quantifiable fashion, measuring tree growth and estimating severity of symptom expression. In rootstock trials with earlier maturing scions (Hamlin orange, Sugar Belle mandarin hybrid, and grapefruit), we have collected data to document yields, fruit and juice quality, and fruit drop, and to correlate the disease rankings with yield performance; there are striking examples of very healthy, albeit infected trees, showing high yields of normal fruit, depending upon the rootstock. In another cooperator’s grove, rooted cuttings of 150 rootstock selections, 7 trees per selection all grafted with Valencia, were planted as intersets in a one-year old grove. The interset trees, now 3.5 years old, were just assessed for HLB, and 30 of the selections were found to be completely free of HLB, whereas the overall HLB infection rate in the block is now approaching 40%. A second field day was just held at the St. Helena grove, highlighting the performance of over 70 rootstock accessions, with some trees approaching 5 years of age performing very well in the face of severe HLB and ACP pressure from nearby groves. Some experimental rootstocks have been found with very low rates of HLB infection (<15%), while the ordinary commercial rootstocks also planted there are approaching 70% infection. Rootstock seedlings of 100 accessions were previously grafted with HLB-positive Valencia budwood, and those growing out normal flush were selected and exposed to hot ACP populations in greenhouse conditions for one month. Under DPI permit, these trees were planted in a high-pressure, unsprayed grove on the east coast; nearly one year after planting, these trees were assessed and nearly 80% of them were found to be free of HLB symptoms. New trials have been planned and materials have been under production with commercial nurserymen, with plans to plant in early 2013.
HLB’s impacts have led to grower-directed interest in advanced production and harvesting systems with the potential for early and sustainable yield, ease of harvest and other management efficiencies. In the absence of a long-term HLB solution, grove life may be only 12-15 years. A different production approach is required, and higher density plantings with smaller trees managed with intensive cultural systems may be a solution. This project will identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Though funding became available 1 November 2012, the project was built on many years of previous efforts in rootstock development and field trial testing. Existing field trials were monitored for tree growth and disease incidence, including a portion of the St. Helena project planted with dwarfing selections, and a 40-acre Hamlin/Valencia cooperative [GFC] rootstock trial with trees planted between 300-500/acre. The latter planting is 3 years old, and yield data were collected on the Hamlin portion of the planting. The best yield was only ca. 0.5 boxes per tree across a whole bed planted to one rootstock, in this case C-35 citrange. An informal field day was held at this planting for scientists only, to engage other researchers in participating in the opportunity to assess field performance of these rootstocks from other perspectives. Observations of severity and frequency of HLB symptoms were recorded on the dwarfing rootstocks at the St. Helena planting, prior to recording a third year of yield data in the coming months. Seed trees for selected dwarfing rootstocks, already showing good performance, are being propagated, to support expanded trials in the future.
This three-year project is to continue the search and evaluation of citrus tree survivors found under high pressure of HLB and its pathogen, on the basis of additional visits to groves in severely HLB-affected production areas, primarily in Florida, but also in areas of southern China that we have visited previously. Past exploration in China has identified three such trees and at least one of these remains free of HLB after several years. The field visits will be conducted by our collaborators and ourselves, when possible; we expect to visit Florida groves seasonally every year, and southern China once within the 3 years. We have previously propagated a few trees from Pineapple budwood collected in Martin County. Two of 5 original source trees were found to be qPCR negative, while > 90% of the other trees in the block were dead. These trees are maintained in a greenhouse at the CREC and are being grown off to sufficient size for use in future experiments. This summer, we visited properties at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program, with the express purpose of identifying particularly healthy appearing trees, that can be found in blocks as HLB symptoms are becoming more widespread and obvious. These trees have been noted and marked on maps, and revisited these specific trees throughout November and December. Some have begun to display more obvious symptoms, but there remain several that seem unaffected. We will continue to monitor these. Additional locations in the Indian River area have been visited recently and we have noted apparently healthy trees for future monitoring. A trip to visit southern China was originally planned for year 2 of the project, but an opportunity arose in Oct/Nov 2012 to return to Jiangxi and Guangxi provinces, locations that have been visited previously as part of an earlier funded project (CRDF #68). Several reports were presented to the industry on these visits, focused on approaches to manage HLB. Southern Jiangxi produces navel oranges, and there are areas where ACP and HLB is intensively managed to maintain infection rates ~1%/year. We revisited these areas, going to the same groves visited earlier and found HLB is still very much under control. We visited other regions in Jiangxi, not seen previously, and found infection rates of 10-30% under less stringent management regimes. We returned to Guangxi and revisited groves we had seen twice before, under very strict ACP/HLB management and found these groves (mandarins and oranges) continue to thrive in spite of severe infections nearby. However, other locations previously seen with low infection rates were now showing substantially higher rates, again a consequence of less stringent management. Our collaborator in the search for escape trees in Guangxi continues to seek out survivors and performs inoculations on these in greenhouse conditions. To date, there have been no HLB-immune selections identified in Guangxi. We were unable to visit with the collaborators in Guangdong province on this trip, so we cannot update on the materials there.
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