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.
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. Funding for this project became available on 18 May, 2012, so no new activities have been initiated at this point. 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 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 have focused visits to 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, for future observations, as the season progresses and symptom expression increases in the autumn. They include commercial varieties on typical rootstocks, as well as on experimental rootstocks from our program, and some other citrus accessions.
We have reported previously on the escape trees that have been identified and assessed in collaboration with colleagues in Guangdong and Guangxi provinces in China. There has been no change in the status of the trees and no additional testing has been carried out. The trees propagated remain in their respective locations. The two trees free of HLB symptoms from Guangdong had been propagated in the greenhouse at the Guangdong Institute of Fruit Tree Research facilities. Some were grafted with HLB-infected budwood in the greenhouse, and others were planted in their research field to assess their reaction to natural inoculation with HLB. Under observations for several months, the propagated trees in the field, surrounded by severe HLB disease and intense inoculum and vector pressure in Guangdong, appeared not to show any HLB symptoms and no pathogen was detected by qPCR. Most of the trees propagated from the individual symptom-free tree found in Guangxi, that were inoculated in a protected greenhouse, were confirmed to be infected and all qPCR positive trees displayed HLB symptoms. Though they apparently were not resistant to inoculation, the question remains as to why the original source tree was not infected and symptom-free; the possibility of vector resistance in the host could be explored further. Unfortunately, the two scientists assigned to this project in Guangdong have moved to new positions; we are in discussions with the GIFTR director regarding the future status of the materials and the project.
Two trees were found growing in HLB-ravaged orchards in Guangdong and one in Guangxi province, which were free of HLB symptoms, while other trees planted at the same time were either dead or declining. The trees in Guangdong were propagated at the Guangdong Institute of Fruit Tree Research (GIFTR), and were repeatedly tested by inoculation with HLB-affected budwood . The original source trees were tested multiple times by qPCR and remained negative for HLB over time, as did the propagations inoculated in the greenhouse. One original source tree was destroyed during highway expansion. Several propagations of one selection were replanted in an infected orchard location at the GIFTR, under high inoculum and vector pressure, but they remained HLB-negative throughout the project. Their current status is unknown as the scientists working on the project with us have moved to other places. The HLB-escape tree found in Guangxi was transplanted to a protected location at the Guangxi Citrus Research Institute (GCRI). Several propagations were made from this tree and inoculated with HLB-infected budwood; most of the re-propagated trees exhibited HLB symptoms and were found to be qPCR positive. In conclusion, HLB-survivor trees were found through our collaborators in China, they were inoculated in greenhouses, and some remained symptom free and qPCR negative; one selection remained so in a field planting, throughout the duration of the project. To expand further our search for survivors, and to continue to learn about Chinese citrus industry adjustments in response to HLB, we established contact with a citrus extension specialist in the Fujian Provincial Academy of Agricultural Sciences. A valuable side benefit of the project was the opportunity to survey regions where HLB is severe and widespread; in doing so we visited groves that appeared to be unaffected by HLB though surrounded by severely declining groves. These surprising locations were seen both in Guangxi and Guangdong; these were mostly mandarin plantings but we also saw healthy sweet oranges in Guangxi. We investigated the management programs which enabled them to survive > 8-10 years in good health. We interviewed growers, pathologists, horticulturists, and entomologists associated with these healthy groves. We visited the Pinghe County pummelo production area in Fujian province, where we saw HLB managed through good psyllid control, good nutrition and subsequent tree health, and natural tolerance of the pummelo variety. We revisited specific orchards we saw previously in Guangdong and Guangxi in healthy condition in 2008, despite widespread infection of neighboring plots. One orchard in Guangdong was devastated with HLB in 2010, and were informed that fruit prices for this variety were very low and the grower had stopped taking care of his trees. Orchards revisited in Guangxi were a different matter. These orchards not only maintained their healthy condition, but were substantially more productive and healthier in appearance than two years earlier. These observations confirm the utility and effectiveness of the management strategy that was developed and employed under direction of the extension entomologist at the GCRI. Although located in different provinces several hundred miles apart, the key elements outlined to us were the same. These include critically timed and thorough pesticide applications, use of pathogen-free planting materials, and maintenance of tree health through good nutrition; when tree removal was in order, it was performed only once a year and after intensive psyllid eradication measures were taken. We have reported on our experiences and observations throughout China in several articles in The Citrus Industry, at many grower group meetings throughout Florida, as well as in California and Brazil. We learned that there may be potential to find mutants of commercial cultivars that are not affected by HLB in the field, though there remain many questions about underlying mechanisms. Nonetheless, there is justification to continue such efforts in China, as well as now in Florida. And also importantly, we learned many of the approaches that are employed to manage HLB in regions of China where the disease has been endemic for more than 100 years, and provided some degree of hope for the same in Florida.
Two trees have been found growing in HLB-ravaged orchards in Guangdong and one other in Guangxi province, that appeared to be free of HLB symptoms, while all other trees planted at the same time were either dead or declining, and replants likewise were afflicted. The trees from Guangdong were propagated at the Guangdong Institute of Fruit Tree Research facilities, the original source trees have been tested three times after propagation using standard RT-PCR protocols, and they remain PCR negative for HLB. Unfortunately we have learned now that one of the original source trees was destroyed as the orchard was removed for highway expansion, but fortunately the propagated trees remain alive. Repeated qPCR tests on the propagated trees have shown them to be HLB-negative. These greenhouse grown trees were inoculated with HLB-infected budwood in early spring 2010, but until now symptoms have been observed. The symptom-free tree found in Guangxi, which was transplanted to the Guangxi CRC, is housed under screen and pushed to produce additional propagation materials. Previously produced test trees that were inoculated remain symptom free and qPCR negative, according to reports from our collaborator.
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