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. 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. So far, the 1/8th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
Two experiments were undertaken to evaluate the effectiveness of reflective mulch to repel ACP, reduce incidence of HLB, and improve growth of newly planted citrus compared to standard practices. The first trial planted 5 May 2013 at SWFREC consists of 24, 250 ft. rows of ‘Ray Ruby’ grapefruit on ‘smooth flat Seville’ divided into 8 main plots, half receiving organic amendments since 1993, most recently with 12 tons/ac composted horticultural waste applied in a 6 ft swath to the plant drill. The plot had been underlain with drain tile and was flat except for a 6 in beds 36 inches wide on 18 ft centers covered with polyethylene film mulch with irrigation provided through two drip tape lines. Each 3-row plot is divided into 2 subplots: whiteface or metalized mulch. One of 160 leaf samples collected 28 Jan 2014 tested positive for HLB came white mulch compost plot. A second round of HLB leaf samples were collected on June 25 which are now in the process of being analyzed. Of 258 ACP found on sticky cards, 133 were from white mulch+compost, 78 white mulch no compost, 30 in metallized mulch compost and 17 in metalized mulch no compost. Only 13 infested flush were found in trees on metalized mulch compared to 30 infested on white mulch no compost and 81 on white mulch with compost (N=17,638). Trees in in compost plots had 22% more flush shoots than trees without compost, with no effect of mulch type on flush. Two growth measurements indicated no significant differences between mulch types but larger trees in composted plots compared to no compost. Insecticide drenches are applied as needed as well as other standard grove maintenance sprays. The second trial was initiated at the Florida Research Center for Agricultural Sustainability in Vero Beach: Land prep was synchronized with tree planting and reflective mulch installation on a 15 acre site. Beds were initially mowed and new weed growth was treated with a systemic herbicide application followed by a contact herbicide. Fill sand was applied to low areas within the tree line to prevent standing water. Finally, beds were subjected to 2 passes with a rotovator and rolled twice to pack and stabilize the soil. A randomized complete design was used with 4 replicates and 3 treatments: beds covered with metalized polyethylene mulch, beds covered with urban plant debris (UPD), and bare ground. Mulch (6 ft. wide) was installed centered on the surveyed tree line using a plastic laying machine that buried the sides of the plastic leaving 5 ft. exposed. (‘Ray Ruby’ on ‘sour orange’ trees were planted at 12 ft. x 25 ft. tree spacing (145 trees/Ac) on 17 March 2014. Containerized trees (pretreated at the nursery with a neonicotinoid) were ‘mudded in’ according to commercial practices. After planting, the irrigation system was installed with two drippers (2 g/hr each) per tree arranged on each side within 6 in. of the tree trunk. The compost treatment was then applied as a 5 ft. wide by 3.5 in. deep layer UPD. Fertigation with a liquid 6-0-8 (with minors) was included all irrigation events. Imidacloprid was applied by soil drench for ACP control within 2 weeks of planting. Typical commercial young tree caretaking was continued including weed control, fertigation, and canopy sprays of insecticides, fungicides and foliar fertilizers. A second neonicotinoid (Belay 2.13 SC) trunk drench application was made for ACP control 65 days after planting. Field data collected included volumetric water content (%VMC) of soil and trunk caliper measurements for each tree.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July 2012 on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3.5 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. To date, 2125 psyllids have been found on sticky cards of which greater than 70% are in no-mulch plots while only 11% have been found in plots that receive insecticides. Only 2% of the ACP found on sticky cards come from plots with both mulch and insecticides. Thus far over 61,000 flush have been observed of which 7,934 were infested with ACP and 1685 were infested with Aphids. ACP and aphid infested flush are predominately in no-mulch plots with more than 65% of the total. Very few infested flush have been found in plots receiving insecticides (513 ACP and 558 aphid infested flush) while very few infested flush (100 ACP and 178 aphid infested flush) have been found in plots with both mulch and insecticide applications. Leaf samples for HLB testing were collected mid-January 2014 of which there were 20 positive samples none of which were from the mulched treatments while 12 were from the no mulch foliar nutrition without insecticide plots. All other non-mulch treatments were similar. The most recent HLB leaf sample was collected on June 16, 2014 but results are not yet available. Growth measurements made 2014 by measuring trunk diameter showed results of which are different from previous reports. Trees on mulch and not receiving soil drenches of systemic insecticide are 20% larger than on bare ground whether or not they are receiving foliar nutrition. However, mulch did not improve growth of trees treated with insecticide; actually the contrary. This may be due to failure of the irrigation system during the month of May resulted in worse drought stress in trees on mulch compared to bare ground trees that profited more from rainfall. Normal grove care operations continued which included one herbicide application in June of glyphosate, Kocide is sprayed monthly for control of canker, and one application of AgriMek to reduce ACP and other citrus pest populations to more realistic levels in all treatments. Verimark was applied as a drench in all insecticide plots late in June as well as a Nurpid application in May.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. ‘Hamlin’ orange on ‘Carrizo’ citrange rootstock was planted 3-4 July on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.643 degrees S. -81.455 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3.5 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 July 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 August. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. 2,655 ACP have been found on sticky cards of which greater than 70% are in no-mulch plots while only 14% have been found in plots that receive insecticide drenches. Only 3% of the ACP found on sticky cards come from plots with both mulch and insecticides. Thus far over 72,270 flush shoots have been observed of which 8,386 were infested with ACP and 1,737 were infested with aphids. Greater than 70% of ACP and aphid infested flush were found in no-mulch plots. Only 599 ACP and 559 aphid infested shoots were found in plots receiving insecticides with 122 ACP and 191 aphid infested shoots found in trees on both mulch treated with insecticide. Leaf samples for HLB testing were collected July 2014 of which there were 55 positive samples 12 of which were trees on mulch,10 from trees treated with insecticide with only 1 positive tree on mulch and drenched with insecticide. The most recent HLB leaf sample for nutrient analysis was collected on 16 June 2014 but results are not yet available. Growth measurements were made 1 July 2014 by measuring trunk area cross section. Trees on mulch and receiving foliar nutrition are now 20% larger than the no mulch no foliar nutrient control. However, trees receiving insecticide drench both with or without foliar nutrition but without mulch were larger than with mulch. The reason may be due to an irrigation system failure during the month of May which impacted trees on mulch more than those not on mulch which made better use of rainfall. Treatments applied: July 9: Foliar nutrition went out monthly Griffin Green 1gpa, August 5: 1 lb Fortress and 1 gal N-Sure in a 50 gallon volume, August 21: the entire field was sprayed by Duda with foliar nutrition 6-0-8 10 gpa, September 3: 1 lb Fortress and 1 gal N-Sure August in a 50 gallon volume. Normal grove care operations continued which included one herbicide application in September of glyphosate, Kocide sprayed monthly for canker control, and one application of Intrepid for leafminer control.
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.
The overall goal of this project is to 1) determine the overall effects of ACPS/open hydroponics growing systems on drought susceptibility and 2) the efficacy of plant growth regulators on mitigating the effects of preharvest fruit drop resulting from HLB. During the past quarter field data was collected for trees growing under traditional grower irrigation systems compared to ACPS “open hydroponics”. Additional data on the vulnerability to drought symptoms was also collected during this quarter on the two different experimental groups. The data are now being analyzed for a formal report/publication. Data collection will continue through the summer with support by the Horticultural Sciences Ph.D. student funded by this project. Preharvest fruit drop data from the 4-acre plant growth regulator trial in Lake Alfred are in the final stages of analysis. The anatomical data from citrus trees in the different experimental blocks are also in the final stages of sectioning and analysis. As part of an extension of this project a greenhouse study has been initiated to test the efficacy of 2,4-D and other plant growth regulators on root health in small trees with and without HLB. The plants have been potted, graft inoculated, and their initial root mass measured prior to the first applications of the PGR treatments. These trees will be evaluated regularly over the next six months for changes in growth and physiology in response to the PGRs and HLB. Additional field sites were selected in the Ft. Pierce area for expansion of the project to evaluate the efficacy of 2,4-D on preharvest fruit drop in grapefruit cultivars. The field experiment will begin this summer and preharvest fruit drop counts will begin starting in August or September.
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.
In this quarter over 12 tetracycline derivatives were designed and synthesized and examined for antimicrobial activity against the causative agent of HLB (citrus greening), as measured against the surrogate laboratory strain Liberibacter crescens. Synthetic modification of the starting tetracycline(s) have yielded separate series of compounds that are more potent (3 orders of magnitude) than EPA registered oxytetracycline, and semisynthesis has yielded extremely potent compounds following specific structure-versus-activity relationships. In one series, molecular descriptors describing increased activity against HLB have emerged, and further chemical changes will result in the most potent derivatives against HLB while remaining inactive against human microbial pathogens and displaying “non-antibiotic” activity. These in vitro studies against the surrogate strain have delineated superior compounds that are currently the subject of greenhouse studies of Liberibacter infection in young and infected citrus trees. Currently, three compounds are being evaluated in controlled studies of Liberibacter infection at the USDA, in the Shatters and Powell laboratories, whereby young trees will be treated with the test compounds and the bacterial titer of the trees determined after dosing using different conditions and using differing formulations. It is expected that bacterial infection levels would be reduced and readily measured by quantitative PCR over time, and the dosage modes and concentrations adjusted in further studies to optimize anti-HLB activity. In other studies being conducted in the Gonzalez laboratory (UofF) three compounds, including the most active recently determined from in vitro studies, have been synthetically scaled up for further testing in greenhouse studies complementing those ongoing in greenhouse field trials. As further improvements to the compounds and their activity against HLB occur, other compounds are planned for synthesis to further increase their potency specifically against HLB. The most potent compound will continue to progress through greenhouse assays and field studies as planned to determine the most potent and ideal agricultural agent to treat HLB and to begin field studies for EPA compound registration.
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.
In May 2014 we initiated a bimonthly survey of 4 flatwoods groves and 8 ridge sites in Hardee, Desoto and Highlands counties where soil acidification or irrigation water was implemented 1-1.5 years ago. All the blocks are Valenicia orange on Swingle or Carrizo, roostocks sensitive to excess bicarbonates. The survey assesses seasonal changes in soil pH, root mass density and Phytophthora populations to quantify the response to bicarbonate management, as well as, root growth dynamics of HLB-affected trees. It is expected that seasonal root flushes have been considerably altered from the root growth cycle of healthy tree. The results so far indicate that acidification has dropped rhizosphere pH about 1 unit from the baseline measured last year, and the root mass density and tree health has responded positively. As expected with a resurgence of root production, Phytophthora populations have increased in all 4 flatwoods locations and 3 of the 8 ridge sites.
The goal of this project is to understand the biology of HLB by identifying key components and processes involved in disease development. We identified four secreted proteins (also called effectors) from the causative agent, Candidatus Liberibacter asiaticus (CLas), which are consistently, and in some cases, highly expressed in infected citrus trees. We have generated antibodies against these CLas-specific proteins and developed serological detection methods for HLB. This method is currently under extensive evaluation using field and greenhouse trees. We are also using these effectors as molecular probes to identify their targets in citrus because effectors have been shown to perform key virulence functions during bacterial infection. This project will reveal important information of HLB pathogenesis, therefore providing guidance for disease management. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first year of this project, we cloned the four CLas effector genes into an Y2H bait vector, transformed them into the yeast strain AH109, and confirmed that the effectors are highly expressed in the yeast without self activation activities. Therefore, these constructs are appropriate for Y2H screens. In the past quarter (April to June, 2014), our main progress include: 1) Finished the construction of a citrus cDNA library, which is currently used for Y2H screening. We collected RNA samples from asymptomatic and symptomatic tissues of HLB-infected sweet orange leaves. These RNA samples were mixed with RNA extracted from healthy tissues to ensure that we would be able to cover as many genes as possible. A cDNA library was constructed and further normalized to reduce the level of cDNAs representing frequent transcripts using double strand specific nuclease (Evrogen). The cDNA was also size-fractionated to achieve an average insert size of >1 kb, with additional enrichment for long cDNAs that tend to represent full length transcripts. The cDNA library was transformed to a target complexity of about 3 millions of primary clones. Normalization of the library was confirmed with sequencing of 50 randomly picked colonies, which exhibited increase in complexity measured by diversity of transcripts. We are now in the process of Y2H screening using this cDNA library and the four CLas effectors as the baits. 2) Determined the subcellular localization of the effectors in plant cells. We have made gene expression constructs that produce fusion proteins with each CLas effector gene tagged to two consecutive genes encoding the yellow fluorescence protein (YFP). We used two YFP tags because these CLas effectors are small in size and may therefore diffuse to certain sub-cellular localizations non-specifically. We expressed these fusion proteins in plant cells and examined their localizations using confocal microscope. Our data showed that three of the four proteins can enter plant nucleus. This is interesting because they may manipulate plant physiology by associating with specific plant targets in the nucleus. These results will provide insight into the biological function of the effectors and also provide guidance when we interpret the Y2H data.
Seasonal root sampling continues in two field sites for root density. Root cages have been installed at the second field site (Valencia) to compare the root growth dynamics in healthy and HLB affected trees with two sweet orange scions that have large differences in seasonal carbohydrate dynamics. Sampling has already revealed seasonal variation in root infections and apparent shifts in the root flush cycle caused by Liberibacter. The interaction between root growth and root density in Las infected trees is becoming clearer with continued sampling of root cages in the Hamlin block. The introduction of a Valencia block will provide insight if these dynamics are differ based on differing seasonal carbohydrate demands for fruit development. As more of our healthy trees become PCR positive for Las it is increasingly difficult to find sufficient presumed healthy trees in the field. It is especially hard to find trees of sufficient age for seasonal root sampling. While field sampling continues, it will become more of a description of HLB affected tree decline than a comparison to healthy trees. Therefore, more emphasis will be placed on greenhouse experiments for direct healthy v. HLB comparisons as the project continues. Sampling at a rootstock trial site is underway with a full year of data on the effects of HLB on these new experimental rootstocks. This has already begun to demonstrate how these new rootstock lines respond to Liberibacter infection. The most promising lines have been graft inoculated in the greenhouse and will be transplanted to rhizotrons to monitor root growth and death as soon as the graft success can be confirmed.
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