The goal of this project is to find ways to optimally deploy the superinfecting Citrus tristeza virus (CTV)-based vector to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. Plant material that will be used in this project has been prepared in our greenhouse. Using plant material and inoculum sources that are already available, we are conducting initial experiments to examine the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. To examine the effect of preexisting CTV infections on multiplication of the superinfecting vector in inoculated citrus trees, we first graft-inoculated sweet orange trees with the T36 or T30 isolate of CTV, the isolates that were propagated in our greenhouse, as well as with CTV-infected material obtained from field. Trees with developed CTV infection along with uninfected control trees were challenged by graft-inoculation with the superinfecting vector carrying a GFP gene. The latter protein is used as a marker protein in this assay, which production represents a measure of vector multiplication. The trees are now awaiting further examination for which tissue samples from the challenged trees will be observed under the fluorescence microscope to evaluate the ability of the vector to superinfect trees that were earlier infected with the other isolates of the virus. Levels of GFP fluorescence will be monitored and compared between samples from trees with and without preexisting CTV infection. Another objective is to select rootstock/scion combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the foreign protein of interest from this vector. For this purpose, we are preparing trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla. The plants will be used for the experiments similar to the experiments described above.
2012 field trials using the developed moving greenhouse system showed that the 5-h canopy solar heating treatments have variable heating periods. The time needed to reach a temperature inside the covered canopy of 45-50 ‘C or higher, were low in early fall compared to summer treatments. Also, temperature profiles of 5-h treatment period revealed that the upper section of the canopies (> 1.5 m) heated much faster than lower section. Thus, to increase the rate of heating and to maintain adequate temperatures throughout the enclosed canopy during the treatment period, the moving greenhouse system is being modified to incorporate supplemental heating and forced convection air flow system. The major materials needed for the modifications such as two 3000 Watt infrared radiant heaters and portable generator to power the heating units and air circulating fans were purchased. A change in budget request was submitted in October 2013 to procure the portable generator unit. Because citrus trees acclimatize to the climate of each season, during the cold months between November and March, no further thermal treatments can be carried out. Hence, no further experimental results are reported. Experiments and data collection will continue during late spring and summer of 2013. Late spring/summer trails would ensure minimal to no damage to the plant physiology due to artificial heating
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 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 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 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. Additional plantings are welcome from the research community.
Dr. Guixia Hao, who has extensive experience in plant transformation and molecular biology, began working on this project 9/23/2012. New constructs have been used to transform citrus scions including 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 (designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. 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 and will be used in transformations next quarter. A series of transgenics scions produced in the last several years, continue to move forward in the testing pipeline.
The main aim of this project is to identify proteins that are implicated in the recognition between Candidatus Liberibacter asiaticus (CLas) and the insect (Asian Citrus Psyllid, ACP) cells in the gut and Salivary glands. This information will greatly help in understanding the specific interaction that facilitates the circulation within the insect. Clas can invade the epicedial cells in the gut to the hemocoel and penetrate the salivary glades to be inoculated in a new plant. Using protein overlay assay, we have identified insect-proteins that bind specifically to proteins from CLas. In this approach we screened first several CLas-antibodies for the specificity by western blots. We also purified IgGs from the chosen antibodies using Protein-A column and FPLC system. Kindly we received the crude antibodies from Dr. Helvecio de Coletta-Filho. Towards detecting the receptors in the ACP, we found seven proteins that are potentially receptors for the CLAS when we used the total proteins from ACP. Using proteins from the midgut, four proteins have been detected suggesting that, the other three are located in the salivary glandes. All proteins were analyzed by LC-MS. In our current trials, we are willing to identify the CLas-surface proteins that recognize the receptor in the insect using a modified protein overlay assay that we established in our lab. The data we obtained is currently subject to a publication in preparation.
Construct optimization: We have engineered several new constructs for transformation in citrus genotypes. These include a ProBs314TBB-avrBs3: avrGf2 and a ProBs34TBB-avrBs3: avrGf2. In transient assays, these constructs elicited an earlier and stronger HR than constructs carrying avrGf1. We have also designed a construct which utilizes a portion of the promoter region of a citrus gene that demonstrates strong binding activity by PthA4 homologues. Transient assays demonstrate higher levels of activation than other constructs. Six new constructs are being used for stable transformation The constructs vary based upon the promoters used, the number of copies of the avrGf2 gene (single or multiple), the presence or absence of a terminator – nopaline synthase terminator (NOS T) upstream of the promoter and the plasmid used. Transformation summary In vitro germination experiments are ongoing with citrus varieties ‘Duncan’ grapefruit, ‘Ruby Red’ grapefruit and ‘Pineapple’ sweet orange for future epicotyl experiments. To date both epicotyl and cotyledon transformation experiments have been carried out with ‘Duncan’ grapefruit and ‘Pineapple’ sweet orange segments and the 6 new constructs designed with the avrGf2 gene. Sweet orange epicotyl and cotyledon transformation experiments have been carried out and a total of 1, 005 and 554 segments, respectively transformed with 5 of the 6 constructs. On the other hand 6 constructs have been used to transform altogether 3,623 grapefruit epicotyl segments and 5 constructs to transform 527 cotyledon segments in the transformation experiments. Shoots regenerated from transformed segments of sweet orange (53) and grapefruit (19) have been placed on rooting media. Putative sweet orange and grapefruit transgenic plants, 44 and 19 respectively originated from the regenerated shoots placed on rooting media have been placed in soil for acclimatization and will be tested via PCR for confirmation of integration of the transgene.
In the last few months, we continued to detect and identify the receptor and ligand. To ascertain the binding of CLas proteins to one immobilized ACP protein, the protein overlay assay was carried out on protein blots issued from 2D-SDS-PAGE. Non CLas-carrying insect total proteins were separated by 2D-SDS-PAGE. After 2D-SDS-PAGE, far-Western blot experiment was carried out. Comparisons between the stained electrophoretic profiles of ACP proteins in the gel and results of far-Western blot experiments on the membrane allowed the unambiguous election of protein spots from 2-DE gels for LC-MS/MS analysis. Some ACP proteins (receptor) were identified, which could play an important role in Clas adhesion to ACP cell. The function of these proteins was analyzed with bioinformatics. These genes were cloned, proteins were expressed, antibody against these proteins were made. Next, we expect to find the proteins of Clas (ligand) that adhesion to ACP cell using antibodies against identified ACP proteins, and certify the proteins interaction between ACP proteins (receptor) and Clas proteins (ligand). Overall, our research work is carried out according to project plan
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, 4. Provide economic analysis of costs and projected benefits and 5. Extend results to clientele. The experiment was planted 3-4 July on a 10-acre block on 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 plot is split into two subplots, mulch and no mulch. Mulch is metalized (aluminized/reflective) polyethylene film shown in preliminary evaluations to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. Flush inspection commenced and sticky cards were placed in the block on 13 Aug. Sticky cards are observed and replaced every other week for ACP and other common citrus pests. About 10 psyllids have been found on sticky card of which greater than 2/3 are in no mulch plots. In addition more ACP have been found on sticky cards in plots that do not have chemical control than plots that receive insecticides. Up to 10 young flush shoots are in inspected every 2 weeks on 15 trees in each of the 32 subplots. To date one ACP nymph has been found on flush. The first leaf samples were collected on October 17 for HLB testing of which one tested positive and is now undergoing a retest to insure the result is accurate. Monthly foliar nutrition applications were suspended after the early November spray in preparation for the winter dormant season. Leaf samples were collected November 7 for nutrient analysis. Normal grove care operations. These include one application of Intrepid for leaf miner control, one application of glyphosate for weed control in mulched plots, one application of glyphosate and Solicam in unmulched plots and Kocide once to control canker. All trees were desuckered and any low hanging limbs pruned to reduce the chance of herbicide damage. Some trees needed staking to prevent canopy break so new ties and stakes were added. Drip emitters were beginning to clog so all emitters were replaced in November with a clog resistant model as well as installing flush valves in December. Over the night of December 22-23 a freeze was experienced but the flood irrigation plan was executed so that no damage was incurred by the trees. New water sensor probes were tested and prepared for soil moisture data collection.
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 2,750 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. 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 one week, 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 second quarter of the project, during which we have established the infrastructure for the screening program. A technician dedicated to the project has been hired, two small greenhouses for rearing psyllids have been completed and are functioning well, and 18 individually caged CLas-infected plants are being used to rear ACP for infestations. Psyllids will be available for challenging test plants in January. This screening program supports two USHRL projects funded by CRDF for transforming citrus.
The research team has continued to work with growers to collect data on yield, enhanced nutrient programs, and soil and leaf analyses. Initial analyses have been conducted. However, the current results are limited because only four growers have provided data thus far. As more data become available, the relationship between production and enhanced nutritional programs, if any, will become clearer.
The research team has continued to work with the growers to obtain information on the use of enhanced nutrient programs, soil and leaf analyses and yield. Two additional growers have provided data, and efforts to encourage participation from other growers continue. Data are being reformatted so that analyses can be conducted.
A talented postdoctoral fellow has been hired for the project and he has quickly begun making progress on research goals. Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. A. Mutagenesis of Arabidopsis EFR Conditions for optimal PCR random mutagenesis have been established and performed on the ectodomain of EFR. A library of approximately 1×106 clones, ready for transformation and screening, has been produced. A screening protocol has been established, whereby pools of 10 A. tumefaciens clones and infiltrated into N. benthamiana, and then scored for ROS induction in response to elf18-CLas. Screening has been initiated and we intend to screen approximately 3000 clones per week over the next three months. B. Natural variants of EFR Initial screening of a five species and cultivars of Brassicaceae has been performed, and we intend to obtain and screen a larger collection of species from several different genera for additional screening. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. The PAMP receptors XA21 (from monocots) and EFR (from dicots) have been used to construct chimeric PAMP receptors. EFR-XA21 constructs have been produced to test the effectiveness of the XA21 cytoplasmic domain in signaling in dicots. This construct produces a ROS burst in response to elf18, to a similar degree as wild type EFR, when expressed transiently in N. benthamiana. Conversly, XA21-EFR and XA21 constructs have been produced and tested for responsiveness to ax21 in N. benthamiana. Thus far, a significant response has not been observed in the ROS burst assay; most likely due to a known issue of poor activity of the synthetic peptide (personal communication from Pam Ronald’s lab). Yet importantly, extracts from Xcv produced significant ROS burst with both XA21 and XA21-EFR constructs. Further evaluation of ax21 responsiveness using extracts from Xanthomonas euvesicatoria 85-10 (formerly, Xanthomonas campestris pv. vesicatoria) wild-type, or ax21 and raxST knockout strains (provided by Pam Ronald’s lab) that will enable us to conclude definitively on the functionality of XA21 and XA21-EFR is in progress. Transgenic Arabidopsis plants are being produced with XA21 or XA21-EFR to assess resistance to Xanthomonas campestris pv. campestris 8004 in dicots.
Our objective is to determine how Asian Citrus Psyllid (ACP) behavior is affected in response to infection of trees with the bacterium, Candidatis Liberibacter asiaticus (Las) that causes huanglongbing (HLB). We have been comparing ACP response to healthy (uninfected) versus diseased (infected) citrus. In previous experiments, we have determined that ACP adults initially settle on Las-infected plants as compared with uninfected plants. We hypothesized that while the Las-infected plants are initially attractive to ACP, after prolonged feeding, the ACP experience imbalanced nutrition and choose to seek a better host; therefore, moving to nearby uninfected plants. This may serve as a mechanism to spread the pathogen that causes HLB. In the previous quarter, we determined that ACP setting behavior may be affected by the age of the C. Las infection. Because of this, we used plants that were <5 month (young) or >12 months (old) following PCR confirmation of infection in settling choice assays. The choice scenarios we are testing include: control vs old infection, control vs, young infection, and young vs old infection. We have completed 4 replications of these settling experiments and are currently working on a fifth replication. Our results indicate the following: 1) When comparing response to the control vs old-infected plants, the ACP settle evenly between these treatments on the first day; however, at seven days ACP move to and preferentially colonize the infected plants. 2) When comparing the response to the control vs young-infected plants, the ACP settle on the young-infected plants and remain on this treatment for the duration of the 7-day experiment. 3) When comparing the response to young-infected vs old-infected plants, the ACP appear to settle evenly and stay evenly settled between these two treatments for the duration of the 7-day experiment. We will continue to conduct these settling choice tests until we have at least five replications. As an additional experiment, we will conduct similar settling bioassays with young-infected plants vs control plants in the presence of absence of large quantities of methyl salicylate (MeSA) to determine if exposure of ACP to large amounts of MeSA can mask the attractive odor of infected plants. The idea is to mask the main odorant we believe is responsible for attracting ACP to infected plants (MeSA) and thus develop a strategy to reduce pathogen spread. Also, we are in the beginning stages of developing a mathematical model to describe how the effect of infection of citrus plants, which subsequently affects behavior of ACP, may play a role in spread of the pathogen under field conditions.
In recent seasons, concurrent with freeze and drought episodes, symptomatic HLB-infected trees were much more affected by the extremes of temperature and moisture than trees without HLB. Symptoms exhibited by the stressed trees were excessive leaf loss and premature fruit drop even when HLB-infected trees were managed with enhanced nutritional programs which are thought to improve tree health of HLB-infected trees. This stress intolerance may be due to a loss of fibrous roots. To assess root status of HLB-affected trees, blocks of 2,307 three-yr-old Hamlin orange trees and 2,693 four-yr-old Valencia orange trees were surveyed visually and by real time PCR (PCR) to determine Las infection status. The incidence of Las-infected trees (pre-symptomatic PCR+, visually negative and symptomatic PCR+, visually positive) trees was 89% for the Hamlin block and 88% for the Valencia block. HLB+ trees had a 30 and 37% reduction in fibrous root mass density for pre-symptomatic and symptomatic trees, respectively, compared to HLB ‘ trees. In a second survey, 10- to 25-yr-old Valencia trees were identified within 3-6 months of canopy expression as HLB symptomatic (PCR+, visually positive) or non-symptomatic (PCR-, visually negative) in orchards located in the central ridge, south-central and southwest flatwoods. Pairs of HLB+ and HLB- trees were evaluated for PCR status, fibrous root mass density and Phytophthora nicotianae progagules in the rhizosphere soil. HLB+ trees had 27-40% lower fibrous root mass density and in one location higher P. nicotianae per root but Phytophthora populations per cm3 soil were high on both HLB+ and HLB- trees. Fibrous root loss results primarily from HLB damage which may be interacting with P. nicotianae.The root loss due to HLB is equal to or greater than 27% in our surveys of young and mature trees. In Brazil, Bassanezi and co-workers measured 973 mature trees of early, mid and late season sweet orange varieties and developed a model for crop loss in relation to visual canopy symptoms. Based on their model, trees in the early stages of HLB canopy decline are predicted to lose 30% of their crop, the same magnitude as the fibrous root loss we measured. An assessment of crop loss for groves in Florida treated with an enhanced nutritional program demonstrates similar magnitude of crop loss (23-39%) for HLB-affected Valencia trees. HLB-affected trees in these well managed Florida groves did not further decline in yield from 2009 to 2011 compared to the trees with HLB symptoms. We presume this is because additional loss of fibrous root density had not occurred since the initial onset of HLB symptoms 4 years prior.
Objective 1 (To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface). Assays were performed to determine the ability of citrus bacterial canker (CBC) strains to respond to different stimuli. Cluster analysis showed an association between carbon source utilization and chemotaxis response. CBC strains were separated according to their host range. An in silico study of genes involved in chemotaxis was performed. Sequences for methyl-accepting chemotaxis proteins (MCPs) were conserved in all the xanthomonas strains evaluated. However, some MCPs were only present in a particular group of Xanthomonas strains. MCPs for the strains could be grouped according to host range demonstrating the connection with differential chemotaxis response. Chemotaxis response to different fractions from Chinese cabbage, Key lime and sweet orange leaves was compared. Chemotaxis response was greatest for apoplastic fluids, leaf extracts showed a moderate activity, and leaf washes showed no activity. Apoplast fluid from sweet orange most stimulated movement in wide host range canker strain 306, cabbage fluid did the same for X. campestris. Key lime apoplast fluid most affected X. alfalfae subsp. citrumelonis. Narrow host range strains of CBC (Aw and A*) showed little response to any of the leaf fractions from any of the plant species. Objective 2 (To investigate bifofilm formation and composition and its relationship with bacterial motility structures in different CBC strains and comparison to non canker causing xanthomonads). Protein analysis showed no qualitative differences in bacteria appendages among the Xanthomonas strains. Alterations in gene expression of type IV pilus, and flagellum components are under evaluation with qPCR primers for assay of bacterial colonies at different stages of motility swarming, planktonic or biofilm-forming.
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