Work has been continuing on the development of a construct using the FT3 cDNA insert and an FMV promoter. This construct will eventually be used to test the efficacy of the FT3 cDNA as compared to the genomic DNA construct currently being used. Over the past several months, extensive testing was conducted to establish a more effective disinfestation technique for use on seeds and other explant tissue. This technique should allow for the continuous use of seed for transformation, even many months after their initial collection. Transformation of Carrizo has picked up following the most recent harvest of seed. These transformants will be used in the experiments examining the effects of GA and day length on FT phenotype. This is month 7 of the in vivo tracking of FT1, FT2, and FT3 and samples are continuing to be collected and processed. These data will be evaluated at the end of the year-long trial to compare month-to-month variations in gene expression. The FT3 protein that was commercially synthesized has finally arrived and experiments with direct application of the protein will be commencing shortly.
In the previous period ‘Duncan’ grapefruit (considered susceptible to HLB) and ‘Sun Chu Sha’ mandarin (considered moderately tolerant to HLB) were inoculated with Flagellin 22 (flg22). Tissue samples were collected before inoculation (time 0) and at 6, 24, 72 and 120 hours post infiltration with flg22. Total RNA was extracted from all the samples (3 replicates of each) and we determined and analyzed gene expression using comparative Ct real time PCR. Genes associated with SAR and PAMP-triggered immunity (PTI) as well as genes in the salicylic acid and jasmonic acid biosynthetic pathways were studied. So far during this period we have studied the expression of 11 genes (EDS1, EDS5, ICS1, NDR1, NPR1, NPR3, PAL1, PR1, R13032, RAR1, SGT1). The results have been run through a Q-test for outliers and are in the process of being statistically analyzed using JMP Genomics. Expression levels of additional genes will also be added to the study.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 60 different peptides for activity against HLB. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids.
The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. During this quarter we accomplished: ‘ Finished 2012 survey of 19 groves, encompassing 32 blocks sampled. The total area sampled corresponded to 850 acres in 7 counties of Florida. ‘ During 2012, 855 isolates were collected. The number of cultivars were Lee, Minneola, Murcott, Orlando and Sunburst. ‘ Out of 855 isolates, 336 were tested for pathogenicity, and 274 were confirmed as pathogenic (82%). For each pathogenic isolate, monoconidial cultures were done. ‘ Using the RZ-based microtiter assay, 163 isolates were screened for fungicide sensitivity to azoxystrobin and pyraclostrobin. Forty percent of the isolates tested in 2012 were found to be resistant to strobilurins. ‘ DNA extraction of 100 isolates was done in order to identify the point mutation that corresponds with the resistant phenotype. ‘ A greenhouse experiment was conducted to test the ability of resistant and sensitive isolates to infect tangerine plants previously applied with a full rate (15 fl oz) of Abound (azoxystrobin). During this experiment 10 different isolates were used (5 sensitive and 5 resistant). After 5 days of inoculation, the numbers of lessions were counted on individual leaves per treatment-isolate combination. A significant difference (p<0.001) was found between sensitive and resistant isolates, means that resistant isolates were able to infect plants previously covered with the full rate of fungicide.
We are examining how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. The first objective of this project is to examine initiation of HLB infection after psyllid inoculation to investigate how introduction of the pathogenic bacterium into different types of flushes of a tree affects establishment of infection. We have conducted electron microscopy examination of the sites on the leaves of citrus plants where psyllids carrying the HLB bacteria fed. Psyllids were allowed to feed on the plants for 7 days and after one more week the sites where they have fed were used for microscopy observations. Our results demonstrated that even at so early stages of infection the bacteria could be already visualized in the initial sites of introduction. To examine what types of flushes are more susceptible to psyllid inoculation with the HLB bacteria, we exposed sweet orange and grapefruit plants that have young growing flushes and plants that have only matured flushes to HLB-infected psyllids. The leaves on which the psyllids fed were analyzed by PCR to see if the HLB bacterium could be detected soon after the exposure of leaves to infected psyllids. As a result in these experiments, we were able to detect presence of the bacterium fairly early after the initial exposure, approximately between two weeks and one month (similar to our results from electron microscopy studies). Plants exposed to infected psyllids have been transferred to greenhouse and further monitored for the development of infection. We have repeated this experiment several times and now are analyzing and comparing infection rates of plants with young flushes versus plants with only matured flushes. According to our data, both young and mature flushes could be inoculated by psyllids, yet inoculation efficiency of mature flushes is significantly lower. To characterize inoculum sources of the bacterium available for psyllids within an infected tree, we are evaluating the proportion of psyllids that acquired the bacterium after their exposure to different types of flushes during infection development and their ability to transmit infection to new trees. We conducted several trials in which healthy psyllids were placed on either a young growing flush or an older symptomatic flush of an infected tree using small traps made up of mesh material and after 21 days psyllids were analyzed by PCR with HLB-specific primers. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. These plants were monitored for the development of infection. Analysis of numbers of plants that became infected upon inoculation with psyllids fed on different types of flushes revealed that more receptor plants that were inoculated by psyllids kept on young flushes became infected and less proportion of receptor plants inoculated with psyllids kept on old flushes became infected with HLB. The next objective is to examine psyllid transmission rates from and to citrus varieties that are highly tolerant to HLB. We have propagated 6 different varieties of citrus: Valencia sweet orange, Duncan grapefruit, Persian lime, Eureka lemon, Carrizo citrange, and Poncirus trifoliata. Those varieties represent plants with different degrees of susceptibility to HLB. Currently these plants are being exposed to HLB-infected psyllids. After 1-month exposure, plants were moved to greenhouse and monitored for the development of HLB infection. The first four varieties showed the highest infection rates, while only about 10% of Carrizo citrange and Poncirus trifoliate became infected. The results obtained in this project are now being prepared for a publication.
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.
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