Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks in airpots are growing well in our RES system, with some plants reaching 8 feet in height. Additional transgenics were propagated onto additional new rootstocks expected to reduce juvenility, including the somatic hybrid Amblycarpa + Flying Dragon. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. Experiments to efficiently stack promising transgenes are underway. The first transformation experiments using the two-transgene Gateway based cloned construct combining our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene) were initiated, and so far 30 putative transgenic lines of the sweet orange cultivars Hamlin and Valencia have been regenerated. These plantlets have been micrografted to Carrizo rootstock. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance.A construct containing CEMA gene stacked with the NPR1 gene has been constructed. Also, another vector containing a AttacinE gene stacked with the NPR1 gene is also under construction. Correlation of transgene expression with disease resistance response: More than 150 transgenic lines with different genes have been analyzed using ELISA by either C-myc or LIMA antibody (which also works for CEME) to measure transgene expression. As expected, significant differences were observed in our transgenic plants. Correlations between the data obtained from ELISA and other molecular data with HLB challenge response data are underway. Transgenic lines examined by ELISA include 40 lines with NPR-1, 50 lines with LIMA, and 9 lines with CEME. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free consumer-friendly transformation): We have finished construction of several parts of the T-DNA region of a pCAMBIA0390 derived binary vector for cre-lox based marker-free selection. A fusion codA-hptII gene driven by the d35S promoter have been constructed and a cre gene driven by a glucocorticoid-responsive elements promoter have also been constructed and cloned into a pUC based vector. We are experiencing problems cloning the glucocorticoid receptor gene driven by a constitutive mirabilis mosaic virus promoter as all sequenced clones have mutations and/or deletions in them. Work is underway to rectify this.
Monthly monitoring of all the alternative host plants continued this quarter. Transmission rate data were the same as found in last quarter on the 6 citrus relatives tested. Severinia buxifolia (Sb), Calamondin (Cal), Zanthoxylum fagara (Zf), Citropsis gilletiana (Cg), Choisya spps (Ch), and Esenbeckia runyonii (Er), were all found to be alternative hosts of Candidatus Liberibacter asiaticus. Bacterial populations in Zanthozylum fagara, Citropsis gillentiana, Choisa spp. and Esenbeckia runyonii maintained for a relatively short period of time, or maintained at a low population level (Cq value around 35). This followed the same trend found with Murraya paniculata. Las persistency in the plant, percentage of psyllids with Las and Cq values in the psyllids was summarized. Monthly monitoring of the live bacterial population dynamics in Severinia buxifolia and Valencia sweet orange by PMA-qPCR ended in April 2012. The data of the last month showed that Severinia and Valencia both contained high levels of live bacteria around 10^7, and 3×10^7 bacteria/gram tissue, respectively. The total population was similar to the these levels in both Severinia and Valencia.
Three different psyllid transmission tests using all of the alternative host plants were completed except for tests using Amyris since all transmission attempts from HLB infected citrus to Amyris was negative (no PCR positives or symptomatic plants) Monthly monitoring of the bacterial population dynamics in Severinia buxifolia by PMA-qPCR showed that the live bacterial population level dropped in January 2012 to about 10^6 bacteria/gram tissue, and then came back to the high level in February and March (about 10^7 bacteria/gram tissue), while total population fluctuated from 10^7 to 1.2×10^8 bacteria/gram tissue. Monthly monitoring of bacterial population dynamics in Valencia sweet orange showed that the live bacterial population also dropped in January 2012 to about 10^7 bacteria/gram tissue, and then came back to the high level in February and March (about 4×10^7 bacteria/gram tissue). The total population fluctuated from 10^7 to 10^8 bacteria/gram tissue.
In alternative hosts tested in psyllid transmission experiments, positive PCR transmission results from citrus to alternative host were obtained for the following species: Severinia buxifolia (Sb), Calamondin (Cal), Zanthoxylum fagara (Zf), Citropsis gilletiana (Cg), Choisya spps (Ch), and Esenbeckia runyonii (Er). No eggs or nymphs were found on any of these alternative hosts (except for Sb and Cal), and further transmission tests (three reps were done for each) are still under investigation. The psyllid feeding behavior and living status on those plants was recorded since the psyllids seemed not to readily feed on some of the plants even when force fed. Monthly monitoring of the live bacterial population dynamics in Severinia buxifolia by PMA-qPCR showed that the live bacterial population remained high (about 10^7 bacteria/gram tissue) through this time period, while total population fluctuated from 10^7 to 10^8 bacteria/gram tissue. Monthly monitoring of live bacterial population dynamics in Valencia sweet orange by PMA-qPCR showed that the live bacterial population remained on the high level (about 3×10^7 bacteria/gram tissue) through these three months, while total population was about the same as live ones in these three months, 3.2×10^7 bacteria/gram tissue.
HLB in Florida is associated with bacterial pathogen Candidatus Liberibacter asiaticus (Las) and transmitted by Asian citrus psyllid (Diaphorina citri) to citrus. Some plant species have been listed as hosts of the psyllid and/or the associated bacterium based on field samples, but their status as alternative hosts has never been throughly studied. In this work the following rutaceous plants were investigated: Murraya paniculata (orange jasamine), Murraya (Bergera) koenegii, Murraya exotica, Severinia buxifolia, Citrofortunella microcarpa (Calomondin), Citropsis gilletiana, Esenbeckia runyonii, Zanthoxylum fagara, Choisya aztec ‘Pearl’, Choisya ternata ‘Sundance’, Helietta parvifolia, and Amyris texana, were studied to investigate their alternative host status. Possible transmission pathways for each plant were tested with repeated psyllid transmission experiments as well as grafting where compatible. After inoculation, plants were monitored for symptom development and tested by real-time PCR (qPCR). At the USDA, ARS, FDWSRU, Ft. Detrick, MD, psyllid transmission tests, Asian citrus psyllids transmitted Las to M. paniculata and M. exotica, but not M. koneigii. Disease symptoms did not develop in Murraya plants, and positive infections were determined by PCR. Back-inoculations from M. paniculata to sweet orange was successful however there was some variability in infection rates, titer, and the Las bacterium did not persist in M. paniculata (published). At the Texas A&M Citrus Center, Weslaco a collection of eight rutaceous species were established and included two Amyris species (torch wood), Zanthozylum fagara (lime pricklyash), Helietta parvifolia (baretta), Esenbeckia berlandia, Casimiroa tetrameria and two Choisya species (Mexican orange) and were tested as hosts for the psyllid. Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica all were found to be feeding hosts for the psyllid. Egg laying was found on torchwood and egg laying and nymphal development were found on C. ternata. At CREC we experimentally demonstrated the alternative host status of several plant species, of which 6 plants (Calomondin, C. gilletiana, E. runyonii, Z. fagara, C. aztec and C. ternata) were 1st time reported as hosts of Las. S. buxifolia, Calomondin, C. gilletiana, E. runyonii, Z. fagara, C. aztec and C. ternata were infected by Las although the transmissibility varied and was based on bacterial persistency and psyllid activities. At the USDA, ARS, Beltsville quarantine work with dodder as an alternative host to study the plant infection process and for its use with plants that are not graft compatible with citrus was done and published. qPCR has limitations since it does not differentiate live bacterial genomes (LBG)and dead bacterial cells. Propidium monoazide (PMA), a novel DNA-binding dye, has been used with many bacterial pathogens to effectively remove DNA from dead cells, but no applications on uncultured bacteria like Las have been reported. In our work we devised PMA-qPCR protocols and optmized them to work with plant and psyllid materials. They were then used to determine LBG in various studies, such as establishing correlation between LBG and microscopic counting, checking the reactions of different citrus plants to Las infection, and checking the connection between LBG and leaf symptom expression. Lastly, the LBG dynamics inside HLB positive citrus and non-citrus hosts was monitored monthly through a 20-month period, and a seasonal development pattern was observed in both hosts. The optimized PMA-qPCR developed provides an accurate way to determine LBG in plant hosts of Las, which should benefit various HLB research and serve as a crucial component in HLB management. This work has been accepted for publication.
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.
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