The heat-treated trees are continually being monitored for physiological and visual changes. Physiological tests include stomatal conductance readings, water potential readings, and leaf anatomy samples. Pictures are taken monthly to monitor the tree’s recovery from the heat treatment as well as to monitor any signs of reinfection. Recently, more tests have been done to assess the effectiveness of the heat treatment. Average fruit diameter, average fruit set, average leaf size, and average leaf area index (LAI) were measured in the 36 treated trees as well as 9 control trees for comparison. Physiological indices: average fruit diameter, average fruit set, and average leaf size can show how the heat treatment has affected the disease as well as the tree itself. If the bacteria are successfully reduced or eliminated, the physiological indices should increase after treatment. The average LAI quantifies the leaf density of the tree canopy. In a HLB-infected tree, LAI decreases as the disease progressed. The data from these tests are currently being analyzed. The chlorophyll fluorescence is also being measured. This measurement is a good indicator of overall tree health. The cross-sections of leaf petioles have been returned from the lab and are currently being stained for analysis. Once staining is completed, the phloem area can be measured. The phloem cells of an infected citrus tree tend to collapse, causing the overall phloem area to be reduced and blocking nutrient transport throughout the tree. Examining the phloem area before and after treatment could provide more evidence to support the success of heat treatment.
Data have been collected from five growers. We are developing and evaluating models for Valencia and Hamlin oranges at low and high levels of HLB.
For this quarter, we continue data collection on the trial set-up in February 2010 to evaluate the interaction between nitrogen and hedging date. The field trial was a factorial experiment with 3 nitrogen levels (0, 50 and 100 lb N/ac) and three hedging treatments namely non-hedged trees, early hedging in February, and late hedging in April. These 9 treatments were split into subplots with one subplot sprayed with a recommended insecticide at the beginning of the flush cycle for psyllid control and the other subplot left unsprayed. One spray application was done during this quarter with imidacloprid (Provado at 20 oz/ac) just prior to the major flush cycle of early July. We continued the monitoring of various developmental stages of psyllids on flush shoots. During this quarter, all non-hedged trees at different nitrogen levels and with no pesticide application harbored significantly more psyllids than the other treatment. Flush production during the July flush cycle was more important in non-hedged trees relative to trees hedged in February and April, in contrast to what was observed in the previous flush cycle in May. Relative to non-fertilized trees, trees that received nitrogen had significantly more psyllids. Specifically, the non-hedged trees that received nitrogen had 3 to 5-fold more psyllids than the non-fertilized ones. Application of imidacloprid to trees was very effective at controlling psyllid populations independently of hedging or nitrogen treatment. Hardly any psyllid eggs, nymphs or adults could be collected from all plots that received Provado treatment.
Due to its reduced genome, Candidatus Liberibacter asiaticus has only a few transcriptional regulators. We hypothesized that natural and/or synthetic chemicals can interact with these transcription factors to interfere with regulatory activities, resulting in decreased tolerance to the stress conditions encountered in the phloem. During the first year of this proposal, we focused on the identification of small molecules that bind and/or modify Ca. L. asiaticus transcriptional regulators. Each regulator was cloned, purified, screened against a chemical library of 1200 small molecule. We identified several small molecules that interact with CLIBASIA_01180 and CLIBASIA_01510. Thermal denaturation experiments were then performed with each of the identified small molecules, at numerous concentrations, to confirm the effects of these ligands in vitro.. The second year of this proposal focused on the in vitro and most importantly, in vivo experiments, to prove the specificity of the chemicals identified. For CLIBASIA_01510, seventeen chemicals were identified that increased the mid-transition temperature during thermal denaturation assays. CLIBASIA_01510 is homologous to proteins involved in gene regulation through direct interaction with the beta subunit of the RNA polymerase, not by direct binding to DNA. Based on these observations, the effect of chemicals identified in the screen, were tested in vivo using a bacterial two-hybrid system. We also determined the optimal growth media and growth phase to obtain tight protein:protein interactions. We then examined the effects of each ligand. Two chemicals that were identified during the chemical screens were found to disrupt the CLIBASIA_01510:RpoB interaction. Further experiments, using the chemical scaffold as a guide, identified another chemical that binds to the complex with higher affinity. Using Liberibacter crescens, we found that the administration of the chemical decreased mRNA expression of CLIBASIA_01510 homologs. The decrease in mRNA expression was positively correlated with decreased stress tolerance. The protein CLIBASIA_01810 is a transcriptional regulator that modulates proteins involved in cell wall synthesis. During year 2 of this project, our primary focus was testing the effects of the identified chemicals on the survival of Ca. L. asiaticus in infected orange seedlings. These experiments were performed using leaves from 18 month old infected seedlings (kindly provided by Dr. Svetlana Folimonova at Lake Alfred). To this end, a new protocol for the efficient extraction of Ca. L. asiaticus mRNA was established. Brief exposure to the selected chemicals substantially reduced the metabolic capacity of Ca. L. asiaticus. We observed a significant decrease in the transcription of genes regulated by CLIBASA_01810, as well as a decrease in the abundance of 16S RNA genes. The effects of each chemical were confirmed in L. crescens, where modifications to the cell wall were found to lower tolerance to stress conditions. These exciting results suggest longer periods of treatment could result in the complete elimination of the pathogen.
The Core Citrus Transformation Facility (CCTF) continues to serve the community of researchers exploring ways to improve Citrus plants and make them tolerant/resistant to diseases. CCTF does its service by producing transgenic material. Within the last quarter, the CCTF facility worked on producing transgenic plants of the following combinations: produced the following transgenic citrus plants (transgene in parenthesis): Mexican lime (pHK vector); Duncan grapefruit (ELP3 gene); Duncan (MKK7 gene); Duncan plants (p7 gene); Duncan (p10 gene); Mexican lime and Hamlin (p33 gene); Duncan (SUC-CitNPR1 gene); Duncan (pWG19-5 vector); Duncan plants (pWG20-7 vector); Duncan (pWG21-1 vector); Duncan (pWG22-1 vector); Duncan (pWG24-13 vector); and Duncan (pWG25-13 vector). All of these plants are for researchers funded by CRDF in the battle against HLB and canker.
Candidatus Liberibacter asiaticus (CLas) the causal agent of Citrus Greening is transmitted from plant to plant by Asian citrus psyllid (ACP). CLas colonizes its insect vector and is transmitted in circulative propagative manner. The bacteria multiply within the insect vector hemolymph. We noticed that the bacteria also form biofilm on the gut surface. In general bacteria need a cell-to-cell signaling system (Quorum sensing) in order to form a biofilm. Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components cell-to-cell communication systems. But the genome lacks the second components; luxI that produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We confirmed the functionality of LuxR by expressing in E. coli and the acquisition of different AHLs We detect AHLs in the insect vector (psyllid) healthy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. Using different bacterial biosensor, we partly identify these AHLs (number of Carbon). CLas biofilm formation on the surface of insect Gut confirms the presence of cell-to-cell communication in insect while the planktonic state of CLas in plant indicate the absence of this communication. In plant, we found molecules that bind to LuxR but inactive its function (plant defense). We try now to characterize these molecule and study their effect on biofilm formation inside insect. We use purified molecule to feed infected insect through artificial diet system. We produced citrus plants that express LuxR protein in the phloem sap in order to test 1- If the acquired LuxR proteins in insect interfere with the biofilm formation in insect (cure the insect from CLas) 2- if the expression of LuxR in plant induce biofilm formation (localize the infection in plant) Interestingly, we found that feeding infected ACP with CLas on the LUXR expressing plants reduce the bacterial populations in insect and reduced the infection rate significantly. This result strongly indicates that we can target this system to interfere with the insect transmission and the spread of Disease. The main aim of this project is to express molecules in plant that interfere the growth of CLas in insect by feeding.
Third quarterly report for the project #00101004 During the previous three months, operation of the Mature Tissue Transformation Laboratory was (MTTL) steady. Despite some interruptions in the labor force attendance, the lab continued on its path to become fully organized and ready for the first incoming orders. Seven co-incubation experiments were performed within this period. Four with material from Valencia plants where 2380 explants were used. Two with the material from Hamlin plants where 750 explants were used. And finally, one experiment was done with 240 explants from Pineapple orange plants. The data were analyzed from one experiment performed in the previous reporting period and from four experiments performed in this reporting period. Here are the results: #exp explants used shoots tested (+) shoots Val 13 510 22 0 Val 14 500 752 23 Val 15 820 187 4 Val 16 590 976 37 Ham 16 270 150 5 In accordance with previously made decision to use the Agrobacterium strain harboring binary vector with the GFP gene within the T-DNA borders we were able to estimate the success rate in transformation experiments. In those experiments that ‘worked’ (where explants were not contaminated and produced sizeable population of shoots), transformation rate was about 3%. This should allow production of transgenic plants at a reasonable pace.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Several of the transgenic plants have reached the top of the scaffold, and the apical stems have been trained to grow down (expected to encourage early flowering). The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Another rootstock with strong potential to influence juvenility was identified (Nova+HBP x sour orange + Flying Dragon). Seeds have been planted. Experiments to efficiently stack promising transgenes are underway. Experiments to efficiently stack promising transgenes are underway. Transgenic sweet orange plants containing a construct with CEME gene (AMP) stacked with the NPR1 (SAR inducer) gene have been evaluated. We have recovered 10 transgenic lines that contain both genes incorporated into the genome. We have also transformed our newly released sweet orange somaclone OLL#8 with this construct. Also, constructs containing the AttacinE gene stacked with the NPR1 gene and the CEMA gene stacked with the NPR1 gene have been produced, and transformation of OLL#8 and Valencia Sweet Orange is currently underway. Correlation of transgene expression with disease resistance response: Western blot analysis for plants containing LIMA and GNA are nearly completed, data is showing a strong correlation between transgene expression and desired phenotype. This supports the dogma that fairly large populations of transgenic plants are necessary (for each transgene/cultivar) to obtain adequate transgene expression while maintaining cultivar integrity. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free or ‘all plant’ consumer-friendly transformation): 1. In efforts to reduce transgene mediated metabolic load on the plant, we have transformed Hamlin suspension cultures with constructs containing our reporter gene (grape anthocyanin gene) driven by either an embryo-specific Carrot DC3 promoter or an embryo-specific Arabidopsis At2S2 promoter. It is expected that plants obtained from these constructs will not produce the reporter protein once a transgenic plant has been selected. Currently putatively transgenic embryos have germinated and are being grown to size for analysis. 2. The binary vector for an inducible cre-lox based marker free selection is under construction. We anticipate transformation experiments with this vector in the following quarter. Targeted transgene expression: ‘ additional transgenic plants of Duncan, Carrizo, Pineapple, Hamlin, and Valencia (produced with Agrobacterium-mediated transformation) containing the LIMA gene (AMP) controlled by AtSUS2 promoter (phloem specific) have been propagated by micrografting. Plant characterization and molecular analysis on these plants will begin the next quarter. In greenhouse evaluation (Southern Gardens w/ Mike Irey) of transgenic plants exposed to HLB positive psyllids, we observed several transgenic LIMA and NPR1 lines driven by a phloem specific AtSUC2 phloem-limited promoter to be HLB tolerant. Most of these lines were negative to qPCR after 2 years of evaluation and did not demonstrate visible disease symptoms.
For this quarter, we continue data collection on the trial set-up in February 2010 to evaluate the interaction between nitrogen and hedging date. The field trial was a factorial experiment with 3 nitrogen levels (0, 50 and 100 lb N/ac) and three hedging treatments namely non-hedged trees, early hedging in February and late hedging in April. These 9 treatments were split into subplots with one subplot sprayed with a recommended insecticide at the beginning of the flush cycle for psyllid control and the other subplot left unsprayed. One spray application was done during this quarter with imidacloprid (Provado at 20 oz/ac) just prior to the major flush cycle of early July. We continued the monitoring of various developmental stages of psyllids on flush shoots. During this quarter, all non-hedged trees at different nitrogen levels and no pesticide application harbored significantly more psyllids than the other treatment. Flush production during the July flush cycle was more important in non-hedged trees relative to trees hedged in February and April in contrast to what was observed in the previous flush cycle in May. Relative to non-fertilized trees, trees that received nitrogen had significantly more psyllids. Specifically, the non-hedged trees that received nitrogen had 3 to 5-fold more psyllids than the non-fertilized ones. Application of imidacloprid to trees was very effective at controlling psyllid populations independently of hedging or nitrogen treatment. Hardly any psyllid eggs, nymphs or adults could be collected from all plots that received Provado treatment.
Several anti-NodT scFv single-chain antibodies are now in hand. The anti-NodT antibody with the highest affinity for the target 30 amino-acid peptide has been selected and has been expressed successfully in E. coli. The plant gene expression construct consisting of the 35S Cauliflower Mosaic Virus promoter driving expression of a fusion of the flowering locus T protein with best-performing anti-NodT scFv protein has been produced. We are now in the process of transferring this 35S::FLT-scFv expression cassette into the pTLAB citrus transformation construct. We have been working on this construct for several months during January – April 2013, but still have not completed this step, which has proven to be unexpectedly difficult. We anticipate that we will be able to complete this step during the next quarter, using some alternate techniques. Once the construct is completed, production of transgenic plants will begin.
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 75 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. 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. Preliminary results suggest that the RNAi approach against psyllids will work. We also continue to screen transgenic plants for other labs.
Function of individual X. citri transcription activator like effectors (TALEs): The activity and specificity of specific X. citri TALE proteins PthA1-4 have been tested using two different approaches. In one approach, activity was tested transiently using a reporter assay in Duncan grapefruit leaves. In these assays, plants were co-inoculated with a reporter construct consisting of a 14 TALE binding element (EBE) version of the Bs3 promoter driving the GUSi reporter gene together with Agrobacterium containing individual pthA genes or combinations of genes. Our results from these studies show that on its own Xc PthA4 is the most effective activator of gene expression, however co-inoculation with other individual proteins increases expression. We also used a second approach in which stable transgenic Nicotiana benthiamiana plants containing a 4 EBE promoter:GUS construct were inoculated with individual citrus TALEs introduced via X. campestris pv. campestris. We then quantified activity using GUS leaf disc staining and fluorescence MUG assays. In both assays, we could observe that individual TALEs did trigger expression of the GUS gene, so long as the corresponding EBE was present. This system also permitted us to examine the activity of pthA genes from a range of strains, including the sequenced Brazilian A 306 strain, A44 from Argentina, a typical A strain from Miami, an unusual strain isolated from Etrog in Florida,and a C strain designated #93 Brazil. TALEs from these strains triggered expression of the construct when matching EBEs were present. These data show that the promoter constructs are functioning as designed, with specificity for individual TALEs from a wide range of strains. We also now have a number of assays to evaluate TALE-promoter interactions to evaluate the roles of TALEs individually and in combination. Transformation and production of stable citrus lines: We have experienced difficulty in recovering functional stable transgenic citrus with from our transformation efforts. We have obtained many transformants, but to date we have not identified a line with a functional, intact transgene construct. In response, we are pursuing several alternative approaches to obtain stable transgenics, including further examination and testing of original constructs, preparation of new constructs in alternative vectors, additional controls, optimization of the transformation protocol, and new methods of transformation. We continue to regularly set up transformation experiments and analyze transformants by PCR, sequencing and pathogen testing. Given the successful function of constructs in transient reporter and disease resistance assays and the success of stable transformation with other constructs, we expect to recover functioning stable transformants through continued optimization of the transformation process.
Thermal treatment has been regarded as an effort to prolong the productivity of citrus trees infected by the Huanglongbing disease (HLB). A commonly known method of thermotherapy application in the grove is to cover or tent a tree, allowing solar radiation to raise the air temperature under the cover which subsequently raises the temperature of the tree. Two specific objectives were addressed: 1) to develop a simple in-field system that collects solar energy to raise tree canopy temperature, and 2) to extend the productive life of HLB-infected trees using in-field solar thermal treatment. In accomplishing objective 1, two thermal treating systems were developed. The first system was in the form of a mobile enclosure which covers individual trees with greenhouse plastic mounted onto a metal frame. In this system, temperature rise under the cover occurs solely by solar radiation. The second system was an improvement on the first system and used additional supplemental heat to make the temperature rise faster as well as provide more consistency in maintaining the intended temperature. Supplemental heat was provided by infrared heaters attached to the lower parts of the cover s frame. In accomplishing objective 2, the second system was used in an experiment with nine different treatments on mature citrus trees. The treatments varied in their temperature and duration. The temperature and duration combinations used were 45 C at 60, 120, and 180 minutes, 50 C at 40, 80, and 120 minutes, and 55 C at 20, 40, 60 minutes. The parameters used to evaluate the effect of the treatments on the trees were yield, juice quality, physiological markers (leaf water potential, stomatal conductance, and phloem area), and tree health markers (chlorophyll fluorescence, fruit set, fruit diameter, leaf area, and PCR analysis). It was found that treatments at 55 C regardless of the tested duration gave the best results. For the trees treated at this temperature, there was an increase in phloem area 30 DAT, fruit set, fruit size, and leaf areas had the highest values, and the PCR analysis showed a decrease in the number of CLas copies on the leaves after treatment. One of the major challenges of this project was the lack of reliable technique for detecting live to death CLas in plant as a results of heat treatment. The high chance of reinfection of treated heat trees also added to uncertainty of the effects of the treatment towards the disease and the plant. Due to this, it is difficult to conclude how thermal treatment is actually influencing HLB-infected citrus trees. Future work will include further investigation in determining how the treatment actually affects the disease in the plant and the plant itself. Additional future work also includes continued use of supplemental heat, primarily steam, and development of systems to treat both the canopy and the roots of the tree.
A new, rugged, heating tunnel prototype that incorporates two infrared radiant heaters and four fans was built. The heating and air circulation system has been integrated with an adjustable temperature control switch for automatic control of the temperature. As mentioned in our early report there was a lack of repeatability of live bacteria PCR analyses due to both intrinsic variability in the leaves and experimental variability due to the large number of steps required for DNA extraction procedures. Therefore, to assess the effect of the 2012 heating treatments on tree health at the time of fruit harvest, i.e. April 2013, we assessed overall appearance of the trees, and determined fruit yield and juice quality. We also determined the number of new shoots, new leaves and the mean leaf surface area in selected portions of treated and control trees. The main findings are summarized below. Qualitative observations: The trees that were treated in 2012 were those that showed the most prominent symptoms of all the trees in this research project. The trees treated in July of 2012 reached a temperature of at least 45 ‘C for at least 4 h. In April 2013, the treated trees looked healthier than the surrounding, untreated trees that now show severe symptoms. This suggests that the thermal treatment was effective in mitigating the progress of the disease. Because of cold weather, trees treated in September of 2012 did not reach 45 ‘C for as long as those treated in July of 2012. Trees treated in September of 2012 did not appear to be as healthy as the trees treated in July, but appeared healthier than trees that were infected and were untreated (symptomatic control). Overall, asymptomatic (healthy control) trees looked healthier than treated or untreated trees. A large amount of fruit drop was observed for all trees. Quantitative results: Data from the April 2013 harvest showed that the average fruit yield for the healthy control trees was 217 lb/tree. Trees treated in July yielded an average of 200 lb/tree. Symptomatic control trees had a mean yield of 162 lb/tree. Although yield variability was high, (standard deviations in the order of 50 lb) these results suggest that treated trees had a yield close to that of healthy trees.Healthy control trees had the same mean soluble solids content (SSC) of 12.0 ‘Brix as trees that were treated in 2012. In contrast, untreated symptomatic control and trees that had a mild heat treatment had a mean SSC of 11.3 and 11.0 ‘Brix, respectively. Mean new leaf surface area of healthy trees (16.6 cm2) was slightly greater than that of treated trees (13.4 cm2). In contrast, the mean surface area of new leaves from untreated trees (7.6 cm2) was considerably smaller (about one half) than that of healthy or treated trees. This suggests that symptomatic trees suffering stress conditions might have slower canopy foliage development compared to healthy trees. Also, thermally treated trees have good health/vigor compared to symptomatic control trees. Primarily, it was observed that healthy trees have well-developed medium to dense foliage from past seasons; whereas, symptomatic control trees have thin foliage. The number of new shoots in a 0.5 x 0.5 m frame was on average 30 for healthy control trees, 56 for untreated symptomatic controls, and 42 for treated trees. In conclusion, results from 2012 experiments suggest that thermal treatment is effective in mitigating the effects of HLB. It is anticipated that the use of fans and electric heaters will result in more uniform and faster tree treatments.
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 the 2008-2012 fungicide sensitivity tests. During this trimester, 182 isolates were tested for sensitivity to azoxystrobin and pyraclostrobin using the resazurin-based microtiter assay. ‘ Out of 182 isolates tested, 55 were resistant while the remaining isolates were sensitive. ‘ In summary, from 2008 to 2012 survey, 817 isolates were tested for QoI fungicide sensitivity. The number of resistant isolates were 471 (57.6% of the total isolates) ‘ DNA extraction of 123 isolates was done in order to identify the point mutation that corresponds with the resistant phenotype. ‘ In total, 235 isolates were DNA extracted from the 2008-2012 survey, including eight isolates from our baseline collection. ‘ RFLP-PCR analyses were performed using the cytochrome b gene amplification of the 235 isolates to identify the G143A mutation in resistant isolates. ‘ In summary, 161 isolates were resistant and all of them carried the G143A mutation. In contrast, the remaining 74 isolates were sensitive and none of them had the G143A point mutation. ‘ We found two genotypes associated with the partial structure of the cytochrome b gene, named profile I and profile II. ‘ Two experiments were conducted using detached leaves from 4 cultivars using 10 isolates (5 R and 5 S). In vitro inoculations were performed to evaluate some fitness parameters related with incubation period and number of lesions per cm2 in individual detached leaves. ‘ In vitro fitness components were established using the same group of isolates described above (5 R and 5 S). Fitness parameters evaluated were: mycelium growth, sporulation and conidium germination. ‘ A second experiment under greenhouse conditions was conducted to test the ability to infect tangerine plants previously applied with a full rate (15 fl oz) of Abound (azoxystrobin). During this experiment 10 different isolates were used (the same isolates used in the experiments described above). After 5 days of incubation, the numbers of lesions 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 treated with the full rate of fungicide. ‘ A second experiment using the spiroplate method was done to test boscalid sensitivity from 16 different isolates. ‘ The paper ‘Distribution of QoI resistance in population of tangerine-infecting Alternaria alternata in Florida’ was finished and ready to be submitted in Plant Disease Journal within the week.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
