Citrus blight continues to be a major economic problem in citrus groves in Florida. Thousands of trees each year succumb to citrus blight, with estimated losses at over $60 million per year. The disease can occur on all common citrus cultivars, and Carrizo citrange are especially susceptible. Early symptoms are zinc deficiency in the leaves which may disappear, zinc accumulation in the phloem and eventually high zinc levels in the xylem. Blockage of xylem tissues with amorphous plugs follows with reduced water uptake. The causal agent of citrus blight is unknown. However, symptoms and all of the characteristics associated with citrus blight can be reproduced by root graft inoculations. Therefore in a project previously funded by CRDF we used NGS RNA sequencing protocols to look for novel viruses in roots of sweet orange with blight, but not present in roots of healthy trees, or trees affected by HLB. We identified several related endogenous pararetroviruses related to Petunia Vein Clearing Virus (PVCV) using a collection of 10 RNA libraries prepared from 10 different root samples collected from healthy trees or those with blight or HLB. The objectives of the proposed work are the following: 1. Generate a complete genome sequence for CBAPRV. This has been completed. 2. Develop a highly specific RT-PCR assay that can determine when CBAPRV is active. This has been completed. 3. Use this assay to screen a large number of trees from blight affected areas in Florida. This is largely completed. 4. Transmission tests to determine if CBAPRV is the causal agent of citrus blight. This is underway. In the quarter just ending we have focused our efforts on the remaining objectives. During this quarter we have been validating the CBAPRV assay that was developed, and further sampling blight affected areas in Florida to assess the correlation between CBAPRV and blight affected trees. At this point, there is a strong but not 100% correlation between the presence of CBAPRV RNA (the hallmark of active exogenous virus) and the onset of blight. 94% of the trees that were identified as affected by citrus blight have active viral CBAPRV RNA in leaves and roots, and none of the non-blighted trees have evidence of CBAPRV RNA. This is a strong correlation, but the 6% of trees that have been identified as blighted without the presence of CBAPRV suggests two possibilities: 1) the presence of the active virus is associated with the stresses of blighted trees, but the virus is not the causal agent of the disease, or 2) the method of sampling for CBAPRV RNA is not 100% effective. To assess which of these possible explanations is correct we are moving on to attempted transmissions of the virus. There is no evidence that the virus is insect transmitted, so we have begun to work in two directions: 1) grafting of virus present tissue onto a variety of rootstocks and scions, and 2) attempts at identification and purification of viral particles that could be used to establish modified Koch’s postulates. The final quarter of work will focus exclusively on these efforts. Some additional testing of field materials collected in August of 2016 will also be completed.
We have proposed targeting specific regulators of key phage encoded virulence genes (such as the Las peroxidase) as well as the peroxidase itself and key regulators of the (lethal) phage lytic cycle. Objective 1 is control of HLB using the putative Las chromosomally encoded LexA-like repressor protein LC1 (Clibasia_01645), potentially a key phage lytic cycle regulator. We had previously shown that LC1 binds specifically to its own promoter as well as to a key lytic phage SC1 promoter region midway between the divergent late and early gene promoter regions. Bioinformatics revealed several putative sites for LC1 binding. Electrophoretic mobility shift assays demonstrated that LC1 binds to its own bidirectional promoter and to another DNA region between SC1_gp125 and SC1_gp130 genes on SC1. Moreover, SC1_gp125 is predicted to encode a C2-like repressor (LC2), therefore, if functionally confirmed as a repressor, LC2 would constitute a second potential target implicated in lytic cycle activation. To evaluate the potential activity of the LC1 and LC2 repressors, promoter::GFP reporter constructs were transformed into Liberibacter crescens (Lcr) and E. coli. SC1_gp125 (LC2) promoter activity was significantly higher than SC1_gp130 and even the strong LacZ promoter. These promoter::GFP reporters were cotransformed with vectors expressing the full-length LC1, truncated LC1 and LC2 repressors (all driven by LacZ promoter) in Lcr. Not only did LC2 self-repress its own promoter, but both full length and truncated LC1 repressors also repressed the LC2 promoter. Inactivation of either repressor would likely lead to Las cell lysis. Objective 2 is control of HLB using a repressor protein of unknown identity from psyllids as target. We previously reported functional repression of a Las phage lytic cycle holin promoter by a predicted Wolbachia repressor protein found in aqueous psyllid extracts. The in vitro translated Wolbachia protein partially repressed the holin::GUS reporter in a dose-independent manner, as compared to the aqueous extract from the psyllid, thus indicating that complete suppression of holin promoter requires an additional partner. Heat-inactivated psyllid extract was unable to enhance the Wolbachia repressor- induced suppression of holin promoter activity, confirming the heat labile proteinaceous nature of the additional psyllid-sourced partner. Including Objective 1 results, we have identified 3 protein repressors and target promoters that are being used for high-throughput screening of combinatorial libraries for chemicals that might be used to activate the phage lytic cycle in Las, both in Las-infected psyllids and citrus. Objective 3 is control of HLB using the Las phage peroxidase and Las lytic cycle activator(s) as targets. Bacteria use a variety of enzymes, including peroxidase, peroxiredoxin, catalase, and bifunctional catalase/peroxidases, to degrade Reactive Oxygen Species (ROS). In addition to the previously identified secreted peroxidase, we recently identified and cloned two chromosomally encoded peroxiredoxins, one secreted. Lcr transformed with the BCP-like peroxiredoxin showed a significant (200 fold) increase in tolerance to 100 M hydrogen peroxide, as compared to a marginal 4-5 fold tolerance provided by either the PR2 peroxiredoxin or SC2 peroxidase. Most importantly, BCP-like peroxiredoxin augmented Lcr tolerance to 50 M tert-butyl hydroperoxide (tBOOH; an organic oxidizing agent) by nearly 1000 fold. Las BCP-like peroxiredoxin may represent a second critical secreted effector, this one conserved across all pathogenic Liberibacter species (even in the phageless Las strains), that functions to suppress host symptoms.
The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of September 2016, we have processed more than 40,850 grower samples. For the 2016 calendar year to date, we’ve received 3,531 samples from growers, which is on track for a calendar year total exceeding 2015 levels. The 3,995 growers samples processed during 2015 represented a 46% increase in the number of grower’s samples over the previous calendar year, which in itself had seen a 37% increase over 2013 numbers. These increases are likely due to the increased efforts to mitigate the HLB-associated tree stresses. Growers in this area, and most other regions, currently have one or more HLB mitigation program that they are evaluating. These growers are using the HLB lab to evaluate the effectiveness of their efforts. Another evidence of increased grower usage of the lab is seen in the fact that 60% of the individuals who submitted samples during 2015 were new clients who had not previously submitted samples. So far, new clients comprise 54% of submitters in 2016. Additionally, more than 44,100 samples have been received for research for the entire period of diagnostic service, supported by grant funding of individual researchers. This brings the grand total to more than 85,000 plant samples processed. Grower samples are typically processed and reports returned within a two to four week time period. For this report, focusing on the quarter from July through September 2016, there were 306 growers samples processed in addition to research samples and psyllids. The HLB Diagnostic Lab continues to offer the service of detection of CLas in psyllids as funded in this grant. Current methods of sample processing have become streamlined and therefore seen no change in procedure.
The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of September 2016, we have processed more than 40,850 grower samples. For the 2016 calendar year to date, we’ve received 3,531 samples from growers, which is on track for a calendar year total exceeding 2015 levels. The 3,995 growers samples processed during 2015 represented a 46% increase in the number of grower’s samples over the previous calendar year, which in itself had seen a 37% increase over 2013 numbers. These increases are likely due to the increased efforts to mitigate the HLB-associated tree stresses. Growers in this area, and most other regions, currently have one or more HLB mitigation program that they are evaluating. These growers are using the HLB lab to evaluate the effectiveness of their efforts. Another evidence of increased grower usage of the lab is seen in the fact that 60% of the individuals who submitted samples during 2015 were new clients who had not previously submitted samples. So far, new clients comprise 54% of submitters in 2016. Additionally, more than 44,100 samples have been received for research for the entire period of diagnostic service, supported by grant funding of individual researchers. This brings the grand total to more than 85,000 plant samples processed. Grower samples are typically processed and reports returned within a two to four week time period. For this report, focusing on the quarter from July through September 2016, there were 306 growers samples processed in addition to research samples and psyllids. The HLB Diagnostic Lab continues to offer the service of detection of CLas in psyllids as funded in this grant. Current methods of sample processing have become streamlined and therefore seen no change in procedure.
Las appears to have acquired key genes for plant adaptation by way of its phage and these phage genes are highly regulated; repressed in psyllids, and derepressed only in plants. We have proposed targeting specific regulators of key phage encoded virulence genes (such as the Las peroxidase) as well as the peroxidase itself and key regulators of the (lethal) phage lytic cycle. Objective 1 is control of HLB using the putative Las LexA-like repressor protein, potentially a key phage lytic cycle regulator. We had previously shown that this chromosomally encoded phage repressor (Clibasia_01645) binds specifically to its own promoter as well as to an SC1 promoter region midway between the divergent lytic cycle (late gene) and early gene promoter regions. Four different green fluorescent protein (GFP) reporter constructs were made using both the chromosomally encoded, bidirectional promoter regions of the phage repressor cloned in both directions, and the phage encoded late and early gene (divergent) promoter regions. In Lcr, phage late (lytic) gene promoter activity was quantified and was significantly (7X) higher than the early gene or lacZ promoter (used as a control), both of which were strongly and comparably expressed. The LexA-like repressor promoter exhibited low but significant expression levels, as expected. This work established a baseline for the reporter assays in Lcr using repressor expression plasmids. For repressor expression, 1) the full length chromosomal LexA (CLIBASIA_01645) repressor gene, 2) a naturally occurring truncated version of the LexA repressor gene, and 3) a phage encoded C2-like repressor gene were all cloned to be driven by the constitutive LacZ promoter to examine transcription repression of GFP following co-transformation with the GFP containing promoter reporter constructs in Lcr. These experiments are underway. Objective 2 is control of HLB using a repressor protein of unknown identity from psyllids as target. We previously reported functional repression of a Las phage lytic cycle holin promoter by a predicted Wolbachia repressor protein found in aqueous psyllid extracts. This Wolbachia gene was cloned in an expression vector and used for in vitro protein synthesis. The expressed Wolbachia protein was added to an Lcr culture expressing the holin promoter/GUS reporter and exhibited partial but strong inhibition of the holin promoter. This work provides confirmation of a specific, now identified repressor target, and also indicated that complete suppression of holin promoter activity required an additional partner. Objective 3 is control of HLB using the Las phage peroxidase and Las lytic cycle activator(s) as targets. Bacteria use a variety of enzymes, including peroxidase, peroxiredoxin, catalase, and bifunctional catalase/peroxidases, to degrade Reactive Oxygen Species (ROS). We identified and cloned two chromosomally encoded peroxiredoxins, one secreted, in addition to the phage peroxidase. These peroxiredoxins are highly conserved among all Liberibacters (including the Las Ishi strain which does not have a phage). Three separate experiments to interrupt either or both genes in Lcr were unsuccessful, indicating that we will have to use a rescue plasmid that can be cured in order to conduct the small molecule repression screen on any of these targets.
This is a three-year continuing project, terminated in Aug. 31, 2016. The overall objective was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. Based on our optimized nano delivery system and our screened effective antimicrobials from our previous funded projects (CRDF#584 and #617), a total of 14 antimicrobials were formulated in nano emulsions and applied on the HLB-affected potted plants in the greenhouse by foliar spray and bark-painting, including two agricultural antibiotics {Validoxyamine (VA) and Zhongshengmycin (ZS)}, seven antimicrobial compounds {Sulfadimethoxine Sodium (SDX), Silver Nitrate (SN), Silver Phosphite (SP), EBI-602, Actidione (ACT), p-Cymene (PCY) and Carvacrol (Carv)}, two antibiotics {(Oxytetracycline (OXY) and Streptomycin (Strep) }, combination of ACT and VA (Act+VA), and two positive controls {(Ampicillin (AMP) and Penicillin (Pen) }. The results indicated that the nanoemulsion formulation enhanced the therapeutic efficiency of the above antimicrobials against Las bacterium. We also screened two adjuvants and optimized one formulation to improve the effectiveness of Pen by foliar spray. More than 180 HLB-affected citrus trees were treated by combining thermotherapy, chemotherapy and nutrients. The thermotherapy was carried out by steam at 125~128 F for 120 seconds or 180 seconds, respectively. The chemotherapy treatments included EBI-602, Silver nanoparticle and CARV, using Pen as the positive control. The Nutrient treatment was additional micronutrient nutrition beyond the normal fertilization. According to the Field Trial Tree Evaluation Methods developed by CRDF, we investigated tree canopy, tree health, fruit drop and fruit quality as well as Las bacterial titers by real-time PCR. The tree canopy decline index (DI) was compared between the treated and control plants. The two-year results showed that PEN was the more effective to control Las bacterium than EBI-602, silver nanoparticle or CARV. Thermotherapy and additional nutrition promoted citrus growth and vigor, especially in the severe HLB-affected trees, whereas Las bacterial titers returned to original levels after a short-term decrease by heat-treatment. The disease severity index (SDI) decreased by 6% after application with PEN, followed by EBI-602 (4%), CARV (3.5%) and silver nanoparticle (1.3%). The integrated practices (antimicrobial treatment coupled with heat treatment and nutrition fertilization) decreased the fruit drop by 10~20 %, increased the fruit and juice weight by 3~13 %, and decreased the ratio of brix to acid by 0.2~5.0 %. However, this project was terminated in Aug. 31, 2016. Thereby, we could not get the second year data of fruit drop, fruit quality and yield. In addition to keep on the field trials of our previous enhanced projects, more than three-year s results indicated that PEN was also the most effective antimicrobial in eliminating the Las bacterium by gravity bag infusion. Due to the larger molecular weight and less solubility in water, VA, SDX, PCY and CARV were not very effective when applied by gravity bag infusion. The outcomes of this project will have potentials to go forward to solve the immediate problems that Florida citrus faces. A total of three publications have been published in Crop Protection, PLoS ONE and Journal of Applied Microbiology.
This project is a continuation of the funding that has been provided to Southern Gardens Citrus (SGC) to provide growers, researchers and private companies a laboratory to detect the pathogen that causes huanglongbing (syn. citrus greening). For the fourth quarter of the funding period (4/1/2016-6/30/2016), 8,931samples were processed. Of those 7,897 were plant samples and 1034 were psyllid samples. For year 1 of the funding, a total of 32,949 samples were run of which 30,693 were plant samples and 2,256 were psyllid samples. As mentioned in a previous report a procedure has been developed to extract DNA from roots that still allows the use of the Qiagen robot but removes the inhibitors that are a problem with root samples. For year 2 of the funding period, we plan on adapting a PMA protocol to allow a live/dead determination with the qPCR procedure. As has been the trend in the last couple of years, most of the samples coming in the lab are research samples from private, university and CRDF trials.
The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of June 2016, we have processed more than 40,500 grower samples. For the 2016 calendar year to date, we’ve received 3,288 samples from growers, which is on track for a calendar year total exceeding 2015 levels. The 3,995 growers samples processed during 2015 represented a 46% increase in the number of grower’s samples over the previous calendar year, which in itself had seen a 37% increase over 2013 numbers. These increases are likely due to the increased efforts to mitigate the HLB-associated tree stresses. Growers in this area, and most other regions, currently have one or more HLB mitigation program that they are evaluating. These growers are using the HLB lab to evaluate the effectiveness of their efforts. Another evidence of increased grower usage of the lab is seen in the fact that 60% of the individuals who submitted samples during 2015 were new clients who had not previously submitted samples. So far, new clients comprise 48% of submitters in 2016. Additionally, more than 44,100 samples have been received for research for the entire period of diagnostic service, supported by grant funding of individual researchers. This brings the grand total to more than 84,750 plant samples processed. Grower samples are typically processed and reports returned within a two to four week time period. For this report, focusing on the quarter from April through June 2016, there were 2,370 growers samples processed in addition to research samples and psyllids. This quarter completed the first year of the grant, with a grand total of 5,401 growers samples being processed from July 2015-June 2016. These numbers are significantly higher than the expected increases in sample volume, which may indicate that the second half of the grant could see even higher numbers of growers samples submitted. The HLB Diagnostic Lab continues to offer the service of detection of CLas in psyllids as funded in this grant. Current methods of sample processing have become streamlined and therefore seen no change in procedure.
The objective of this research will 1) characterize Pr-D (FP3) and its role and disease suppression; 2) investigate the dynamics of the prophages/phages in Las bacteria by revealing the variations in gene expression and recombination; and 3) identify critical elements, such as heat and chemical stress that facilitates lytic activities of the prophages. In addition, we will demonstrate whether or not the cross protection using mild strains of Las bacteria will work for the HLB pathosystem along with quantitative detection protocols for prophage-based strain differentiation. We have detected the bacterial transcripts in mixed eukaryotic/ prokaryotic samples at set time points throughout a typical course of thermotherapy treatment. Overall, the analysis revealed that, depending upon the time at which the samples was taken, between 4% and 9% of the total predicted genes for Las appear to be differentially regulated during the thermotherapy process compared to a sample taken at time zero. These genes provide initial evidence of how the bacteria itself is modifying its transcriptional activity in response to the increase in temperature. Although a majority of the regulated genes found are defined as hypothetical, several do have a predicted function and their contributions to the effects of heat therapy are now under investigation. Based on RNA-seq data, we further verified some of the transcriptome using Las infected citrus plants after heat treatment at 5 time points: T0/T3/T6/T9/T12 hours. Comparing the effects of heat stress among the samples, we identified differentiation on gene expression level between the plants treated with heat, at all set points T3/T6/T9/T12, compared to no treatment T0. We found genes that constantly overexpressed and downregulated in Las bacteria after heat treatment. For example, superoxide dismutase gene was overexpressed after 3/6/9 and 12 hours after heat treatment. Interestingly, the dnaK, heat shock proteins gene was also overexpressed. While some genes were consistently down regulated in the samples of T3/T6/T9 and T12 treatments, some were only downregulated after 12 hours of heat treatment. These genes are in particular related to motility, such us flgF-Flagellar basal-body rod protein, flagellar C-ring protein, and components of type IV pilus. In addition, some factors that promote growth and protein synthesis were downregulated, they are methionyl-tRNA synthetase, RNA polymerase sigma factor RpoD, and DNA-directed RNA polymerase subunit. These results indicates that the heat treatment had a strong effect on Las bacteria gene regulation, and possibly induced the lytic cycle, reducing growth and motility of Las bacteria after heat treatment. Through these analyses, we may have identified key genes involved in the lytic induction after heat stress that can be eventually manipulated to induce lytic pathways. Based on the variations of Las prophages/phages, we recognized certain molecular mechanisms behind the symptom variations and their association with “mild strains” of Las bacteria and host tolerance/resistance. Construction of a transcriptional reporter system is also currently in progress for the final verification of the genes identified as being involved in stress response to heat in plants subjected to thermotherapy. This system will also allow future experimentation to rapidly identify other catalysts that can produce the same reduction in bacterial numbers as thermotherapy. Purification of the ~10Kb FP3 region has been achieved from both periwinkle and citrus, though the amount purified from citrus appears to be less than that from periwinkle (as would be expected from the lower bacterial titer found in citrus). A library as been constructed from the DNA resulting from the FP3 purification for sequencing of this important region.
This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermo-therapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (April 2016 to June 2016), three foliar sprays of the antimicrobial chemicals (Pen, Pcy, Carv and EBI-602) were applied at a two week interval for all treated trees from March to May, 2016. For gravity bag infusion, two refill applications of Pen, SD and SDX were conducted at two week interval in April, 2016. According to the Field Trial Tree Evaluation Methods developed by CRDF, we investigated tree canopy, tree health, fruit drop and fruit quality and Las bacterial titers by real-time PCR. Fruit quality tests were done on the field trail of combination of chemotherapy, thermo-therapy and nutrient therapy. A total of 50 fruit were harvested from the 3 trees in each trial replicate. Tests were run on 20 fruit from each sample for size, peel color, puncture resistance, fruit weight, juice weight, brix and acid. The tree canopy decline index (DI) was compared between the treated and control plants. Eight mature leaves with petioles from each of the treated and control trees were sampled around the canopy for PCR test. The preliminary results indicated that:1) the integrated practices (antimicrobial treatment coupled with heat treatment and nutrition fertilization) could decrease the fruit drop, increase the fruit and juice weight, and decrease the ratio of brix to acid; 2) compared to the control plants, all antimicrobials reduced the Las bacterial titers, especially PEN. 3) Both SD and Pen reduced the DSI through two years application; 4) two new adjuvants (Bio and MF200) improved the effectiveness of Pen by foliar spray; 5) Ten antimicrobials were prepared in two different concentrations of the nano formulations (0.1 % and 1.0 %) in the greenhouse test. The Ct values kept over 36.o in the PEN-treatment. In next quarter, we will keep on our application and prepare the final reports. One papers has been published in
Citrus blight continues to be a major economic problem in citrus groves in Florida. Thousands of trees each year succumb to citrus blight, with estimated losses at over $60 million per year. The disease can occur on all common citrus cultivars, and Carrizo citrange are especially susceptible. Early symptoms are zinc deficiency in the leaves which may disappear, zinc accumulation in the phloem and eventually high zinc levels in the xylem. Blockage of xylem tissues with amorphous plugs follows with reduced water uptake. The causal agent of citrus blight is unknown. However, symptoms and all of the characteristics associated with citrus blight can be reproduced by root graft inoculations. Therefore in a project previously funded by CRDF we used NGS RNA sequencing protocols to look for novel viruses in roots of sweet orange with blight, but not present in roots of healthy trees, or trees affected by HLB. We identified several related endogenous pararetroviruses related to Petunia Vein Clearing Virus (PVCV) using a collection of 10 RNA libraries prepared from 10 different root samples collected from healthy trees or those with blight or HLB. In the quarter just ending we have focused our efforts on the remaining objectives: generating complete genome sequences for any and all active blight associated pararetroviruses and developing a active virus specific assay comprehensive enough to detect all blight associated pararetroviruses. As of the last quarter we had successfully generated a complete genome sequence for a blight associated active pararetrovirus, we are prepared to hand the sequence data to CRDF. In addition to the original genome sequence we have continued sequencing other pararetrovirus isolates from additional trees and geographic locations to determine levels of diversity. Six other complete genome sequences are underway. Also in our last quarter we had tested multiple assays for the active pararetrovirus on a large sample set to determine which of the assays was specific only to active pararetrovirus while still being inclusive enough to detect all active pararetroviruses. A single assay was developed that was very effective in detecting all active pararetrovirus samples in the 2015 sampling, this will meet the objective of the project. As a final check, an additional set of samples was taken in the summer of 2016 for testing and validating the new assay. Finally, as an additional bit of work to try to establish that pararetroviruses were infect fully biologically active in blighted trees we have consulted with a team of experts in the pararetrovirus community to modify existing virus purification protocols to develop a means to extract viral particles that could be used to establish modified Koch’s postulates.
This project is a continuation of the funding that has been provided to SGC to provide an HLB detection laboratory that is available to growers and researchers throughout the state. For the third quarter (1/1/2016 – 3/31/2016), 8,618 samples were processed. Of those, 8,336 were plant samples and 282 were psyllid samples. To date for the funding period, a total of 24,018 samples have been run of which 22,796 were plant samples and 1222 were psyllid samples. SGC has developed a procedure to extract DNA from roots and has been running a limited number of samples for selected experiments. The procedure adds an additional step to the DNA extraction procedure (and about $1.50 more to the cost) but it still allows us to use robotic extraction and the Qiagen magnetic bead chemistry.
Accumulating evidence confirms our working hypotheses that Las acquired key genes for plant adaptation by way of its phage and that these phage genes are highly regulated; off in psyllids, and on only in plants. We have proposed targeting specific regulators of key phage encoded virulence genes (such as the Las peroxidase) as well as key regulators of the (lethal) phage lytic cycle. Direct targeting of the Las peroxidase enzyme itself is also proposed. Objective 1 is control of HLB using the putative Las LexA-like repressor protein, potentially a key phage lytic cycle regulator. We had previously shown that this chromosomally encoded phage repressor (Clibasia_01645) binds specifically to its own promoter as well as to an SC1 promoter region midway between the divergent lytic cycle (late gene) and early gene promoter regions. Such binding is characteristic of a chromosomally encoded phage regulator/repressor, but did not prove functional repression of a phage gene. In order to demonstrate actual repression, multiple DNA constructs in two cross compatible bacterial vectors were made for use in L. crescens (Lcr) as a proxy host for Las. Lcr is missing all SC1 and SC2 prophage genes. Lcr-compatible vectors were used to 1) express the LexA repressor, and 2) separately express the multiple potential targets of the repressor using promoter activity reporter constructs. Four different GFP reporter constructs were made by translationally fusing an enhanced green flourescence protein, GFP with both 1) the chromosomally encoded, bidirectional promoter regions of the phage repressor in both directions, and 2) the phage encoded promoter regions, again in both directions. Only the phage SC1_gp125 promoter (lytic gene direction) GFP construct exhibited strong fluorescence in E. coli, whereas the remaining 3 constructs were largely inactive in E. coli. This confirmed our previously published work indicating that Liberibacter promoters don’t function as well in E. coli as they do in Liberibacters. This also indicated strong constitutive activity of the lytic cycle promoter in the absence of repression. Objective 2 is control of HLB using a repressor protein of unknown identity from psyllids as target. We previously reported functional repression of a Las phage lytic cycle holin promoter by an unknown repressor protein from aqueous psyllid extracts. The repressor protein has now been identified by liquid chromatography mass spectroscopy to be comprised at least in part, of a protein encoded by Wolbachia, a bacterial endosymbiont commonly found infecting psyllids, including all psyllids carrying Las in Florida. This repressor protein was also found to be uniquely encoded by Wolbachia found in Asian citrus psyllids and absent in Wolbachia found in the fruit fly (Drosphila). Objective 3 is control of HLB using the Las phage peroxidase and Las lytic cycle activator(s) as targets. Bacteria use a variety of enzymes, some secreted, to degrade Reactive Oxygen Species (ROS), including peroxidase, peroxiredoxin, catalase, and bifunctional catalase/peroxidases. We identified two chromosomally encoded Las and Lcr peroxiredoxins, one secreted, in addition to the phage peroxidase. Hence, a small molecule based chemical repression screen for loss of peroxidase activity would be unlikely to work in their presence. These peroxiredoxins are highly conserved among all Liberibacters (including the Las Ishi strain which does not have a phage). We have cloned the peroxiredoxins and are in the process of creating Lcr deletion mutants of the corresponding genes in order to conduct the small molecule repression screen on the best target.
The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of March 2016, we have processed more than 38,000 grower samples. For the 2016 calendar year to date, we’ve received 917 samples from growers, which is on track towards a calendar year total very close to 2015 levels. The 3,995 growers samples processed during 2015 represented a 46% increase in the number of grower’s samples over the previous calendar year, which in itself had seen a 37% increase over 2013 numbers. These increases are likely due to the increased efforts to mitigate the HLB-associated tree stresses. Growers in this area, and most other regions, currently have one or more HLB mitigation program that they are evaluating. These growers are using the HLB lab to evaluate the effectiveness of their efforts. Another evidence of increased grower usage of the lab is seen in the fact that 60% of the individuals who submitted samples during 2015 were new clients who had not previously submitted samples. So far, new clients comprise 46% of submitters in 2016. Additionally, nearly 43,800 samples have been received for research for the entire period of diagnostic service, supported by grant funding of individual researchers. This brings the grand total to more than 81,800 plant samples processed. Grower samples are typically processed and reports returned within a two to four week time period. For this report, focusing on the quarter from March 2016, there were 855 growers samples processed and 1,071 research. Since the start of the current grant in July 2015, the lab has received 3,093 growers samples, which is even higher than the expected increases in sample volume. The HLB Diagnostic Lab continues to offer the service of detection of CLas in psyllids as funded in this grant. Current methods of sample processing have become streamlined and therefore seen no change in procedure.
The overall objective of this research program is to develop an effective and sustainable phage-based biocontrol system for citrus canker caused by Xanthomonas axonopodis pv. citri (Xac). Our approach is to develop a pool of virulent (lytic) phages and antibacterial particles called ‘tailocins’ that can be implemented in the field as an alternative to copper for control of citrus canker. In greenhouse trials we have demonstrated that both phage and tailocin cocktails are effective in reducing disease symptoms with one treatment. Tailocins are protein assemblages that function like the tails of phages, by adsorbing to the bacterial cell and then puncturing the cell envelope. Unlike phages, tailocins do not have a capsid and thus inject no DNA, instead relying on the membrane puncturing activity to kill the cell. Like phages, tailocins use tail fibers to recognize specific receptors on the target cell surface. Tailocins are thus potent and specific lethal nanoparticles. Our current efforts have focused on further characterization of both Xac phages and tailocins. Phage CCP504 (Podophage) propagated on homologous host (Xac Block22) had an observed adsorption rate constants of 1.7 X ^-10 ml cell^-1 min^-1, whereas CCP513 (Siphophage) propagated on the same host had an observed adsorption rate constants of 8 X ^-11 ml cell^-1 min^-1. Ongoing studies will determine burst size for each of the phages. Two non-type IV pilus dependent phages that form plaques on Xac are currently being characterized. Since the phages and tailocins will be exposed to natural sunlight when deployed as control agents we have initiated studies to assess the effect of UVC, UVB and UVA light on activity. Initial studies indicate that phage CPP504 and phage CCP513 are extremely sensitive to UVC, less sensitive to UVB and even less to UVA, as indicated by log reduction(s) in activity. Tailocins XT-1 and XT-4 lost less than 10 fold activity when exposed to UVB at energy levels that causes 100-1,000 fold reduction in phage activity. Our previous results indicate that tailocins can be effective in reducing disease as a foliar spray and the observed insensitivity to UVB warrants continued studies and the search for diverse tailocins with activity against Xac.
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