Quaternary Ammonium Compounds (Quat) is a powerful antimicrobial agent. However, Quat cannot be directly applied to citrus plants as a film forming fungicide/bactericide as it exhibits severe phytotoxicity and poor rainfastness. This research uses a silica based Quat delivery system (Fixed-Quat). Fixed-Quat material is non-phytotoxic and it demonstrated good rainfastness. In the previous reporting period, based on the promising feedback from 2014 Citrus Canker Trials, Fixed-Quat A-II nanogel formulation was synthesized. EPA approved ( For Food Use only) raw materials were used for the synthesis of Fixed-Quat A-II material. In this reporting period, further optimization of Fixed-Quat A-II nanogel was conducted. A new batch of stable Fixed-Quat A-II nanogel was synthesized with Quat concentration of 13,500 ppm (demonstrated industrial feasibility of producing concentrated formulations). For use as sanitizing and flocculating agents EPA requires a maximum quat concentration of 200ppm (end use concentration). Safety of the Fixed-Quat A-II nanogel was evaluated by phytotoxicity studies carried out in a Panasonic Environmental Test Chamber (Model MLR- 352H) which allowed for controlled day/night cycling temperatures, light intensity and humidity to simulate summer weather conditions (biocide application season). The composition and interactions between the components was confirmed using Fourier transform infrared spectroscopy (FTIR). Studies conducted on Vinca sp and Hierloom Tomato revealed no sign of plant injury even as high as 500 ppm Quat for Vinca and 1000 ppm for Tomato. Antimicrobial studies of Fixed-Quat A-II nanogel was conducted against Xanthomonas alfalfae subsp. citrumelonis (Citrus Canker Surrogate), Pseudomonas syringae pv syringae, a gram negative causative agent of bacterial speck in citrus and tomato sp and Clavibacter michiganensis subsp michiganensis, a gram positive causative agent of canker and systemic infections in tomato. Studies were conducted to determine the Minimum Inhibitory Concentration (MIC) and compared against Kocide 3000 and copper sulfate. MICs of Fixed-Quat A-II were found to be 0.6-1.25 g/mL for X. alfalfae, 1.25 g/mL for P. syringae and 2.5 g/mL for C. michiganensis.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, June 2015. This report covers the second funding period of the Huanglongbing Diagnostic Laboratory’s service, July 2012-June 2015. The HLB Diagnostic Laboratory has been operational at UF-IFAS-SWFREC since February 2008. Since the opening of the lab, there has been continued development of techniques, protocols, and efficiency. The lab has been in operation for over 7 years, and as of June 30, 2015, has processed more than 35,000 growers samples. Additionally, nearly 41,200 samples have been received for research, which are supported by grant funding of individual researchers, bringing the total to more than 76,200 plant samples processed. Grower samples are typically processed and reports returned within a 2-4 week time period. During the final quarter, April-June 2015, 849 samples were received from growers, bringing the 2015 year-to-date total to 1819 samples. This is already two-thirds the total number of growers samples received during the entirety of 2014, which had seen a 37% increase over 2013, and continues the trend seen in recent years of increasing growers samples. This was the busiest second quarter for growers samples since 2011. The increased number is likely due to the increased efforts to mitigate the HLB-associated tree stresses and evaluating different management strategies, as well as continued replanting of trees. During this quarter, there were 259 research samples processed, bringing the second quarter total to 1,108 and the year-to-date sample total to 4,606. The 2012-2015 grant period saw a grand total of 25,919 plant samples processed, with 18,604 research samples. Of the 7,315 growers samples, more than half of that number came in the third year (July 2014-June 2015), which surpassed levels of the final year seen in the previous grant (July 2011-June 2012), the highest since the peak in 2009-10. Objectives for the funding were to continue providing timely diagnostic services, free of charge, to growers and to assist with research efforts, especially in light of growers persisting need for testing citrus samples and interest in psyllid testing to assist in monitoring and assessing efficacy of HLB management strategies. Flexibility in providing any service to growers has been part of this lab’s mission, which has been demonstrated when we participated in evaluating commercial HLB detection kits and our having been requested to quantify live vs. dead bacterial cells. In addition to leaf tissue and psyllid, we have also recently processed plant samples from roots, fruit, peduncle, bark, etc., and occasionally non-host plants with suspicious symptoms. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant. Techniques, Protocols and Research Current methods of sample processing have become streamlined and therefore seen little change. Protocols have remained in place for detection of HLB in Asian Citrus Psyllid, as well as for quantification of HLB in both plant and psyllid samples. The research needs of dozens researchers are provided through the infrastructure of this lab, while the researchers contribute resources to cover the costs of their samples.
For the period covered by this funding cycle, 25,547 have been run by the SGDL. For comparison purposes, the estimated sample load was 25,000 in the original proposal. Of the samples for the period, 92% of the samples (21,928) were plant samples, 7% were psyllid samples (1588) and 8% (2,031) were either juice or leaf samples submitted by juice processors. Although it is somewhat difficult to track, an effort was made to try to determine the end use of the samples that were submitted. Based on the numbers where a reasonable guess could be made, 8.7% the non.psyllid samples were grower samples presumably for disease diagnosis, 10.4% of the samples were processor samples (either juice or leaf samples for juice analyses), and 80.9% of the samples were research samples from trials. Of the research samples, 88.5% of the samples were from private submissions (i.e. chemical companies, trial managers, private companies, etc.), 7.8% were from universities and 3.7% were from federal agencies. Based on the recent sample submissions, the trend toward research samples is continuing. Specifically many of the samples are now coming from trial managers that are either selecting trial sites, using the samples for data collection, or to validate data from other sources.
One of the primary goals of this work is to identify a small molecule treatment that can be used to activate the phage lytic cycle genes encoded by Las prophage, thus bringing about the death of Las bacteria carrying these prophage. The majority of Candidatus Liberibacter asiaticus (Las) strains described carry bacteriophage similar to SC1 and SC2 of Las UF506. Lytic phage particles are formed and have been reported in both periwinkles and in citrus. We previously reported that relative mRNA expression levels of prophage late genes SC2-gp095 (“peroxidase”), SC2-gp100 (“glutathione peroxidase”) and particularly SC1-gp110 (‘holin’) were much higher in periwinkle than in citrus. We also reported that both the prophage holin (SC1_gp110) and endolysin (SC1_gp035) were functional, and that strong expression of the holin gene alone in Las cells would be sufficient to kill the cells, whether or not phage particles were formed. This could be part of the explanation as to why Las has to date not been cultured (Fleites et al. 2014). The holin promoter (a “late” phage gene promoter that is switched on only when the lytic cycle is activated) is strongly suppressed by psyllid extracts. Identification of the repressor protein is underway. We now report that the peroxidase on SC2 is a reactive oxygen species (ROS) scavenger and confirm that it has peroxidase activity. SC2-gp095 was cloned in a shuttle vector and transformed into L. crescens (Lcr). Transformed Lcr cells showed 20-25% enhanced resistance to hydrogen peroxide on agar plates and 47% higher enzymatic activity than Lcr. Non-classical secretion potential was confirmed by enzymatic and Western blot analyses in Lcr. Transient expression of SC2_gp095 in planta resulted in strong transcriptional down-regulation of RbohB, the key gatekeeper of the H2O2 mediated defense signaling in plants, helping explain the surprisingly long incubation period (years) before HLB symptoms appear in Las-infected citrus. In psyllids, stringent control of all lytic cycle genes, including SC1 excision and replication (early genes) as well as the lysis genes and the peroxidase (late genes), appears to be critical to prevent Las from being detected by the insect innate immune system. Upon examination of SC1 and SC2 promoter regions, we found strong matches to a predicted Las chromosomally encoded repressor, and driving the lytic cycle (late) genes on SC1 and the peroxidase (late) gene on SC2. This likely ties these phage genes into the bacterial SOS response, which is activated by stress, particularly DNA damage. This is the second phage promoter / repressor region we have identified, and we are working to confirm the functionality of the predicted Las repressor. We have sequenced five PCR amplified UF506 repressor genes from citrus, and all encode a full-length protein. In contrast, all eight sequences derived from UF506 infected periwinkle had a truncated version of the repressor, indicating Las survival in periwinkle depends on this mutation. If so, this repressor represents an excellent molecular target for control. Even if only a small amount of phage late gene expression is activated in psyllids, it would likely trigger a strong insect innate immune response that can become systemic and result in aborting the infection process.
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 if 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 propagated more Las-infected periwinkle and citrus plants that contain high titers of prophage/phage FP3, which will be used for isolation and characterization of prophage/phage FP3. Phages from infected periwinkle tissues have been enriched using a refined phage purification pipeline consisting of differential centrifugations and PEG precipitation. The partially purified phage preparation showed an enrichment of phage DNA by 20~50 folds and a trace amount of Las genomic DNA as determined by PCR. While phage-like particles were observed under electron microscope, the images were not conclusive due to the large amount of contaminating host materials present. Further purification will be carried with sucrose density gradient and/or CsCl equilibrium density gradient centrifugation to separate the Las bacteriophages from contaminating host materials. Different varieties of citrus plants inoculated with a mild strain have been evaluated in greenhouse. Intriguingly, different varieties showed different response to the “mild stains/isolates”. However, in a given variety, the mild strain status was maintained after three consecutive propagation. We are evaluating the factors that affect the symptoms and titers and determining if a mild strain can be maintained in major commercial citrus varieties. We have developed a digital PCR (dPCR) system for early detection of HLB and tracking of lysogenic and lytic activities of the Las prophage/phage. We show that as few as 1 to 2 copies of the targeted DNA molecules per microliter can be detected, with the prophage probe providing the best sensitivity. With this tool, we were able to detect the lytic activities of the Las bacteriophage in some sample. For example, 20 times more phage DNA than bacterial DNA was present in sample EL L2VC2 than other samples, indicating active induction and replication of the Las phage in the infected citrus trees. New primers were designed to amplify the Las phage region iFP3. These new primers contained a thiophosphate modification between the last two nucleotides at the 3′ end to increase their stability during rolling circle amplification (RCA). Samples from a Las positive citrus plant, a Las positive periwinkle plant, and a Las negative control plant were used for RCA. After several attempts at amplification via commercially available RCA kits, a protocol that amplified the DNA approximately 7 fold was developed. The specificity of the product obtained with the designed primers is currently being tested. In addition, Las-infected plants were subjected to heat stress using the same parameters as those currently used in thermotherapy. Bacterial RNA was purified from these samples using a novel technique that we newly developed with depletion of microbial 23S and 16S rRNA for use in RNA-Seq analysis. Overall both the quantity and purity were measured and the samples are being sent for RNA-Seq analysis. The development of this technique will provide a more rapid approach than originally planned since the effects of heat stress on all of the phage-encoded genes can be investigated simultaneously. Data obtained via RNA-Seq will also be broader than that obtained from looking at the phage encoded genes individually through PCR-based assays, and thus may also lead to insights related to the regulation of the phage encoded genes through trans-acting factors.
In the past year, Fixed-Quat A nanogel material was developed successfully using EPA approved materials. This material demonstrated no phytotoxicity under controlled laboratory conditions while providing strong antimicrobial properties against Xanthomonas alfalfae and other laboratory model bacteria. Later, the material was delivered for 2014 citrus canker trial. This material was sprayed on ‘Ray Ruby’ grapefruit with 3 week intervals at two rates (100 ppm and 200 ppm). Trees were sprayed throughout the ‘canker’ season from March to October. Results from the trial exhibited high efficacy against canker. While untreated control trees exhibited a total canker infection of 63%, trees sprayed with 100 ppm of Fixed-Quat A nanogel exhibited only 24% infection and those trees sprayed with 200 ppm of Fixed-Quat exhibited an impressive efficacy (infection reduced to 15%). In the same trial, commercial products Kocide 3000 and Magna-Bon displayed 16% and 18% infection respectively at standard applications rates. In this reporting period, feedback from the 2014 canker trial was taken into consideration to generate a new and improved Fixed-Quat A-II nanogel from environmentally safe, EPA approved for “Food Use” and industrially-attractive raw materials. The new nanogel material was synthesized with a concentration as high as 12,000 ppm to allow for easy transport and to allow for easier storage in bulk quantities. The Fixed-Quat A-II nanogel’s safety was tested by phytotoxicity studies carried out in a Panasonic Environmental Test Chamber (Model MLR- 352H) which allowed for controlled day/night cycling temperatures, light intensity and humidity to simulate summer weather conditions (biocide application season). Studies conducted on Vinca sp, an ornamental plant revealed no sign of plant injury even as high as 900 ppm of Quat. Antimicrobial studies of Fixed-Quat A-II nanogel was conducted against Xanthamonas alfalfae subsp. citrumelonis (Citrus Canker Surrogate). The MIC of new Fixed-Quat A-II nanogel was found to be 0.8 ‘ 1.6 ppm. The morphology and particle size of Fixed-Quat A-II nanogel was examined using Scanning Electron Microscopy (SEM) and indicated that Fixed-Quat A-II nanogel existed as a large layered micron size composite material. This demonstrates the film forming capability of Fixed-Quat nanogel. The size and composition of Fixed-Quat A-II was additionally studied using UV-Vis Spectroscopy and Dynamic Light Scattering (DLS). DLS confirmed that Fixed-Quat A-II exists as a large multi-micron composite while UV-Vis suggested interactions between the composite components. A batch of Fixed-Quat A-II nanogel (~3.0 gallon; 10,000 ppm) was delivered for 2015 citrus canker field trial.
This project was to study the characteristics of Liberibacter-like bacteria which have variations in the region commonly used for diagnosis by qPCR. Nearly all of these Liberibacter-like bacteria were found in citrus relatives; for example: Atalantia, Balsamocitrus, Calodendron, and Aegle genera. Nine of these isolates were grafted onto rough lemon seedlings in a greenhouse at Ft. Pierce. Most of the seedlings showed symptoms several months after grafting, but the bark patch used for grafting did not survive for more than a new weeks. Once the bark patch died, the Liberibacter-like bacteria did not persist in the host rough lemon. Two isolates did persist for a longer period of time, but these isolates test nearly identical to Candidatus Liberibacter asiaticus (Las). The host range plants were inoculated with these two isolates, and subsequently challenged using the type strain of Las. While this project is officially ending, these challenged plants will be monitored to determine if the symptoms if HLB are attenuated by either of the isolates. Using information developed from our study of the genomes of these Liberibacter-like bacteria, we were able to identify and are in the process of characterizing a fourth species of Candidatus Liberibacter, ‘Ca. L. caribbeanus’ found in Colombia, South America. The identification of bacterial genomic regions that are conserved among all four species will be useful for the development of better detection methodologies for Liberibacter species.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, April 2015. The lab has been in operation for more than 8 years, and as of April 2015, we have processed more than 34,500 grower samples. Additionally, more than 40,900 samples have been received for research for the entire period of diagnostic service supported by grant funding of individual researchers, totaling more than 75,500 samples processed. Grower samples are typically processed and reports returned within a two to four week time period. Numbers specific to this report are 1353 samples received from growers. This amount of samples for just the first quarter equals nearly 50% the total number of growers samples received during the entirety of last calendar year, which represents a significant increase from previous years. This was the busiest first quarter for growers samples since 2011. The increased number is likely due to the increased efforts to mitigate the HLB-associated tree stresses. Grower 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. During this quarter, there were 2528 research samples processed, bringing the first quarter total to 3881. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
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 completed the correlation studies for the blight related pararetrovirus. For the correlation study leaves and roots were collected from over 50 trees from five geographically distinct locations. The majority of these trees were identified as being blight affected by water uptake testing, but putatively healthy trees were also sampled. In some cases, bark tissue from trunks was also collected for testing. RNA extractions were completed for all samples from all trees, and the presence of active pararetrovirus was assessed using the two primer sets selected in the optimization study from the previous quarter. Every tree that showed diminished water take up using the syringe injection test was positive for citrus blight associated pararetrovirus DNA. In the previous quarter the root samples were tests, and all but 1 tree tested positive for pararetroviral RNA. In this quarter those results were confirmed again using more stringent treatments to eliminate genomic DNA. In addition, leaf samples from all 50 trees (along with healthy controls) were tested for the presence of active pararetrovirus RNA. Active pararetrovirus RNA was found in all blight affected tree leaf samples and none of the healthy control samples. At this stage we consider that we have met the goals of this objective, and there are two important conclusions. First, there is a very strong correlation between the reduced water uptake via the syringe test and the presence of active citrus blight associated pararetrovirus. Secondly, the active pararetrovirus can be found in leaf samples, eliminating the need for laborious root sampling as part of blight surveys. Our efforts now turn towards the remaining project objectives. Progress continues to be made towards generation of a complete genome sequence for the blight associated pararetrovirus. A primer walking strategy is being implemented to extend known sequences in both directions to generate a full genome. Sequencing efforts are being complicated by the need for complete removal of genomic DNA from the template RNA. In addition, the sequence data clearly indicates that there are more than one active citrus blight associated pararetrovirus. The active viruses are more closely related to each other than they are to the non-active endogenous pararetroviruses, and genome sequence has been generated for roughly 50 % of the genome at this point.
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. Progress towards the generation of a complete genome sequence for the blight associated pararetroviruses was stymied by the discovery of a variable region in the genome. The previously developed primer walking strategy that was implemented to generate a full genome was insufficient to cross this variable region and additional approaches were needed to complete the 5′ and 3′ ends of the sequence. An adapted strategy was employed to generate higher levels of coverage in variable regions of the genome, and an inverted PCR strategy will be developed to complete the 5′ and 3′ ends. As was suspected, the sequence data clearly indicates that there are three different active citrus blight associated pararetroviruses with clear differences in sequence identity. The active viruses are more closely related to each other than they are to the non-active endogenous pararetroviruses, and genome sequence has been generated for roughly 90% of the genome at this point. As we move into the next quarter, we hope to align the active pararetrovirus sequences with the non-active pararetrovirus sequences, to identify regions and PCR primers that will amplify from all active but no inactive pararetroviruses. These primers will then be tested on the 50 tree sample set to determine specificity, sensitivity and inclusivity as a usable blight assay.
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 built on this start by using the same ten RNA libraries to compare global gene expression in the roots of trees with blight or HLB as compared to the healthy controls. These analyses were performed using citrus genome coding sequences and mapping the short RNA reads with BowTie to the coding sequences. Each Blight library was compared to the healthy control library separately. The numbers of short RNA reads that mapped to each known citrus coding sequence were tabulated. The differences in expression were determined using Gfold, a program that can identify differentially expressed genes in such datasets without replications, by using the Poisson distribution. We have found large numbers of genes are differentially expressed in blight vs healthy roots. These include genes known to be involved in response of plants to both disease and stress. We also have identified proteins containing NAC domain-2 that are up regulated in roots with blight. These protein are active in the secondary strengthening of cell walls and when they are over expressed polyphenolic compounds accumulate in the lumen of xylem vessels. This could lead to the occlusion of the xylem vessels seen in blight. We have designed primers for qPCR of these genes that appear to be differentially expressed to validate the differences by qPCR using each RNA library as true replicates.
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 expanded the correlation studies for the blight related pararetrovirus. Eight sets of primers for reverse transcription-PCR detection of viral RNA (an indicator of virus replication and biological activity) were tested on the original set of blight affected, HLB infected and healthy citrus trees. Two sets of primers were identified that were the most specific to the blight related pararetrovirus. These were applied to an additional 8 samples from two different Florida locations with blight problems. As with the original testing, the blight associated pararetrovirus RNA was only found in blight affected trees, and was present in all trees identified as blight affected via water uptake testing. More significantly, it was determined that the RNA from the blight associated pararetrovirus was present in both the roots and canopy of affected trees, indicating that blight affected trees may be identifiable using a leaf based test. Based on these results, an additional sampling was conducted to complete the correlation objective. Leaves and roots were collected from over 50 trees from five geographically distinct locations. The majority of these trees were identified as being blight affected by water uptake testing, but putatively healthy trees were also sampled. In some cases, bark tissue from trunks was also collected for testing. RNA extractions are underway as a first step to completing the first objective. In addition, progress is being made towards generation of a complete genome sequence for the blight associated pararetrovirus. A primer walking strategy is being implemented to extend known sequences in both directions to generate a full genome.
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 expanded the correlation studies for the blight related pararetrovirus. At the end of the last quarter leaves and roots were collected from over 50 trees from five geographically distinct locations. The majority of these trees were identified as being blight affected by water uptake testing, but putatively healthy trees were also sampled. In some cases, bark tissue from trunks was also collected for testing. RNA extractions were completed for all samples from all trees, and the presence of active pararetrovirus was assessed using the two primer sets selected in the optimization study from the previous quarter. Every tree that showed diminished water take up using the syringe injection test was positive for citrus blight associated pararetrovirus DNA. When the RNA extractions were treated with DNAse to eliminate potential genomic DNA sources of citrus blight associated pararetrovirus, all but 1 tree tested positive for pararetroviral RNA. However, no reverse transcription negative controls suggest that some samples still had low levels of genomic DNA, and these results need to be confirmed. Still, in initial analysis, there is a very strong correlation between the reduced water uptake via the syringe test and the presence of active citrus blight associated pararetrovirus. In addition, progress is being made towards generation of a complete genome sequence for the blight associated pararetrovirus. A primer walking strategy is being implemented to extend known sequences in both directions to generate a full genome. Sequencing efforts are being complicated by the need for complete removal of genomic DNA from the template RNA. In addition, the screening of multiple trees has indicated that there may be more than one active citrus blight associated pararetrovirus. The active viruses are more closely related to each other than they are to the non-active endogenous pararetroviruses.
This project provides the infrastructure to support six CRB-funded projects that investigate multiple methods of detection of Candidatus Liberibacter asiaticus (CLas), the causal agent of huanglongbing (HLB), in asymptomatic plants or in psyllids. Early detection of CLas in asymptomatic plants uses a systems approach that includes transcriptomics, proteomics, and metabolomics. Detection of CLas using digital drop PCR uses preserved psyllids and CLas-infected plant material from colonies maintained in the Contained Research Facility. Colonies of both CLas-negative and CLas-positive Asian citrus psyllids and a culture of CLas (Hacienda Heights strain) are maintained for use in the above studies. The first set of studies on the early detection of CLas in asymptomatic plants using the systems approach was conducted using Washington navels, Lisbon lemons and Tango mandarins on Carrizo rootstock. The plants were assigned to one of the following treatments: Control-control (no manipulation); graft-control (grafted with clean scion with 3 grafts per plant); and HLB-graft (grafted with scion that tested positive by qPCR for CLas and grafted with 3 grafts per plant). The plants used to get CLas-infected plant material had the following Ct values: Washington navel ‘ 30.17; Lisbon lemon ‘ 24.00; and Tango mandarin ‘ 27.39. The clean scion used for the graft-control all had Ct values of 40. The plants were grafted on the following days ‘ February 25, 2014 ‘ navel graft-control and mandarin graft-control; February 26, 2014 ‘ lemon graft-control, and navel ‘ HLB-graft; and February 27, 2104 ‘ lemon HLB-graft, and mandarin HLB-graft. All plants were housed in the same greenhouse under the same environmental conditions (80’F, 16L: 8D). The plants were sampled regularly by each group, and samples for qPCR analysis were taken at approximately monthly intervals. The qPCR samples were taken such that they did not interfere with sampling by any of the research groups. The plant tissue was held in a -80 freezer until it was sent to the Citrus Research Board Laboratory for qPCR testing using USDA-APHIS standard methods. The number of plants becoming CLas-positive and the time to the first CLas-positive reading by qPCR varied with cultivar. Of the 22 lemons graft-inoculated with CLas, 10 plants became CLas positive after 319 days. For the navels, 15 plants out of 19 inoculated plants became positive after 320 days. Of the 16 mandarins graft inoculated with CLas, only 2 had at least one positive Ct value, and 2 had suspect Ct values. However, the sample immediately following the positive or suspect value gave a negative value. A small number of these plants are still being held and sampled in an attempt to get consistent positive Ct values. The time to the first Ct value that indicated the plant was CLas positive was the shortest for the lemons, followed by the navels, and then the mandarins. The mean days to the first positive Ct value for lemons was 121.1 days (‘17.424); for the navels, 160.93 days (‘ 14.36); and for the mandarins, 267 days (‘ 10.99). A second set of experimental plants has been generated and include 25 Washington navels, 42 Lisbon lemons, and 31 Tango mandarins, all on Carrizo rootstock. These plants will be subjected to one of the following treatments: control-control (no manipulation); CLas-negative psyllid feeding; and CLas-positive psyllid feeding. The psyllids will be caged on the plants and allowed to feed for 10 days, after which the psyllids will be removed. All plants will be treated with insecticides to insure removal of the psyllids. The insect inoculation is expected to occur in April 2015. A third set of experimental plants have been started, and there are 236 Carrizo plants waiting to be grafted with the appropriate scion in September 2015.
Dec 2014 The objectives of this project are to optimize Guignardia citricarpa ascospore extraction procedures and qPCR with automated extraction system, determine if prototype passive ascospore traps will capture a sufficient number of Guignardia citricarpa ascospores to be an effective monitoring tool and monitor for G. citricarpa ascospores in six locations around state. We have continued to deploy the slides in the traps but there are very few spores on the Burkhard and new traps to compare counts. Through out the fall there were nearly no spores with less than 10 per slide. There has been little reason to see if qPCR would work because the limit of detection would likely not be met.
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