During the first year we have made progress in developing the non radioactive digexigenin labelled probes for the detaection of Candidatus Liberibacter asiaticus in citrus tissues and psyllid vector. We used both digoxigenin-labeled Plabeled PCR probes as well as RNA probes. we produced digoxigenin-labeled PCR products using specific primers for the OMP, RNA polymerase beta subunit, DNA polymerase region, the r-DNA region, and the 23S and 16S ribosomal RNA intergenic regions. initially dot blot hybridizations were carried out using the known amount of HLB specific DNA from the infected plants. A linear relationship was observed with amount of input DNA and the degree of hybridization. Citrus plants from the green house infected with HLB pathogen were used as the source plants for obtaining the needed tissue for the blots and to isolate nucleic acid template necessary for the amplification of CLas specific DNA. Primers were designed based on the CLas sequence information and amplified specific amplicons from the DNA isolated from HLB infected plants, and amplification was not observed from the healthy plants. In the initial tissue blot experiments we did not observe hybridization signals specific for HLB. We used healthy plants grown under similar conditions as controls. The reason probably is the low titer of pathogen and/or the non uniform distribution of the pathogen in the infected tissues. It is also possible that the PCR probes were not of sufficient specific activity so as to detect low titer of the pathogen. Therefore, the amplified regions of Las were also cloned in the transcription vector, and digoxigenin labeled strand specific RNA probes were generated by transcription. However, use of the high specific activity RNA probes did not improve hybridization of the tissue blot and occasionally non specific hybridization was also observed in healthy tissue blots. This suggested less than optimal hybridization regimen. We have subsequently focused on optimizing the hybridization conditions and use of multiple probes in an effort to increase the extent of signal. These changes have substantially improved detection of Las in citrus tissues. In addition to tissue blots of the stem sections, we have used the midrib and petiole region imprints from the leaves of infected citrus on nylon membranes. The hybridization observed with the midrib imprints showed much clear signals compared to stem imprints. In forthcoming experiments we will use imprints of the leaf midrib, petiole and stem from new flushes of infected citrus since the psyllids preferentially feed and acquire the pathogen from such tissues. We also imprint on the membrane the inside surface of the bark which contains phloem tissue in which the HLB pathogen is located. Although this procedure is not optimized, we believe this to be very useful since the distribution of Las in infected tissue could be easily documented. The second area of our focus is on the detection of Las in psyllid vector by tissue blots (squash blot) on nylon membranes. At the outset, a procedure for isolating the nucleic acid from single psyllid was optimized, and we have been able to amplify CLas specific amplicons from single infected psyllids using pairs of Las specific primers. Conditions of amplifications were optimized with different primer pairs and now we have been able to amplify HLB specific amplicons without non-specific bands in PCR. In initial studies of whole psyllid tissue blots, hybridization signal was also observed with healthy psyllids (psyllid colony from the healthy psyllid containment facility). However use of specific primer pair corresponding to the EFTU gene of CLas has been promising and we will use the probe generated for this gene in tissue blots of psyllids henceforth. The observed background was probably due to the extraneous tissues of the psyllids like wings that attach to the nylon membranes during psyllid squash and very hard to exclude during membrane wash. Therefore we intend to separate the head and the abdomen regions of the psyllids in tissue blots and exclude the wings region to reduce the background signal.
The project has two specific aims. We outlined below the progress made for each of them. Specific Aim 1: Identify sweet orange responses to Huanglongbing disease (HLB) through deep transcriptome profiling using new DNA sequencing technologies. We have performed the bioinformatic analysis of the deep sequencing runs (Illumina Genome Analyzer II, read lengths up to 85 bp pair-ended) for the four types of fruit samples of the experimental design: symptomatic, asymptomatic and apparently healthy from an infected orchard and healthy plants from a disease-free location. We have also completed the deep sequencing runs for the same samples of mature and young leaves: 32-36 million reads were obtained for the immature leaves (95-97% high quality reads after quality trimming) and 32-34 million reads (96-97% high quality reads) obtained for the mature leaves. The bioinformatic analysis of these data is ongoing. In regards with the immature fruit set, the RNA have been extracted and cDNA libraries have been constructed. The runs for these samples are scheduled to take place in the next weeks. For the validation of the mature fruit transcriptome analysis, Taqman Real Time assays have been performed for genes involved in important differentially regulated pathways such as hormone signaling and biosynthesis (gibberellin, ethylene, cytokinin), photosynthesis, transcription factors, terpenoid metabolism. Other assays have been designed to validate the transcriptome results of the mature fruits and related to energy metabolism, hormone, sugar and starch metabolism. In anticipation of the leaf analysis we have initiated the designed of 13 Taqman Real Time assays based on previously published leaf responses to HLB obtained with microarrays. Other Taqman assays will be designed once the leaf samples will be analyzed to validate the transcriptome profile obtained by the deep sequencing. Specific Aim 2: Define and validate gene networks and identify host (sweet orange) response biomarkers regulated by HLB at different stages of infection. We have validated more than 10 Taqman Real Time assays for early HLB-regulated genes developed as biomarkers to distinguish infections at asymptomatic stage in the fruit. These genes belong to sucrose metabolism, photosynthesis, cell wall, lipid, volatile pathways, gibberellin signaling, ethylene response, transcription factors. These biomarkers provided a valuable insight into fruit peel responses to HLB. A significant number of regulated genes in the symptomatic fruits were involved in transport functions. An upregulation of light reactions was observed with the corresponding increased regulation of sucrose and starch metabolism. Genes encoding heat shock proteins appear to be major hubs in a protein-protein network analysis we conducted. The downregulation of these genes would stimulate protein misfolding processes and disrupt source-sink communications. Pathways encoding plant growth regulators like gibberellin and cytokinin were downregulated, whereas ethylene, jasmonate and salicylic acid metabolism and signaling were induced and these could affect fruit metabolism. WRKY, AP2-EREBP and bZIP transcription factors were highly regulated at different stages of disease. Several receptor kinase genes (LRR protein) seemed to be involved in reception of the signal triggering the host response. While cell cycle and organization were downregulated, protein modifications and RNA processing were induced in symptomatic fruits. Interestingly, genes involved in ATP synthesis were upregulated in mature fruits and agreed with previous findings of the presence of an ATP translocase gene in the pathogen genome (Duan et al., 2009). Taqman assays have been designed and analysis are on going to confirm these preliminary results.
This report covers the period January 1, 2010 through March 31, 2010. This project was funded July 1, 2009. Seven coordinating meeting were held during this period. The field personnel, in particular, closely coordinated their activities with the personnel analyzing data. Coordination with the aerial application program through Southeastern Air and the Indian River Citrus league continued as did the mapping of data for subsequent time and space analyses of Asian citrus psyllid populations. Due to the massive amount of data being collected in the field, four Garmin GPS units and five laptop computers were purchased to enable higher staff productivity and more efficient data handling. The extensive trapping program carried out the following activities. 1. Total trees surveyed: Indian River County–2,460; St Lucie County–2, 700; Martin County–900. 2. Traps by county: Indian River 205; St. Lucie, 225; Martin 72. 3. Total traps set and Retrieved: 6,060. 4. Total Psyllids caught: 3,013. By county: Indian River–1,690; St. Lucie 729; Martin 594. To give some perspective on the size of this program 17,000 miles were driven by field personnel during this period. The data analyses from the first aerial application were inconclusive. It was the judgement of the participants that the data from the second application will allow a conclusion as to the efficacy of the aerial program. The data from the second aerial application are now being analyzed. These analyses will be available for the next quarterly report.
Salicylic acid (SA) is the key defense signaling molecule in plants. Increases in SA levels and/signaling lead to enhanced disease resistance against viral, bacterial, and fungal pathogens in many plants. In order to introduce broad resistance in citrus against HLB, citrus canker, and other diseases, we proposed in this study to identify SA-related genes in citrus and manipulate the expression of these genes genetically to elevate SA levels and/or signaling in citrus. Towards this goal, we have three specific objectives in the proposal and have made the following progress with the first-year funding. We believe that this progress has met the expectations in our proposal. Objective 1: Identify genes positively regulating SA-mediated defense in citrus Bioinformatics analysis of the HarvEST:Citrus database and citrus genome database available in the Joint Genome Institute revealed that citrus and the model plant Arabidopsis share significant similarity in their genomic sequences. Thus, most SA regulators that have been characterized in Arabidopsis also exist in citrus. The availability of EST and genome sequences of citrus genes makes it easy for us to clone citrus SA homologs. So far using the RT-PCR approach, we have obtained full-length cDNA sequences for four citrus SA genes, ctNPR1, ctEDS5, ctNDR1 and ctPAD4. We are also in the middle of cloning another four citrus SA genes, ctEDS1, ctSID2, ctALD1, and ctWIN3. Homologs of these SA genes are known to play critical roles in Arabidopsis defense. To provide a more detailed analysis of expression of citrus SA regulators, we collected citrus tissues infected with Ca. L. asiaticus at several time points for RNA extraction. Quantitative RT-PCR will be performed to study expression of these citrus SA genes and other important defense genes in citrus afflicted with the HLB disease. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues We cloned ctNPR1, ctEDS5, ctNDR1 and ctPAD4 to the binary vector pBINplus/ARS and transformed corresponding Arabidopsis mutants and wild type with each of these constructs. We are in the process of selecting T1 Arabidopsis transformants expressing citrus SA homologs and testing some T2 transgenic plants for disease resistance. So far, preliminary data obtained from Arabidopsis expressing ctNDR1 are promising. The Arabidopsis ndr1-1 mutant was previously shown to lack a hypersensitive response and have enhanced disease susceptibility with infection of the Pseudomonas syringae avrRpt2 strain. Overexpressing ctNDR1 in the ndr1-1 mutant rescued these phenotypes conferred by ndr1-1, suggesting that Arabidopsis produces a functional ctNDR1. Summary for other constructs: Plants expressing CtNPR1/pBINplusARS did not show a drastic change of disease resistance with P. syringae infection. We will isolate homozygous lines and plant them side by side with WT and the npr1-1 mutant for a resistance test in the next generation. For plants expressing ctPAD4/pBINplusARS, we obtained T2 seeds and will infect the T2 plants soon with P. syringae. For the ctEDS5/pBINplusARS construct, we were unable to obtain any transgenic lines. This is possibly due to the toxicity of overexpressing ctEDS5 and/or the failure of transformation. We will repeat the transformation process to rule out the latter possibility. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in Citrus Plants of the citrus rootstock US-812 have been transformed with ctNPR1/pBINplusARS and are being grown in the greenhouse in Ft. Pierce in preparation for testing with HLB and citrus canker. The construct ctNDR1/pBINplusARS is in the pipeline for citrus transformation in the next two months, and transgenic citrus plants generated will also be tested with the HLB disease later in the year.
The new microscopes with their superior optics and ergonomic designs have increased productivity. The Leica teaching microscope is used more frequently than expected as each variety that is cleaned up looks a little different. Teaching is a long term and on-going process. By frequent examination of each developing STG, the reasons for success or lack thereof can be determined. This is knowledge is applied to improve subsequent STGing. The new spectrophotometer, a Nanodrop 8000, is used to quantify nucleic acid (NA) content of all samples. An excel spreadsheet with formulas is used to determine what quantity of water needs to be added, if any, to standardize the NA content. Each sample then has the requisite amount of water added to dilute it to the pre-determined concentration in the master plate. Plates for all assays are drawn from this master plate so that the precise amount of NA determined to be optimal for that set of primers is used. Chiefland Foundation source trees were tested for Citrus tatter leaf virus (CTLV) for the first time during the 2009 testing season. Samples were adjusted to the proper titer using the Nanodrop 8000. A SYBR green real-time PCR assay showed that three trees of one little-used variety had CTLV. Several nearby trees had been infected by root grafting. Implementation of this important testing allowed us to immediately remove the diseased trees for STGing and has increased the disease-free state of this valuable resource. During the 2010 testing season we will determine if all CTLV has been removed from the Chiefland Foundation trees. The list of people and the varieties they want to enter into the budwood parent tree indexing and shoot-tip grafting program continues to be long as does the list of varieties that we are working to clean up for the Chiefland Foundation, Florida citrus breeders, and citrus producers. However the improvements to the program are allowing us to get shoot-tip grafted and tested material back to the owner in less than two years in most cases. With improved testing, the Florida citrus industry is provided with budwood tested repeatedly for Citrus greening, Citrus tristeza virus, Citrus leaf blotch virus, Citrus tatter leaf virus (Apple-stem grooving virus), and six viroids. Our Budwood program remains strong and well provided with up-to-date equipment to provide these services thanks to the support of the Florida Citrus Production Research Advisory Council.
Between Oct. 5th and 30th, five experiments were conducted that included Fallglo (1 time), sunburst (2 times), red grapefruit (4 times), and navel oranges (1 time). Fruit were treated on a commercial packingline (3 experiments) or on the Indian River Research and Education Center research line (2 experiments). Treatments included 1) full wash (brush bed + high-pressure wash) + waxing (carnauba), 2) full wash, 3) brush bed only, 4) brush bed with brushes rotating half normal speed, 5) high-pressure wash (HPW) only, 6) HPW for 10 seconds, 7) HPW for 5 seconds, 8) running fruit only over PVC rollers, and 9) a control (not washed or waxed). On the commercial line, fruit remained on the brush bed for ~ 1 min. 10 seconds, and on the full HPW for ~35 seconds. Normal brush rotation speed was ~100 rpm. Fruit were also evaluated for how surface dirt obscured the ability to grade the fruit for canker and other grade defects. Fruit from all treatments were degreened under simulated commercial conditions (5 ppm ethylene, 85F, 95% RH) and color development and weight loss measured almost daily. Fruit were subsequently stored and evaluated for the development of decay and disorders during storage under ambient conditions (~70-75F). In general, all very early season Fallglo fruit were relatively clean and did not need washing for adequate grading. This changed somewhat by the end of October when grapefruit that received more extensive washing (i.e., full washing) was significantly easier to grade compared to unwashed fruit. HPW producing fruit with intermediate gradeability. However, even minimally washed fruit were sufficient for adequate grading. These experiments need to be repeated next season to determine variability in initial fruit cleanliness from year to year. Washing and waxing the fruit gave the greatest inhibition of degreening, almost stopping color development completely. Compared to preliminary results in 2008, results again showed that full washing of fruit on both the brush washer and HPW, or washing on the brush bed along inhibited degreening significantly more than did washing fruit only as they passed over the HPW. Fruit that were not brushed at all, but only passed over rollers experience a slight, but significant delay in color development compared to the control, but the delay was relatively minor compared to the other washing treatments. As the season progresses, fruit exterior surfaces become more soiled with dirt and sooty mold that makes grading more difficult without washing. Experiments are currently underway using late-season red grapefruit, harvested from a block with citrus canker. Unwashed fruit were run past commercial graders and the number of fruit with any canker or surface defects that would justify elimination based on export grade standards were counted. These fruit were then taken to a commercial packinghouse and either left un-washed (control), or washed over 1) the entire line (brush + HPW), 2) the brush line only, or 3) HPW only. Fruit were then evaluated by commercial graders again. Data is still being collected and analyzed from these experiments.
Research results from the first (2007-08) season were successfully completed, the results reported at the Florida State Horticultural Society (FSHS) meetings and at Packinghouse Day and the Indian River Postharvest Workshop in Sept. 2008, a manuscript published in the FSHS proceedings (http://www.fshs.org/; Proc. Fla. State Hort. Soc. 2008. 121:322’325), and subsequent results reported in FCPRAC progress reports. To summarize all experiments and results, preharvest treatments included 1) foliar K (8lb K2O), 2) foliar Mg (6% solution), 3) foliar K + Mg, 4) foliar Vapor Gard’ (1% or 2%), and 5) induced tree water stress by discontinuing irrigation and covering the ground with large plastic sheets. Treatments were conducted in commercial groves on White grapefruit three times, Flame red grapefruit twice, and Valencia oranges once. Fruit were harvested at various times after foliar applications (sometimes weekly), held for 3 to 4 days at ~70F with ~60% RH, washed & sometimes waxed (carnauba), and stored under ambient conditions. Fruit were evaluated at regular intervals for peel breakdown and decay. While foliar treatments did not always significantly reduce peel breakdown, even when the differences were not significant, such applications tended to reduce peel breakdown. In general, foliar K or Mg treatments significantly reduced peel breakdown once each. In one experiment, foliar K actually increased peel breakdown, but the reason for this is unclear. However, a combination of foliar K + Mg or use of an antitranspirant (Vapor Gard’) significantly reduced postharvest peel breakdown more than the control or K or Mg alone. Blocking irrigation and rainfall for 49 days before harvest increased peel breakdown in the first year. In the second year, while differences in peel breakdown were not significantly different, trees under water stress tended to develop twice as much peel breakdown compared to the control. In postharvest experiments, fruit were harvested, held for 3 to 4 days, washed and waxed (carnauba) on a research packingline at the IRREC, and then stored at ambient conditions. Postharvest treatments including 1) holding the fruit for 3 or 4 days after harvest at 30%, 60%, or 100% RH at ~70F, 2) including a fungicide (TBZ or Imazalil) in the wax, 3) not waxing the fruit, or 4) washing, but not rinsing the detergent from the fruit before storage. Exposing fruit to low (30%) RH after harvest dramatically increased peel breakdown and often significantly increased postharvest decay as well. Exposure to intermediate RH occasionally increased peel breakdown compared to the control (100% RH), but results were not consistent and sometimes were not significantly different from the control. Not waxing fruit or rinsing detergent from the fruit usually had no effect on peel breakdown. Interestingly, postharvest treatments with Imazalil occasionally reduced peel breakdown. The reason for this is unclear but worth further investigation.
Funding for this project has enabled us to develop a comprehensive program for ACP management. We are building programs targeting ACP adults using dormant sprays of broad-spectrum insecticides and treatments during the growing season based on scouting. Dormant sprays have evolved into a successful annual area-wide cooperative effort in SW Florida. For the growing season, we have developed a wide range of choices such as (1) recurrent low-volume applications of horticultural oil, (2) wide selection of tested selective and broad spectrum insecticides, and (3) strategies to conserve and augment biological control. A survey of local managers covering 106,148 acres of commercial citrus in SW Florida showed widespread adoption of these management strategies. Our first objective was to assist growers to plan and implement effective strategies for ACP control. All growers surveyed used dormant sprays during the 2008-2009 dormant season in cooperation with a voluntary area-wide program initiated and promoted in cooperation with Gulf Citrus Growers Association and Cooperative Extension. 71,916 acres were sprayed by air in 08/09 and 73,180 in the 09/10 seasons with an additional estimated 30,000 sprayed by ground, virtually all the present commercial acreage in SW Florida. Field counts obtained from DPI-CHRP and growers’ personnel trained by us indicated that populations by air were only 1.5X winter numbers three months into the growing season compared to a 28X increase in untreated blocks. Results of the post application survey will be evaluated this May. Our 2nd objective was to assist in development of efficient monitoring methods. The “tap” sampling method we developed has proved to be an effective and efficient tool for monitoring ACP adults, as well as other pests and beneficial insects. We integrated this and a technique for assessing immature stages into a rapid and easy to use system promoted in Citrus Industry Magazine, fully explained on our website http://swfrec.ifas.ufl.edu/ and used by 95% of surveyed growers. Our 3rd objective was to accelerate testing of new chemical tools for psyllid suppression. We completed four field evaluations of insecticides for ACP control in 2009 and submitted reports for publication in the ESA journal Arthropod Management Tests. These join 17 previously published reports on insecticidal control of ACP available online. We found that foliar applications of spirotetramat, fenproximate + 435 Oil, spirotetramat, or 435 Oil alone during bloom suppressed ACP populations for two months. Our 4th objective was to evaluate the economic feasibility of psyllid management strategies. Two aerial dormant sprays could cost less than $25/ac. The cost of monitoring psyllids using the tap method is about $25 per block up to 100 acres so could be done 20 times per year for as little as $5/ac, and would avoid unnecessary sprays during the growing season that could cost up to $50/ac. Unnecessary applications also cause secondary pest outbreaks of mites, scales and leafminers, reported by 70% of surveyed growers. Our 5th objective was to provide an information bridge between growers and researchers. Experimental results and recommended management programs have been presented at 18 extension meetings across Florida including two production managers meetings and two CCA trainings, as well as the Entomological Society of America, the Florida State Horticultural Society, the Florida Entomological Society, Citrus HLB & Potato Zebra Chip (ZC) in Mc Allen, TX, and the XIII Simposio Internacional de Citricultura. CD Victoria, Mexico. 91% percent of surveyed growers were satisfied at some level by their present management programs and 85% of respondents were monitoring ACP more than 3 times per year. We are working on emphasizing the use of this information to make informed decisions to use insecticides only when needed. (1)Stansly, PA, JA Qureshi, and HA Arevalo. 2009. Why, when and how to monitor and manage Asian citrus psyllid. Citrus Industry 90(3):24-26. (2) Stansly, PA, HA Arevalo, M Zekri, and R Hamel. 2009. Cooperative dormant spray program against Asian citrus psyllid in SW Florida. Citrus Industry 90(10):14-15.
Obj. 1.DNA bar coding in progress using mtCOI primers to amplify a 780 bp fragment from psyllid field and colony collections from various U.S. states and off-shore locations. The purpose of this objective is to examine diversity within and between varied populations in relation to the associated Liberibacter spp. based on 16S rDNA. Over 50 collections are in hand. Obj. 2. qPCR has been applied to detect Ca. Liberibacter presence (or absence) in the psyllid colony cohorts used for light and TEM studies, and by FISH (over different AAPs), for immatures and adults (Roberts, SWFREC, HLB diagnostics lab). The FL psyllid rearing system is fully functional and Ca. Liberibacter-infected and bacterium-free colonies are being maintained routinely. About 200 field-collected adult psyllids testing 10% PCR positive were placed into 2 more cages each with 6 ungrafted plants and 100 psyllids. Grafted plants were held in an air conditioned greenhouse at (28 ‘C 64% RH) and psyllid inoculated plants were held in an air conditioned insectary under similar conditions and 14 h light. All plants tested negative for HLB before they were infected with either method. After about one to one and a half month 85-90% grafted plants tested HLB positive based on PCR conducted on dark green mature leaves, whereas, plants exposed to infected psyllids still tested negative. When grafted plants were pruned to induce new shoots, 31-35% tested HLB positive by PCR. Two weeks later when PCR was conducted on leaves from young shoots 90-100% were positive. Four weeks later 97% tested positive by (dark green mature leaves). Two months after psyllid exposure to infected (PCR +) plants, 83% of the psyllid infested plants tested HLB positive by PCR of the dark green mature leaves. Adult psyllids collected on 8/12/2009 and tested at the SWFREC HLB lab were 61% and 67% HLB positive from budded and psyllid infected plants, respectively. Adult psyllids collected on 8/25/2009 and PCR tested at the USDA-ARS Riverside lab were 36% and 13% HLB positive from budded and psyllid infected plants, respectively. Obj. 3. Because it can be reared year-round and because of quarantine constraints we have adopted Bactericera cockerelli as a surrogate species for Diaphorina citri. All three sections, V1,V2 & V3, of the B. cockerelli ventriculus support bacterial growth and replication that advances into huge colonies in the epithelia and on the outside surfaces as well. The esophagus and external hindgut harbor small numbers on their outside surfaces. Thus focus on transmission is now shifted to the oral region. The salivary glands apparently do not support massive, organ-destroying cultures, however, high titers of bacteria occur in the hemolymph surrounding them. Their role in the terminal stages of transmission is still under study. FISH technique, involving coagulant fixation, adopted from prior works by various authors, have proven unsatisfactory for this species. New techniques are under development, including the rendering of individual organs on slides, and the de-shelling of aldehyde fixed whole bodies for acceptable in-situ localization. Obj. 4. We are investigating whether bacteria gain lateral access to the salivary canal without invading the glands. The most likely site would be near the precibarium, where the maxillary stylets converge (Ullman and McLean 1989) and glandular secretions are present. Studies are underway to determine the configuration of the oral area to aid in interpretations of liberibacter-anatomical TEM visualization.
Our initial efforts in using canines to detect canker started in 2000 but were thwarted by the September 11 attack on the US, which permanently diverted the canine we had been training (it was included in the much needed efforts to detect explosives). However, prior to this dog’s reassignment, our initial research with canines for canker detection demonstrated their ability to detect citrus and citrus canker volatiles/aromas/essences, i.e., minute concentrations of volatile/aromatic compounds, deposited on cloth and other materials as well as directly from the air. A second attempt with USDA, APHIS agriculture detector dogs was cut short by decreasing budgets combined by increasing needs of detection of contraband agricultural products at points of entry into the US. However, our third and ongoing attempt has been much more successful. This most recent canine, ‘Juice’, has demonstrated an ability to detect canker infected citrus fruit with high reliability. The initial success of this project with regards to canker detection, suggests that the same technique may be useful for detection of HLB, perhaps presymptomatic while the disease is still latent. Such a tool would be very useful and is much needed, as it would allow both detection and early removal of presymptomatic (latently infected) trees, which also may be below the titer required for psyllids transmission. Two dogs have been trained to recognize the scent of citrus canker. Dogs were trained on infected versus non-infected trees and fruit. Training of the canines for the detection of canker infected citrus trees was accomplished via a commercial canine training facility in North Florida. As indicated above, ‘Juice’, demonstrated an ability to detect canker infected citrus fruit with high reliability (> 99.3%). Juice was capable of discriminating canker infected from non-infected fruit in 30 runs of 5 blind stations each, with only one false positive and no false negatives (Peruyero and Gottwald, unpublished). Results indicate that canines can detect and differentiate citrus fruit infected with canker from non-infected tissues. The canine was then trained on infected versus non-infected Duncan grapefruit seedlings. Preliminary tests at the canine training facility are very promising. Field trials were conduced in early April at the USHRL farm. Trees (75) were placed in the field in a 3 row by 25 tree/row design. Ten replications were conducted in which the proportion of disease trees ranged from 2-10%. The conditions were sunny but with a 15-20 MPH breeze. Even so, detection highly reliable. Data are presently under analyses. Short movie footage was also taken to document the dogs search and detection behavior. More field trials are scheduled over the next few months to validate the canine’s performance. At the conclusion of the canker field testing (proof of concept) the dog will the be cross-trained for HLB detection and field tests will be conducted to assess performance and detection ability to HLB. This will go beyond the time frame of this grant, but we will pursue it to conclusion utilizing USDA, ARS funding internal.
Our initial efforts in using canines to detect canker started in 2000 but were thwarted by the September 11 attack on the US, which permanently diverted the canine we had been training (it was included in the much needed efforts to detect explosives). However, prior to this dog’s reassignment, our initial research with canines for canker detection demonstrated their ability to detect citrus and citrus canker volatiles/aromas/essences, i.e., minute concentrations of volatile/aromatic compounds, deposited on cloth and other materials as well as directly from the air. A second attempt with USDA, APHIS agriculture detector dogs was cut short by decreasing budgets combined by increasing needs of detection of contraband agricultural products at points of entry into the US. However, our third and ongoing attempt has been much more successful. This most recent canine, ‘Juice’, has demonstrated an ability to detect canker infected citrus fruit with high reliability. The initial success of this project with regards to canker detection, suggests that the same technique may be useful for detection of HLB, perhaps presymptomatic while the disease is still latent. Such a tool would be very useful and is much needed, as it would allow both detection and early removal of presymptomatic (latently infected) trees, which also may be below the titer required for psyllids transmission. Two dogs have been trained to recognize the scent of citrus canker. Dogs were trained on infected versus non-infected trees and fruit. Training of the canines for the detection of canker infected citrus trees was accomplished via a commercial canine training facility in North Florida. As indicated above, ‘Juice’, demonstrated an ability to detect canker infected citrus fruit with high reliability (> 99.3%). Juice was capable of discriminating canker infected from non-infected fruit in 30 runs of 5 blind stations each, with only one false positive and no false negatives (Peruyero and Gottwald, unpublished). Results indicate that canines can detect and differentiate citrus fruit infected with canker from non-infected tissues. The canine was then trained on infected versus non-infected Duncan grapefruit seedlings. Preliminary tests at the canine training facility are very promising. Field trials were conduced in early April at the USHRL farm. Trees (75) were placed in the field in a 3 row by 25 tree/row design. Ten replications were conducted in which the proportion of disease trees ranged from 2-10%. The conditions were sunny but with a 15-20 MPH breeze. Even so, detection highly reliable. Data are presently under analyses. Short movie footage was also taken to document the dogs search and detection behavior. More field trials are scheduled over the next few months to validate the canine’s performance. At the conclusion of the canker field testing (proof of concept) the dog will the be cross-trained for HLB detection and field tests will be conducted to assess performance and detection ability to HLB. This will go beyond the time frame of this grant, but we will pursue it to conclusion utilizing USDA, ARS funding internal.
A series of flexible stochastic models to predict the temporal increase and spatial spread of diseases. The models were initially characterized for citrus canker spreading in plantation and urban (backyard) environments. They have subsequently been extended to HLB in this project. The models can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies. Most attention has been given to spread within plantations, including allowance for proliferation of infection along boundaries in response to vector behavior. The models can readily be extended to consider spread at larger scales including spread through heterogeneous environments up to State-wide scales. We have also considered the effects of uncertainties in the distribution of host crops for example the effects of small areas of crop that may not be recorded but which can act as ‘bridges’ in transmitting disease. The effects of uncertainty in parameter estimates for dispersal parameters and transmission rates have also been included. Additional computer-friendly formulations of the models have also been developed to aid in education of stake-holders to illustrate the effects of uncertainty in predicting future disease spread and the effectiveness of alternative methods of control. Estimation of parameters for dispersal of HLB poses considerable statistical challenges, especially where trees may become infectious before they are symptomatic/detected. Here we use an SEIDR model (Susceptible, Exposed (latently infected but not yet infectious), Infectious but not yet symptomatic/detected, Detected and infectious and Removed trees). Using MCMC methods, and extensive data from Southern Gardens for successive snapshots of the occurrence of symptomatic/detected trees in known populations of susceptible trees, we are able to estimate model parameters for the transmission rates and dispersal kernel for the disease. Current work is focused on the differential effects of host age on epidemiological parameters as well as variability across the plantation. From these it is possible to allow for uncertainty in the parameters as well as variability over time and through space. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. A front-end, a web based version of the model that users can easily access and use is under final development and will be made available later this year. In this version both residential and commercial citrus scenarios can be tested. A wide variety of epidemiological and climate/weather variables have been included and are user selectable and changeable via sliding controls. The simulator then generates epidemics that progress through time and can be visualized on the screen. In addition various disease control/mitigation parameters can be selected and simulations can been visualized to see the effect of these various control strategies. This user selectable “front end” is highly instructive to growers and regulators. The Front end runs simulations one at a time for users to visualize. It is based on a more formal analytical model that can run thousands of simulations based on the same parameters and make more statistically valid predictions for regulatory intervention strategy building and regulatory/industry decision making.
Disease control of citrus huanglongbing (HLB) by interplanting with guava. HLB is the most devastating disease of citrus worldwide and presently threatens the existence of the citrus industry in Florida. In Vietnam guava has been shown to be an effective deterrent to HLB. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring about one year. Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus an unprotected nursery, have been established. Disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) were located in the protected and unprotected plots in June 2009. The guava were established over a year ago and grown to appropriate size as indicated in Vietnam. Trees are assayed for HLB every 60 days, and are in their second assay. Psyllid populations are also being monitored continuously every two weeks within plots to document any repulsion of the vector due to guava. To date no HLB+ plants have been identified in the nursery plantings after multiple assays. Citrus/guava interplantings: 2 commercial plantings with multiple replications each have been established. This has taken considerable time. Guava trees were propagated and grown to transplant size. These were then out planted and grown for a year per Vietnam protocols. One trial was established in a commercial orchard with collaborators in Southern Gardens Citrus. A second trial planting was established at the USHRL Picos Farm in Fort Pierce. Both nursery and field citrus trees are assayed for HLB every 60 days, and have been assayed multiple times. Psyllid populations are also being monitored continuously every two weeks within interplanted plots to document any repulsion of the vector due to guava. Data collection continues and is currently ongoing. A severe frost last winter affected both the USHRL and the Southern Gardens plots causing a delay in the experiment. Damage was extensive in both plots. In the Southern garden plots, damage was more severe and the guava have now been renovated sufficiently that the Southern Gardens plot and plots have now been interplanted with citrus as of November 2009. The damage to the guava was overcome by pruning and replanting of damaged guava trees. Renovation of the USHRL plot was less extensive, and the guava were interplanted with the citrus as of August 2009 in the USHRL Picos Farm plot. A second series of freezes during the 2009/2010 winter season hit both Southern Gardens and Picos plots and both sets of plots have suffered, but both are recovering with pruning and some replanting of guava. To date, 2 HLB+ plants have been identified in the USHRL plots after multiple assays. However this data is insufficient to draw any conclusions as yet. To date no HLB+ plants have been identified in the SG plantings after multiple assays.
The control of citrus sprouts from stumps or remaining roots after tree removal has been a major concern for years within the Florida citrus industry and now more important since the arrival of huanglongbing (HLB) or citrus greening. Sprouts from stumps of clipped trees that are HLB positive can harbor the greening bacterium. This study is conducted to determine effectiveness of Remedy Ultra on the control citrus stump sprouting. This study is examining the impact of delaying application of Remedy on the control of sprout formation. Observations were made on 30-day intervals over an 8-month period after clipping or shearing the tree as the removal method. Studies are being conducted at Lake Placid and Arcadia. Treatments of Remedy Ultra were applied at 25 or 50% solution mixed with diesel fuel at time of clipping, 24, 48 and 72 hours later. At the Lake Placid site, 2, 2, 1, 1, and 1 of the 7 (100%) stumps sprouting at 30, 60, 90, 120 and 150 days after tree removal, respectively. At this site, 4 stumps within the 56 treated stumps had root sprouts but not on the treated stump. All of the root sprouts occurred on trees originally grown on rough lemon rootstock, whereas none of the other rootstocks sprouted at this site. Remedy Ultra was slightly less effective in controlling sprouts when applied at the 25% Remedy/75% diesel than at the 50% rate especially on rough lemon stumps. Delaying application using the lower rate also had a slight negative impact on sprout control. At the Arcadia site, 5 of the 7 untreated stumps sprouted at 60 days after removal with one additional stump sprouting by 90 days. At this site, 86% of the untreated stumps sprouted within 90 days of clipping. Only one stump exhibited root sprouts which occurred within 60 days after treatment regardless of the time of application of the herbicide. The number of sprouts per stump ranged from 1 to 8. Rootstock and trunk diameter have an impact on root or stump sprouting. Larger trunk diameter supports greater sprouting frequency as compared to smaller trunks. Additionally, rootstocks such as rough lemon and Cleopatra mandarin are more prone to sprouting than other rootstocks like sour orange. Historically, some herbicides used for stump treatment have caused injury to adjacent healthy trees by translocation of materials via root grafting to the healthy trees. Resets planted nearby treated citrus stumps were damaged when excessive materials were applied to the soil surface or moved off treated stumps by rain or irrigation. In these studies, no problems were associated with Remedy Ultra damaging adjacent trees.
All field equipment has been ordered and is being tested to ensure that they work as intended. The equipment to measure droplet size being developed by low volume sprayers is a continuing issue. The system developed by Drop Vision has proven to be unacceptable and has been returned to the company. The method used by Drop Vision measures droplet size on glass slides coated with telfon or MgO. Both coating methods will not work where spray mixture contains a higher volume of water and either evaporates or completely covers the slide. Other testing methods are being considered.
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