This report covers the entire period that Huanglongbing Diagnostic Laboratory has been in service because one of the objectives for the funding was to continue to provide seamless service without interruption to growers. This has been achieved as detailed in the report below. The HLB Diagnostic Laboratory located at the University of Florida Institute of Food and Agricultural Science Southwest Florida Research and Education Center (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 fifteen months, and has processed more than 13,000 samples from growers, approximately 4,000 research samples, and 700 screen house samples from the Budwood screenhouse facilities located on the SWFREC property. Since the HLB organism is no longer of the select agent list and reporting requirements have changed, we now supply reports at the request of the USDA and the State of Florida. We exceeded our anticipated yearly load of 10,000 samples. Samples are logged into the computerized database, processed by extracting a specific weight of tissue sample, homogenized, the DNA extracted, PCR amplified, and results are sent to the submitter. The lab has received samples from growers throughout Florida, with the highest number of samples received from Collier, Highlands, and Hendry Counties. There is a slight seasonality to the sample submission volume with respect to harvesting and new growth (flushing) events. To date February, January and March of 2009 has been the months that growers have submitted the most samples. From the currently accumulated data, the HLB lab has a 69% positive sample submission rate, and a 31 % negative submission. Temporally the highest amount of negative sample submissions were in February 2009 at approximately 80%, and the highest amount of positive sample submissions were in March 2009 at approximately 35% (Figure 3). These disease detection rates are not directly indicative of the actual overall field disease levels for HLB since scouting and field sampling are usually selective. Techniques, Protocols and Research We are continuing to update the efficiency of the laboratory by experimenting with a quick efficient extraction kit. We have implemented this kit into everyday operation of the lab and have provided us with exceptionally fast and efficient turnaround time for growers as well as research samples. Also we have developed and are in the beginning stages of implementing a protocol for the detection of HLB in Asian Citrus Psyllid (ACP) with the primary goal of serving research projects within the entomology and plant pathology departments. The HLB Laboratory has one full-time manager and three part-time employees. The current turn around time for processing samples during the off-season is less than two weeks and, at the time of highest sample submission, the turn around time was longer. To improve sensitivity to detect HLB, we are investigating other probe/primer combinations in different areas of Candidatus Liberibacter asiaticus gene and have the ability to detect other greening organisms such as Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. We are also attempting to culture the organism Candidatus Liberibacter either in co-culture or isolate it in pure culture which will then be used to standardize and quantify the detection reaction.
This report covers the entire period that Huanglongbing Diagnostic Laboratory has been in service because one of the objectives for the funding was to continue to provide seamless service without interruption to growers. This has been achieved as detailed in the report below. The HLB Diagnostic Laboratory located at the University of Florida Institute of Food and Agricultural Science Southwest Florida Research and Education Center (UF-IFAS-SWFREC), was establish with funds appropriated by the Florida legislature to serve grower and researcher HLB diagnostic needs in citrus. The lab has been in operation since February 2008, but received samples well before the official opening date. Since the opening of the lab, there has been continued development of techniques, protocols and efficiency. Samples The lab has been in operation for fifteen months, and has received to date more than 13,200 grower samples, approximately 5260 research samples, and 1400 screen house samples from the Budwood facility located on the SWFREC property. The lab has received samples from growers throughout Florida, with the highest number of samples received from growers in Collier, Highlands, and Hendry Counties. There is a slight seasonality to the sample submission volume with respect to harvesting and new growth (flushing) events. Techniques, Protocols and Research We have implemented a new DNA isolation kit into routine operation of the lab and it has provided us with exceptionally fast and efficient turnaround time for growers as well as research samples. This new kit employs magnetic bead technology to effectively and efficiently isolate whole genomic DNA from plant tissue including DNA of the HLB pathogen. We have developed and are in the beginning stages of implementing a protocol for the detection of HLB in Asian Citrus Psyllid (ACP) with the primary goal of serving research projects within the Entomology and Plant Pathology departments with compensation through their programs. To improve sensitivity to detect HLB, we are investigating other probe/primer combinations in different areas of Candidatus Liberibacter asiaticus gene and have the ability to detect other greening organisms such as Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. We are also attempting to culture the organism Candidatus Liberibacter asiaticus either in co-culture or isolate it in pure culture. When successful, the pure culture will be used to calibrate the sensitivity of our lab protocols to determine the minimum number of cells of Liberibacter needed for detection.
The Citrus Greening Bibliographical Database [ http://swfrec.ifas.ufl.edu/hlb/database/ ] went live in January 2009 with the cooperation of the Center for Library Automation at the University of Florida, although funds were not released until 27 Feb 2009. The database has grown from nothing to 1,413 references with over 40% of them linked to the original sources, all of which have been cross-referenced for accuracy. Ninety five percent of the entries are in English, the remaining 5% are in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, and Hebrew. The database includes references from refereed and non-refereed publications, presentations, websites, proceedings, grant reports, and book chapters. All references are related to Huanglongbing (HLB), the associated bacteria (Candidatus Liberibacter spp.), their vectors [Diaphorina citri Kuwayama, Trioza erytrea (Del Guercio)], and the effects of the disease on the tree health. Access to the database and the document links are available to all users at no-cost, and users are invited to provide additional documents. Between the months of March 2009 and May 2009 the database received an average of 789 unique visitors per month, each with an average log-on frequency of 4.4 times per month. These numbers indicate that the database has become an important source of information for a growing number of researchers, growers and other interested clientele. We are continuing to add unique references from researchers all over the world and to link more documents to the current entries. We are planning to maintain this important source of information for years to come, adding new references, keeping it updated for the benefit of the citrus community.
The Citrus Greening Bibliographical Database [ http://swfrec.ifas.ufl.edu/hlb/database/ ] went live in January 2009 with the cooperation of the Center for Library Automation at the University of Florida, although funds were not released until 27 Feb 2009. The database has grown from nothing to 1,413 references with over 40% of them linked to the original sources, all of which have been cross-referenced for accuracy. Ninety five percent of the entries are in English, the remaining 5% are in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, and Hebrew. The database includes references from refereed and non-refereed publications, presentations, websites, proceedings, grant reports, and book chapters. All references are related to Huanglongbing (HLB), the associated bacteria (Candidatus Liberibacter spp.), their vectors [Diaphorina citri Kuwayama, Trioza erytrea (Del Guercio)], and the effects of the disease on the tree health. Access to the database and the document links are available to all users at no-cost, and users are invited to provide additional documents. Between the months of March 2009 and May 2009 the database received an average of 789 unique visitors per month, each with an average log-on frequency of 4.4 times per month. These numbers indicate that the database has become an important source of information for a growing number of researchers, growers and other interested clientele. We are continuing to add unique references from researchers all over the world and to link more documents to the current entries. We are planning to maintain this important source of information for years to come, adding new references, keeping it updated for the benefit of the citrus community.
Last year under another project, we saw reduced infestation of young flush in a replicated trial conducted in a large commercial block of ‘Valencia’ orange in Collier Co. treated every two weeks with 1 gal/acre straight 435 spray oil. This year funds were released on 27 Apr although we reinitiated this experiment with LV applications of oil on 14 Apr for our first objective. The experiment is being conducted in the same block, as last year although we have increased the rate to 2 gal/ac pure FL-435 oil and comparing two LV applicators: a modified London Fogger model 18-20 provided by Chemical Containers and the Proptec P400D. Applications will continue through fall flush at 2-3 week intervals depending on pest pressure. Psyllid populations were below detectable levels until 15 June when preliminary data indicated we may get similar positive results as last year. We have also commenced a separate study on a highly infested, 38-acre block of ‘pineapple’ orange in Glades Co. to compare the London Fogger ‘ applied only to the bed tops with a standard airblast sprayer treating tops and swells. Applications with both sprayers of Delegate (spinetoram) @ 4 oz/ac and Dimethoate (24 oz/ac), both in 2 gal of water were made on 15 June. Preliminary data (2 weeks) indicate that both insecticides worked well with the speed sprayer but neither provided control with the low volume application. Also last year under another project, we saw that ground application of Delegate and Provado (imidacloprid) and Imidan (phosmet) were all effective against the psyllid, but only Imidan of these three worked well by air. This year we are comparing the efficacy of aerial and ground applications of two selective insecticides . Delegate, and Movento (spirotetramat), again against the broad-spectrum Imidan, all with oil. Preliminary results indicate significant differences among insecticides but not between application methods, although the trends favor the ground application for both Imidan and Delegate. Results from these trials have been presented at the Florida State Horticultural Society 2009 annual meeting and will be published in their proceedings.
Objective 1: We conducted tests with nutrient analyses of leaf samples collected from HLB-infected and healthy trees to establish relationships which could be used for (early) diagnosis of HLB. A protocol was established to correct the distortion of leaf nutrient data caused by fluctuations in leaf dry weight (DW). Starch accumulation in symptomatic leaves can significantly alter the interpretation of nutrient status when diagnosed on a DW concentration basis. Conversion of leaf nutrient data to a leaf area basis is one accepted approach which we are using for correcting the influence of DW fluctuation. The Diagnosis and Recommendation Integrated System (DRIS) is another approach to leaf nutrient diagnosis which eliminates any erroneous background noise due to changes in DW. In DRIS, the DW values simply cancel out in a series of nutrient concentration ratios. Thus nutrient interpretation with DRIS considerably reduces the bias from undesirable nutrient concentration or dilution effects due to uncontrollable changes in leaf tissue DW. Furthermore, versions of the DRIS computations are available which can estimate an index of the leaf DW (DWI). In our studies with HLB-infected blotchy mottled leaves where starch accumulation caused DW to increase, the DWI calculated by DRIS was correlated with measured DW. Analytical labs do not routinely report DW for leaf samples, but by using DRIS, we are able to calculate DWI and obtain an estimate of undesirable DW changes which we need to be aware of. Furthermore, DWI may be valuable for diagnosing early onset of HLB in asymptomatic leaves even before PCR methods can detect the Candidatus Liberibacter spp. DNA. Objective 2: An HLB infected commercial citrus grove near Haines City, Florida has been located for field trials to determine the effects of remedial applications of plant nutrients on HLB. The experimental design and treatments are currently being developed in cooperation with the grower and will be reported on in future reports. Objective 3: Seeds of ‘Valencia’ and ‘Hamlin’ sweet orange, and ‘Duncan’ grapefruit have been germinated to produce trees for use in a controlled greenhouse hydroponics study of the effect of specific plant nutrients on HLB expression. The seedlings are currently being grown in sand culture with a complete nutrient solution applied every two days. The seedlings will soon be transferred to larger containers, but maintained in sand culture, to grow up to a sufficient size for budding, at which time a sufficient number of trees will be bud inoculated with HLB. After successful bud inoculation, infected and healthy trees will be transferred to hydroponics culture where specific nutrients (identified through objective 1) will be deleted or supplemented in the nutrient solution to determine their effects on HLB expression and plant growth.
Reducing the amount of vegetative growth produced annually by citrus trees in Florida would reduce the opportunities for Asian citrus psyllid reproduction and thereby the spread of Hunaglongbing. This could be done in citrus without detrimental effects on yield because citrus trees in Florida produce an excess of leaves above that required to support maximum fruit yield. Excess tree growth is routinely controlled through hedging, but little research has been done to examine the effects of hedging on vegetative growth. Recent research in Florida has shown that branch re-growth can be reduced when hedging is performed in fall under Florida conditions because of the onset of cool temperatures. Additionally, research in Florida indicates that late-summer hedging may be able to synchronize a final late-season flush and thus, reduce new flush leaves present during the winter to support over-wintering psyllids. In other fruit crops, such as apple, where excessive vegetative growth can be problematic plant growth regulators (PGRs) are routinely used. However, the use of PGRs in citrus has been limited to influencing fruit development and for maintaining post-harvest quality. PGRs not only control vegetative growth, but also offer the potential to reduce insect pest populations either by reducing pest-required vegetative growth or by altering host plant metabolites or nutrition. In addition, some PGRs have been shown to enhance pesticide efficacy. The PGR prohexadione calcium (Apogee, BASF Corp.) has also been shown to be effective at reducing vegetative growth of potted citrus seedlings under greenhouse conditions. Apogee was capable of reducing extension growth of shoots by more than 50%. Psyllid ovipostioning was also reduced by approximately 50% on Apogee treated seedlings compared to untreated control plants, but variation was large and obscured statistical differences. A 16-acre research block, managed according to standard commercial practices, has been designated at the CREC for further field evaluations of various PGRs and hedging treatments. In addition, plants have been obtained from commercial citrus nurseries to begin greenhouse screening trials to determine efficacy of a variety of different PGRs. The first data from these two trials will be reported in forthcoming reports.
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. Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size. Two nursery sites, a guava protected citrus nursery versus and 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. Trees will be assayed for HLB every 60 days. Psyllid populations will also be monitored. Guava plantings have also been established at three field locations in Florida. Two of these are commercial plantings with collaborators in Southern Gardens Citrus and Consolidated Citrus totaling over 120 acres. Citrus will be interplanted with the guava later this Summer and Fall of 2009 in the commercial plantings. A third planting was established at the USDA, ARS in Fort Pierce, Florida and will be interplanted with citrus in late July early August 2009. The sampling of the plots and monitoring of the guava-effect research will commence immediately following interplanting with citrus and will be conducted every 3 months. Psyllid vectors will also be monitored within plots to document any repulsion of the vector due to guava. Data collection is presently ongoing.
Methods and models for the control of HLB disease of citrus. Citrus huanglongbing (HLB) is the most serious disease of citrus worldwide and presently for the very existence of citrus industry of Florida. The approach is two-fold: First was the examination of the effect of various control strategies on HLB, in control plots, established in 2007. I this phase of the study, five treatments were examined: Minimal control, Insecticide vector control, Roguing, Roguing via PCR+, and Comprehensive. Results indicated that although treatments were significantly different, there was no benefit of any control treatment over another. Small differences were due to plot location, not treatment effects. The test will be repeated, however, the results point to a need for regional control strategy and that small plantings that canÕt control neighbors cannot control the disease. New plots have been established at the USDA, ARS , in Fort Pierce, Florida. Our estimates indicated that for each tree with visual symptoms, there were an average of 13 (range 2-52) that were infected but asymptomatic, i.e., infections that have occurred over the duration of the epidemic but that have not yet expressed symptoms. The second approach is to develop epidemiological models of HLB disease dynamics which improve the understanding of vector-driven disease transmission and analyze disease control policies aimed at disrupting vector population dynamics. In previous and current work we have developed a model for citrus canker. This is the basis upon which we have built a preliminary model for HLB. The HLB simulation model is stochastic based on biological, epidemiological, and meteorological parameters using Markov Chain Monte-Carlo simulation methods and SIR modeling protocols. The model is fit to the HLB data collected in various observed epidemics from Florida and SE Asia by thousands of simulations. Linked-differential equations are used that describe the temporal increase in HLB infected trees and explicitly characterize the population dynamics of the vector. The effectiveness of different disease control measures such as intercropping with guava, roguing and insecticide use will be analyzed via this model and a suite of mathematical tools to identify the most effective strategies. HLB data sets will be correlated with various disease mitigation strategies/events from our epidemiology trials in Florida. The stochastic models allow testing of multiple disease management strategies in thousands of simulated epidemics to determine which will have the optimum effect and in what combination these methods can best be deployed for maximal disease control. The HLB model will continue to be augmented and improved over the next 2 years.
The objectives of this work are to 1) investigate the susceptibility of various rutaceous plant species to Candidatus Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca L. africanus (Laf) and the transmission from these hosts to citrus and 2) using a limited number of these hosts determine if passage through alternative hosts affects the biology (pathogenicity) of the pathogen. This work also will determine if certain alternative hosts are better hosts for the suspected HLB bacterial pathogen and can serve as a reservoir for infection to citrus. During this quarter work was performed at all four investigator locations. At the University of Florida, CREC, alternative plant materials were obtained and PCR tested for Las infection. HLB infected citrus materials to be used for inoculum were propagated in the greenhouse and verified as PCR positive for Las . The materials were used to graft inoculate Severinia buxifolia, Zanthoxylum clava-herculis, Citrus jambhiri and Murraya paniculata. The Murraya (Bergera) koenigii materials were not large or woody enough to graft and were therefore inoculated using infected psyllids. Successful inoculations were done with S. buxifolia, and C. jambhiria. Grafts were not successful with Z. clava-hericulis and are being repeated and there is no data to date on psyllid transmission to B. koenigii. Citrus jambhiri produced symptoms different than those seen in other citrus species. Psyllid transmissions from S. buxifolia to sweet orange and results with the receptor plants is pending. Z. clava-hericulis is not listed as a host of the Asian citrus psyllid, however we have found that the psyllid does feed on this host plant. A graduate student has joined Dr. Brlansky’s research program and will focus his research in this area. At the Texas A&M Citrus Center, Weslaco a collection of eight rutaceous species has been established which include two Amyris species (torch wood), Zanthozylum fagara (lime pricklyash), Helietta parvifolia (baretta), Esenbeckia berlandia, Casimiroa tetrameria and two Choisya species (Mexican orange) and will be tested as hosts for the psyllid. Since HLB is not in Texas some of the plant materials will be shipped to the USDA, ARS, Beltsville, MD for inoculation tests with HLB infected citrus materials. At the USDA, ARS, FDWSRU, Ft. Detrick, MD Murrraya paniculata, M. exotica and M. koenigii were established in quarantine. Two isolates of Las ( from Taiwan and Florida) were obtained from J. S. Hartung at the Exotic Citrus Diseases Collection, USDA, Beltsville, MD and were used in experiments to infect the the plants species listed above. In psyllid transmission tests Asian citrus psyllids transmitted Las to M. paniculata and M. exotica, but not M. koneigii. Disease symptoms rarely developed in Murraya plants, and positive infections were determined by PCR. Back-inoculations from M. paniculata to sweet orange was successful however there was some variability was in infection rates, titer, and persistence of the Las bacterium in M. paniculata. A publication is in preparation. Work will continue acquiring other rutaceous plants and testing them for susceptibility to Las, Lam and Laf.
Transmission of Huanglongbing causal bacteria by psyllid feeding is the natural route of transmission. The causal agent has recently been cultured but pathogenicity tests have not been completed to prove the causal nature of the cultured bacteria. Although the release of our funding was delayed for several weeks, we have initiated a staffing plan and begun experiments. For psyllids to acquire the cultured bacteria we had proposed to use membrane-feeding and/or micro-injection to charge the psyllids with the causal bacteria. We are developing protocols for membrane feeding using dilute (5%, 10%, and 20%) sucrose preparations within the membrane. We found psyllids survived for several days on all dilutions. We are beginning experiments to determine what the psyllids can inject into the membrane during feeding and how much they can obtain from feeding through membranes. We are proposing using 5th instar nymphs and adult psyllids for these studies. We have purchased a micro-injection system and are beginning to test the parameters for successful injection of bacterial culture into the psyllids hemolymph.
Under objective 1 (define rates and formulations of copper sprays for more effective control) one trials with Red grapefruit has been set up in Ft. Pierce, FL and one trial with Hamlin orange in Hardee County, each with 20 treatments of formulations, rates and combinations with other chemical treatments of interest. Under objective 2 (establish the period of fruit susceptibility for grapefruit and oranges), companion trials have been set up looking at whether longer duration of a copper spray program is necessary for control of crop loss. Under objective 3 ( evaluate the use of streptomycin [Firewall] and a polymer delivery system [PDS] to lower the rate and frequency of copper application) three trials (two non-bearing and one bearing) in grapefruit have been initiated to evaluate streptomycin for canker control, and two trials in bearing grapefruit have been initiated to evaluate PDS and the interaction with copper and timing of copper burn. Under objective 4 (To define risk for development of bacterial resistance to copper [Cu] and streptomycin [Sm] in FL citrus groves) one non bearing trial in grapefruit sprayed every 21 days with Kocide 3000 and/or Firewall (Sm) has been monitored since March 2008. No Cu or Sm resistant strains of Xcc have been isolated, however Cu and Sm sprays increased the ratio of epiphytic bacteria with resistance to these chemicals. Overall, the Sm resistant bacterial population is proportionally lower than Cu resistant bacterial population. Under Objective 5 (rapid transfer improved canker management technology to the Florida citrus industry), the 2009-10 canker management recommendations have been published in the Florida Citrus Pest Management Guide and Citrus Industry Magazine. Oral presentations have been delivered to the Florida Citrus Production Managers and at county extension meetings.
Under Objective 1 (define rates and formulations of copper sprays for more effective control) one trial with Red grapefruit has been set up in Ft. Pierce, FL and one trial with Hamlin orange in Hardee County, each with 20 treatments of formulations, rates and combinations with other chemical treatments of interest. Trials have low to moderate disease epidemics this season as a result of a dry spring, moderately wet summer and an absence of tropical storm events. Under Objective 2 (establish the period of fruit susceptibility for grapefruit and oranges), companion trials have been set up looking at whether longer duration of a copper spray program is necessary for control of crop loss. Fruit drop has been quantified in the Hamlin orange plots. Under Objective 3 ( evaluate the use of streptomycin [Firewall] and a polymer delivery system [PDS] to lower the rate and frequency of copper application) three trials (two non-bearing and one bearing) in grapefruit have been initiated to evaluate streptomycin for canker control, and two trials in bearing grapefruit have been initiated to evaluate PDS and the interaction with copper and timing of copper burn. Under Objective 4 (To define risk for development of bacterial resistance to copper [Cu] and streptomycin [Sm] in FL citrus groves) the copper resistance gene has been identified from a resistant Xcc strain from Argentina as CopL, the same resistance in X. vesicatoria the cause of bacterial spot in tomato. Primers constructed based on the gene sequence have been used to screen the remainder of copper resistant strains of Xcc from Argentina and the copper resistant strains of X. alfalfae. pv citrumelonis from Florida citrus nurseries with citrus bacterial spot. All strains screened thus far contain the CopL resistance gene. Under Objective 5 (rapid transfer improved canker management technology to the Florida citrus industry), the 2009-10 canker management recommendations have been published in the Florida Citrus Pest Management Guide and Citrus Industry Magazine. Oral presentations have been delivered to the Florida Citrus Production Managers and at county extension the copper meetings.
The Objectives are to: 1) Test the effect of soil applied imidacloprid as an inducer of SAR on HLB disease incidence in newly planted citrus trees subjected to psyllid mediated infection; and 2) test the effect of imidacloprid on HLB disease progress in newly planted citrus trees that are graft-inoculated with budwood from HLB infected field trees. The following treatments were set up in the USDA-ARS, Picos Farm in Ft. Pierce FL: 1) untreated check, 2) foliar insecticide to control psyllids, 3) soil applied imidacloprid/thiamethoxam to induce SAR, 4) soil applied imidacloprid/thiamethoxam plus the foliar insecticide to induce SAR and control psyllids, 5) HLB bud graft-inoculated untreated check, 6) HLB bud graft-inoculated with soil applied imidacloprid/thiamethoxam to induce SAR. The experimental design is a completely randomized block design with 50 trees per treatment (5 blocks of 10 trees per block). Tree and insecticide treatments commenced in May and HLB graft inoculations will occur in July. A HLB bud-grafted experiment of similar design will be set up in Brazil in spring time (September-October) for southern hemisphere.
Under Objective 1 (evaluate the potential for soil application of the neonicotinoids, imidacloprid and thiamethoxam, and acibenzolar-s-methyl, to provide long-lived SAR control of canker in young trees) two trials in 1 and 3 yr old grapefruit in Ft. Pierce, FL have been set up with 14 treatments in each location. A trial with complementary treatments will be set up in Brazil in the spring (August-September) in the southern hemisphere. Objective 2 (integration of SAR with foliar applications of reduced rates and frequency of copper sprays for control of canker on the most susceptible cultivar in Florida, grapefruit) and objective 3 (evaluation of the complementation of the use of neonicotinoids thiamethoxam and imidacloprid to increase and/or extend insect and disease control) are being addressed within the treatments of the two trials set up in Florida.
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