Funds for this study were received on March 18, 2009. Since these experiments must take place early in the season when the fruit require degreened to meet market requirements, the bulk of the funds will be used on experiments in the fall and early winter when late rains result in pin-point lesions and washing the fruit may inhibit degreening operations. As part of another project, however, we have just built a research grading line that we will also use for this study that will allow evaluation of a number of washing treatments and other variables to develop optimum conditions for grading fruit for canker lesions. Furthermore, preliminary results that supported the current project were presented at the FSHS meetings in June 2009 and a manuscript submitted for publication in the FSHS proceedings. These results showed that inhibition of degreening was significantly less when fruit were washed for only ~15 seconds on a high-pressure washer with brushing compared to longer (1 to 2 min) brush washing treatments. With this information, we will test that ability of even shorter wash treatments or wash treatments without brushing (i.e., only sprays of high pressure water) to clean fresh citrus enough for adequate grading for defects and citrus canker lesions. The variables that will be evaluated include different methods of washing, coloration of the harvested fruit, required degreening time, cleanliness of the fruit, presence of other peel markings (i.e., windscar, melanose, etc.), speed of fruit travel, lighting conditions, the color of the rollers, width of the grading area, and number of graders per unit area.
This is a continuing project for which funding was released this year on 27 April and a comprehensive management scheme has taken shape and been documented: (a) sampling plans for monitoring psyllid populations, (b) preemptive dormant sprays of broad-spectrum insecticides targeting adult psyllids, (c) wide choice of selective and broad-spectrum insecticides to use in the growing season, and (d) strategies for conservation and augmentation of biological control. Objectives and some of the corresponding achievements during this quarter are listed below. 1) Assist growers with planning and evaluation of field tests to develop site specific season-long psyllid control on young and mature trees including active ingredients, timing, application methods, rates etc. – SAR inducers and micro-nutrients did not statistically improve the quantity or quality of the harvest after one year although trends are positive (see Report FCPRAC # 0179) – Aerial application of broad spectrum insecticides reduced ACP adults although ground sprays (speed sprayer) were more effective. LV application of 435 Oil every 2 or 3 weeks at 2 gal./ac. during growing season appear to be have good effects although a single such application with spinetoram or dimethoate was not effective (see Report FCPRAC # 0210). – Area wide dormant spray: 80,000 acres were treated by air and many more by ground in a voluntary program last season promoted by Gulf Citrus Growers Association. A field survey coordinated by SWFREC-Entomology and DPI-CHRP documented greatly reduced psyllid populations compared with the few blocks left untreated. A survey of grower responses is being conducted in coordination with Multi-County Cooperative Extension and plans are to coordinate two dormant sprays this season. 2) Assist in development of efficient monitoring methods to make and evaluate control decisions. Tap samples provide timely information and are 14 times faster than sticky traps. Tap sample and sweep nets gave similar results and took about the same time although taps may be more convenient and less fatiguing (see Report FCPRAC # 0164) 3) Accelerate testing of new chemical tools for psyllid suppression: (a) Laboratory bioassays with new products from Dupont and Gowen are continuing, (b) Foliar application of spirotetramat, and fenproximate with 435 Oil or NIS (spirotetramat) during bloom 2009 suppressed psyllids for 2 months, (c) foliar and soil-applied field testing continues this summer. All field test results are published in Arthropod Management Tests and on our website http://www.imok.ufl.edu/entlab/. 4) Evaluate the economic feasibility of psyllid management strategies. The cost of two aerial dormant sprays is less than $25/ac each and could be as low as $15/acre earch depending on material, volume and distance from the airstrip. The cost of monitoring psyllids using the tap is about $25 per block up to 100 acres so could be done 20 times per year for as little as $5/ac total, depending on block size and would avoid unnecessary sprays during the growing season that could cost up to $50/ac. Details on website. 5) Provide an information bridge between growers and researchers: (a) Conducted 2 scouting workshops, 1 management workshop, and 2 talks given at the Florida State Horticultural Society annual meeting. (b) Published: Stansly, P. A., J. A. Qureshi, and Arevalo, H. A. 2009. Why, when and how to monitor and manage Asian citrus psyllid. Citrus Industry. March 2009. Other publications available at http://swfrec.ifas.ufl.edu/entlab/ and at http://swfrec.ifas.ufl.edu/hlb/database/ (see Report FCPRAC # 0004)
This project is in its second year and has already has provided a comprehensive management strategy that is being used by many growers as we continue to make improvements. Tactics being employed include: (a) sampling plans for monitoring psyllid populations, (b) preemptive dormant sprays of broad-spectrum insecticides targeting adult psyllids, (c) systemic insecticides for young trees (d) a wide choice of tested, selective and broad-spectrum insecticides for use during the growing season based on scouting results, and (d) strategies for conservation and augmentation of biological control. Objectives and some of the corresponding achievements during this quarter are listed below. 1) Assist growers with planning and evaluation of field tests to develop site-specific season-long psyllid control on young and mature trees including active ingredients, timing, application methods, rates etc. – SAR inducers and micro-nutrients did not statistically improve the quantity or quality of the harvest after one year although yields increased by 30% and visual differences of overall tree health were observed (see Report FCPRAC # 0179) – Aerial application of broad spectrum insecticides reduced ACP adults although ground sprays with a speed sprayer were more effective. LV application of 435 Oil every 3 to 4 weeks at 2 gal./ac. during growing season appears to be working although populations were low. A single such application with spinetoram or dimethoate using LV ever other row was much less effective at reducing ACP populations than a speed sprayer running every row (see Report FCPRAC # 0210). – Voluntary area wide spray: 80,000 acres were treated by air and many more by ground in a voluntary program last season promoted by Gulf Citrus Growers Association. A field survey coordinated by SWFREC-Entomology and DPI-CHRP documented greatly reduced psyllid populations compared with the few blocks left untreated. A survey was conducted in coordination with the Multi-County Cooperative Extension to evaluate pest control practices and attitudes regarding area wide control was responded to by 27 growers, managing groves of from 42 to 18,000 acres for a total of 107,000 acres of citrus in SW Florida. Results indicated wide adoption of the comprehensive strategy, with 97% of growers are planning preemptive dormant sprays, 97% using some type of sampling of which 95% used the tap sample, 76% flush observation, 14% Sticky traps, and 9% sweep net, with 85% of growers sampling > 3 times a year. 3) Accelerate testing of new chemical tools for psyllid suppression: (a) Laboratory bioassays with new products from DuPont and Gowen were finalized and some promising products under development, (b) Foliar application of spirotetramat, and fenproximate with 435 Oil or NIS (spirotetramat) during bloom 2009 suppressed psyllids for 2 months, (c) foliar and soil-applied field testing continued this summer. All field test results were published in Arthropod Management Tests and on our website http://www.imok.ufl.edu/entlab/. 4) Evaluate the economic feasibility of psyllid management strategies. The cost of two aerial dormant sprays is less than $25/ac each and could be as low as $15/acre earch depending on material, volume and distance from the airstrip. The cost of monitoring psyllids using the tap is about $25 per block up to 100 acres so could be done 20 times per year for as little as $5/ac total, depending on block size and would avoid unnecessary sprays during the growing season that could cost up to $50/ac. Details on website. 5) Provide an information bridge between growers and researchers: Since June 2009 we have (a) Conducted 2 scouting workshops, given 2 talks given at the Florida Entomological Society, and 5 grower talks, published an article in Citrus Industry and increased the volume of citations for the HLB database to > 1,600 references (see Report FCPRAC # 0004).
This is a continuing project for which funding was released this year on 13 April. For our first objective, we obtained 3 new colonies of Tamarixia radiata, a species specific parasitoid, from south China, North Vietnam and Pakistan last year and now have secured approval from USDA-APHIS-PPQ to release these into the environment. Also, one of us (RN) made another trip to a different part of China although no new colonies were obtained. For our second objective, we have completed a study with the help of Dr. Norman Barr, USDA-ARS Mission TX, to characterize these and other strains genetically so that they can be tracked in the environment. The study confirmed that the different colonies shared the same internal transcribed spacer (ITS) region of nuclear ribosomal DNA, showing that the collections represent a single species. This together with a host range study conducted by Ru Nguyen in quarantine was used to obtain the release permit. The mitochondrial cytochrome c oxidase I (COI) marker was used distinguished the northern Vietnam and Pakistan colonies from each other and from the other colonies. Comparison of COI sequences from field collected populations of Puerto Rico, Guadeloupe, and Texas indicated that Florida is not a likely source of the introduced T. radiata in Puerto Rico but is a likely source of the introduction into Texas. We had already begun to ramp up our colonies of psyllid and the strain of Tamarixia radiata (Objective 1) in preparation for release and evaluation this spring and summer (objective 2). We have over 200 Murraya paniculata plants in our new screen house for this purpose, a new air-conditioned glasshouse for the psyllid colony and another for the parasitoid colony with a backup in a climate-controlled insectary. Parasitoids are being produced at the rate of 600-800 per week. So far this year, 7,050 of these wasps have been released in both conventional and organic commercial citrus groves in Florida. We are also providing information and starter colonies to assist a commercial grove in Florida to mass rear the parasitoid for their use. Parasitism rates are being monitored by rearing field collected nymphs of D. citri and exposing infested sentinel plants in the field to compare with published studies (see below). Nymphs recovered on sentinel plants in the conventional grove were 52% parasitized by T. radiata. Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), an endoparasitoid of D. citri from southern China is also being reared and released throughout the state. A total of 2400, 1325, and 3550 wasps were released during 2007, 2008, and 2009, respectively, in conventional and organic citrus groves and dooryard orange jasmine (Muraya paniculata). A parasitism rate of 6-19% was calculated based on adult emergence from nymphs reared from M. paniculata in May 2008. For objective 3, we released over 0.5 million predatory mites (Amblyseius swirskii) on mature orange trees in a 20 acre organic grove in Lake Wales, FL and are monitoring the populations of mites and psyllids in the grove. Thus, substantial progress was made on all objectives this quarter. Publications (Objective 4) 1. Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. Journal of Economic Entomology (submitted). 2. Qureshi, J. A., M. E. Rogers, D. G. Hall, and P. A. Stansly. 2009. Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology. 102: 247-256. 3. Qureshi, J.A., and Stansly P.A. 2009. Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129’136.
(1) Screening of chemical compounds that eliminate or suppress the Las bacteria in periwinkle using the optimized regeneration system. Antibiotics (penicillin G and streptomycin, oxytetracyclin), a biocide (DBNPA), a fungicide (zineb) and two peptides (D4E1 and D2A21) were tested for their ability to eliminate or suppress Las bacteria and to promote the growth of severe HLB-affected cuttings using the optimized regeneration system. More than 75% of plants could be regenerated from the severe HLB-affected periwinkle cuttings treated with a combination of penicillin and streptomycin (PS). All regenerated plants from the HLB-affected cuttings treated with PS tested negative for Las, even by nested PCR. The Las bacteria were undetected in these regenerated plants with average Ct value of 39.33. When treated with oxytetracyclin or peptides (D4E1 or D2A21), the regenerated plants also tested negative by nested PCR or qPCR with Ct values over 32. However, the regeneration percentage was low (30%). The regenerated control plants treated with water tested positive for Las by PCR and qPCR with low Ct values <26.0, indicating that the bacteria titers were at least 100-fold higher than those in the antibiotic or peptide-treated, regenerated periwinkle. DBNPA can also suppress the Las bacteria. The severe HLB-affected cuttings had a regeneration rate of 33.1 %. The Ct values of the regenerated plants ranged from 27 to 30. The fungicide zineb was not effective in controlling Las bacteria. Whether treated with zineb or not, the Las bacteria can keep reproducing. The Ct value was lower in the zineb-treated, regenerated plants than those treated with antibiotics or peptides, and similar to the water control. Zineb is not very soluable. The water solubility is only 10 mg/L. (HLB-affected periwinkle was treated using PS, oxytetracyclin or DBNPA three times a week by foliar spray (previous treatments were by root soaking). The results were same as for the HLB-affected cuttings treated by root-soaking. Antibiotics PS and oxytetracyclin effectively suppressed or inhibited Las bacteria, and DBNPA partially suppressed Las bacteria. (2) Effect of screened chemical compounds on HLB-affected citrus. Nutrition and HLB development in citrus. The nutrients K, Zn, N, and Ca were tested for their ability to suppress HLB symptoms in citrus. Potassium and Zn, but not N or Ca, delayed HLB symptoms. Neither treatment affected Las bacterium after 9 months. K and Zn reduced titers of Las at 5 months after treatment. Effect of SAR-inducer on HLB development. Seven months after treatment with SAR-inducer (SA and antiguard), grafted, HLB-affected citrus tested positive for Las by PCR with an average Ct value of lower than 28, indicating SAR was not effective in suppressing or inhibiting the Las bacterium. This had also been the case with a perwinkle host. Effect of antibiotics on HLB-affected citrus by root soaking. HLB-affected citrus was soaked in the PS or oxytetracyclin solutions three times a week. One month post-treatment, the HLB-affected citrus tested negative for Las by nested PCR, and undetected by real-time Q-PCR, Oxytetracyclin and water control treatments were positive for Las. (3) Conclusions Antibiotics PS (penicillin and streptomycin) and oxytetracyclin are very effective in control of liberibacter, especially PS. Peptides are also effective, but expensive for field use. Transgenic citrus expressing these peptides may provide effective HLB control. DBNPA can reduce Las titers. Zineb is ineffective.
Funds for this project were not released until 9 June although work commenced at the beginning of April 2009. We are evaluating tap sampling against sticky traps, and sweep net methods for the first objective. A 12-acre commercial block of Valencia oranges was divided into 16 plots, eight treated with insecticides and 8 left untreated. Three sampling methods were assessed: the ‘tap’ sample, the ‘Yellow Corn Rootworm Trap’ (YCRW), and the ‘ACP Trap’. For the tap sample a randomly chosen limb was struck 3 times and fallen psyllids counted from a laminated white sheet of paper held 1 ft below the foliage. Each plot contained six sampling stations located at random, each consisting of a tree with two types of traps hung in the canopy. Traps were collected after 2 weeks, at which time two tap samples were taken from the same tree. The YCRW trap captured significantly more adults per trap per tree during the two-week period (9.3 ‘ 1.2 vs. 1.8 ‘ 0.27 in untreated and treated plot respectively) than did the ‘ACP trap’ (4.78 ‘ 0.59 per trap in untreated plots, and 1.1 ‘ 0.02 in the treated plots). ACP adults counted with the tap sample averaged 0.86 ‘ 0.11 per tree in untreated plots and 0.17 + 0.09 in the treated plots. A bootstrap procedure, which resampled from the database with replacement was used to obtain robust estimates of the standard error of the mean (SEM) and confidence intervals for the mean. Under high density, the SEM:mean ratio was estimated at 8.3% for the tap sample and 8.0%, 8.3% for the YCRW and ACP traps respectively for a sample of 100. Under low density SEM:mean ratios were 34, 10 and 14% for the tap sample, YCRW and ACP Traps respectively. Sticky traps cost approximately $1.00 each, and their deployment, collection and reading takes 14 times longer than the tap sample: an average of 7 minutes per trap (hanging, collecting and reading) compared with 30 seconds per tap sample (average of sampling and recording data from 10 non-continuous trees). On the other hand, they caught about 10 times more psyllids, although this was over a 2 week period whereas data from the tap sample data is available immediately. Tap sampling was also compared with sweep net sampling in eight 2-8 acre blocks of untreated and treated citrus in June at Southwest Florida Research and Extension Center. Two tap or sweep net samples were conducted on the bed and swale sides of each of four trees per eight locations per block. In the untreated block, psyllids averaged 0.6 ‘ 0.1 and 0.8 ‘ 0.2 per two taps or sweeps per tree, respectively. Average time required to conduct two taps or two sweeps was 7 seconds. Psyllid density in the treated blocks was much lower compared to the untreated block and averaged 0.2 ‘ 0.03 and 0.1 ‘ 0.02 adults per two taps or sweeps, respectively, and the mean time required to conduct either sample was 6 seconds. Both tap and sweep net seem to be equally effective for psyllid counts and time at both high and low densities, although average density was less than one adult per two taps or sweeps. However, the sweep net is more work, could be difficult to count at high density, and could spread citrus canker. Technology transfer (third objective): Stansly, P. A., J. A. Qureshi, and Arevalo, H. A. 2009. Why, when and how to monitor and manage Asian citrus psyllid. Citrus Industry. 90 (3): 24-26. Arevalo, H. A., P. A. Stansly, A. B. Fraulo, J. A. Qureshi and L. J. Buss. 2009. Sampling for Asian citrus Psyllid. Field Sheet. SWFREC- University of Florida, http://swfrec.ifas.ufl.edu/entlab/pdf/extension/ACP_sampling_english.pdf. Stansly, P. A., H. A. Arevalo, and J. A. Qureshi. 2009. Scouting citrus for pests and beneficials. . Extension presentation (M. Zekri Organizer). March 19. SWFREC/ Multi-County extension. Stansly, P. A., H. A. Arevalo, and J. A. Qureshi. 2009. Monitoreando Plagas e insectos beneficos en citricos. Extension presentation (M. Zekri Organizer). April 28. SWFREC/ Multi-County extension.
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR Funds for this project were released on 19 March although research began earlier in anticipation of funding. In order to assess seasonal patterns of the pathogen in citrus trees and psyllids in regions of high HLB incidence, a 12 acre block of ‘Valencia’ orange trees was selected at a commercial grove in 2008. The block was divided into 16 plots, half of which were treated with insecticides as needed to suppress psyllid populations while the other 8 plots were left untreated. A sample of psyllid adults collected in the block in November 2008 and analyzed at USDA-ARS Riverside labs contained 21% HLB positive psyllids. Samples of the most symptomatic leaves from every fifth tree were taken on 13 Nov 2008 and analyzed for the presence of Ca. Liberibacter asiaticus at the Huanglongbing Diagnostic Laboratory, Southwest Florida Research and Education Center (SWFREC), Immokalee, FL. DNA was isolated using a commercially available kit (Qiagen; Valencia, Califorina, USA) for plant (DNeasy Plant kit) and psyllids (DNeasy Blood and Tissue kit). Quantitative PCR analysis was conducted using specific TaqMan primer/probe assays for plant (Li et al. 2006) and psyllid (Manjuanth et al. 2008). Thirty-four percent of the samples tested PCR positive. A tree determined to be PCR positive and one PCR negative tree in each plot was trimmed to induce new flush. On 3 June, 10 psyllid adults from our HLB negative colony on orange jasmine (Murraya paniculata) were released on a new shoot that had been caged immediately after trimming. An additional cage was placed on a previously uncaged and psyllid-infested shoot on the same tree. All cages along with branches and psyllids were collected 2 weeks later for PCR analysis. Nineteen percent of shoots caged with psyllids from the HLB negative colony tested positive for HLB on trees, regardless of whether trees had previously tested HLB positive or negative. In contrast, shoots that were naturally infested with psyllids when caged were 63% and 56% positive on previously HLB positive and negative trees, respectively. It would appear that infestation with infected psyllids from the field was over 2.5 times more likely to result in a positive plant sample, whereas the previous history of testing had little bearing. We are processing the adult psyllid samples taken at the same time. We are also caging infested shoots on symptomatic trees at SWFREC and commercial groves and collecting monthly samples of psyllid adults found. Adults emerging from shoots on HLB symptomatic trees caged with negative psyllids during Feb-March 2009 and analyzed at US Sugar and SWFREC were found to be 5% HLB positive by both labs, although psyllids collected at large at the same time and location were 14% positive. Psyllids that were caged on these trees during July-August 2008 were 27% positive according to the USDA-ARS Riverside lab. Therefore, psyllid origin and time of collection are major sources of variation on PCR results that we will need much more data to sort out. As our methods for addressing the first objective are refined as described above, the second objective is being address by the identification of additional sites to survey for psyllids which differ with respect to tree age, variety, and rootstock, and block size as described in grant proposal. A survey to administer to growers for tracking HLB and grove management tactics in the selected sites is in preparation. (Refs: Li, W., J.S. Hartung, and L.Levy. 2006. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. Journal of Microbiological Methods. 66:104-115. Manjuanth, K.L., S.E. Halbert, C. Ramadugu, S. Webb, and R.F. Lee. 2008. Detection of Candidatus Liberibacter asiaticus in Diaphornia citri and its importance in the Management of Citrus Huanglongbing in Florida. Phytopathology. 98(4):387-396.)
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR Funds for this project were released on 19 March. Objective 1. Assess seasonal patterns of pathogen incidence in citrus trees and psyllids in regions of high HLB incidence. A 12 acre block of ‘Valencia’ orange trees was selected at a commercial grove in 2008. A sample of psyllid adults collected in the block in November 2008 and analyzed at USDA-ARS Riverside labs contained 21% HLB positive psyllids. The block was divided into 16 plots, which receive, two levels of micronutrients+SAR, insecticide treatments, or left as control. Plant and psyllid samples are being collected every four months to test for Candidatus Liberibacter asiaticus using PCR. Insecticide applications significantly suppressed psyllids compared to control. No significant differences in the field distribution of HLB in plants were observed using quantitative geostatistical analysis between November 2008 (40% HLB infection) and April 2009 (33% infection). A tree determined to be PCR positive and one PCR negative tree in each plot was trimmed to induce new flush. On 3 June, 10 psyllid adults from HLB negative colony on orange jasmine (Murraya paniculata) were released on a new shoot that had been caged immediately after trimming. An additional cage was placed on a previously uncaged and psyllid-infested shoot on the same tree. All cages along with branches and psyllids were collected 2 weeks later for PCR analysis. Nineteen percent of shoots caged with psyllids from the HLB negative colony tested positive for HLB on trees, regardless of whether trees had previously tested HLB positive or negative. In contrast, shoots that were naturally infested with psyllids when caged were 63% and 56% positive on previously HLB positive and negative trees, respectively. It would appear that infestation with infected psyllids from the field was over 2.5 times more likely to result in a positive plant sample, whereas the previous history of testing had little bearing. However, results were different when the same experiment was repeated in July-August using same plants. Shoots caged with psyllids from HLB negative colony were 64% and 42% HLB positive on previously HLB positive and negative plants, respectively. Whereas, naturally infested caged shoots were 7% and 18% HLB positive on previously HLB positive and negative plants, respectively. Adults emerging from infested shoots caged on HLB symptomatic trees at SWFREC during Feb-March 2009 and analyzed at US Sugar and SWFREC were found to be 5% HLB positive by both labs. Psyllids collected at large at the same time and location were 25% HLB positive, followed by 11% in April. Adults that emerged from infested shoots caged on HLB symptomatic trees were 23% HLB positive according to the USDA-ARS Riverside lab. Adults that emerged in the cages on HLB symptomatic trees from same location during July-August 2008 were 27% positive according to the Riverside lab. Therefore, psyllid origin and time of collection are major sources of variation on PCR results that we will need much more data to sort out. Additional psyllid samples from these experiments are being processed. As our methods for addressing the first objective are refined as described above, the second objective is being addressed by the identification of additional sites to survey for psyllids which differ with respect to tree age, variety, and rootstock, and block size as described in grant proposal. A survey to administer to growers for tracking HLB and grove management tactics in the selected sites is in preparation. Despite the delay in receiving funds, the project is progressing in adherence to timeline and objectives.
Recent studies made useful advances in culturing Ca. Liberibacter spp. (Sechler et al., 2009) but major restrictions remain that leave the study of the bacteria physiology and pathogenicity very limited. In order to efficiently culture Liberibacter, alternative methods need to be explored. Liberibacter is vectored by psyllid insects and is able to proliferate inside the insect. Moreover, it was previously shown that fastidious microorganisms could be grown by co-culturing them with insect cells. Those insect cells acting like feeder cells, providing nutrient complements in a continuous way. We therefore propose to assess the usefulness of insect cell cultures to in vitro culture Candidatus Liberibacter asiaticus (LAS), the Asian form of HLB disease, also found in Florida. LAS inoculums: LAS source material came from infected symptomatic citrus trees from Vietnam. Transmissions from citrus to citrus or from citrus to periwinkles were performed via the insect psyllid Diaphorina citri and subsequent graftings. We systematically tested the inoculums for the presence of LAS by direct PCR. Infection can be localized to only few branches/leaves, so this analysis proved to be essential to be sure the inoculums actually hosted the bacteria. Insect cell cultures: We want to test different insect cell lines, with well defined culture conditions. We thawed various insect cell lines and adapted them to various culture media. We started with testing two commonly used Lepidoptera (Mamestra brassicae and Spodoptera frugiperda) and one Diptera (Drosophila melanogaster) hematocyte cell lines. Commonly used and alternative insect cell culture media were tested in parallel to assess their effect on the bacteria fitness. We tested different ways of preparing the bacterial inoculums from citrus or periwinkle. Periwinkle sap showed a negative impact on Mamestra cells growth. The Drosophila cell line showed a much higher resistance to periwinkle sap. The maceration method was found to be the most appropriate way to release the bacteria in the cell culture medium so far. Detection of LAS in insect cell cultures: We checked for the presence of LAS in inoculated cell cultures by either direct PCR or by nested PCR. While we didnÕt detect the presence of LAS in Mamestra cell cultures (in serum-containing medium), we were able to detect it in a serum-free drosophila cell culture for a couple of weeks after inoculation. Noticeably, LAS was still detectable by direct PCR in a transferred culture after a couple of weeks. Conditions of culture/transfers are assessed to maintain the bacteria for a longer period of time and reach higher bacteria concentrations. Complementation of the Drosophila cell culture medium with various sugars, vitamins or trace elements that could be found in citrus/periwinkle phloem is under investigation. With the same approach, we are analyzing metabolic pathways potentially encoded by the released Liberibacter genome sequences that could be clue to the bacteria growth. Additionally, we are setting-up alternative Drosophila cell line cultures for further inoculations. Liberibacter inoculations of Spodoptera cell cultures are under analysis. We are currently adapting conditions of described LAS qPCR detection to quantify LAS in insect cell cultures.
Objectives of our research proposal are 1) characterizing the microbial community of healthy and Liberibacter-infected citrus phloem tissue by serial-section electron microscopy; 2) localizing antigens and DNA sequences specific to Liberibacter in citrus phloem tissues by immunofluorescence microscopy, immuno-electron microscopy, and electron microscopic in situ hybridization. During the first quarter of this year, we carried out transmission electron microscopy (TEM) analyses of periwinkle phloem tissues infected with Liberibacters by serial sectioning (Objective 1). Periwinkle leaf samples were first examined because periwinkles exhibit high Liberibacter titer, their leaf phloem architecture is simpler than that of citrus plant leaves, and they display starch accumulation symptoms when infected with Liberibacters as citrus trees do. The periwinkle samples were from Dr. Duan’s lab (co-PI). The samples displayed disease symptoms and presence of Liberibacter was verified by PCR prior to TEM imaging. Followings are the main findings. These findings are, however, still preliminary and require further investigation. 1) The plasmodesmata-pore units at the sieve element-companion cell interface are swollen. In the phloem, long distance solute transport is mediated by the sieve tube that is made of continuous linear array of sieve elements. Sieve elements are live cells but they are devoid of most organelles including the nucleus. Their survival is dependent on companion cells that are associated with sieve elements and provide materials required for sieve elements. The primary connections between sieve elements and companion cells are modified plasmodesmata, termed plasmodesmata-pore units (PPUs) and they are thought to be the principal transport pathway into and out of the sieve element, important for phloem loading and unloading. This symplastic transport apparatus comprises a wide channel at the sieve element side that is linked to multiple desmotubules at the companion cell side. The PPU sites in the cell wall are often thickened to accommodate the branching twig-shaped plasmodesmata structure. From our serial section analyses, we observed that PPU units from the sieve elements where Liberibacters inhabit are highly swollen due to insertion of non-filamentous substance around the PPU. The swelling occurs mostly in the companion cell side cell wall, widening the the companion cell side half cell wall 2~3 times thicker than the half cell wall on the sieve element side. We speculate that this alteration in the PPU structure could impede phloem loading as well as normal transport function of the phloem tissue. When compared with uninfected phloem samples and phloem samples infected with phytoplasma, the companion cell wall thickening at the PPUs was most severe in the Liberibacter-infected sieve elements. 2) Bacteria cells are seen exclusively in the sieve elements and two morphologically distinct bacteria cell types were identified. The two groups of bacteria cells were differentiated by their diameters and staining properties. The first group consists of thick (diameters: 225’92 nm) and lightly stained bacteria cells. The second group consists of thin (diameters: 103’16 nm) and darkly stained cells. To determine whether these two groups correspond to different bacterial species, molecular characterization such as immunogold labeling with a Liberibacter-specific antibody or in situ hybridization need to be carried out (Objective 2). We have prepared samples embedded in LR white resin that is compatible with immunogold labeling. We are planning to try two Liberibacter specific antibodies from Dr. Duan.
Objectives of our research proposal are 1) characterizing the microbial community of healthy and Liberibacter-infected citrus phloem tissue by serial-section electron microscopy; 2) localizing antigens and DNA sequences specific to Liberibacter in citrus phloem tissues by immunofluorescence microscopy, immuno-electron microscopy, and electron microscopic in situ hybridization. During the first quarter of this year, we carried out transmission electron microscopy (TEM) analyses of periwinkle phloem tissues infected with Liberibacters by serial sectioning (Objective 1). Periwinkle leaf samples were first examined because periwinkles exhibit high Liberibacter titer, their leaf phloem architecture is simpler than that of citrus plant leaves, and they display starch accumulation symptoms when infected with Liberibacters as citrus trees do. The periwinkle samples were from Dr. Duan’s lab (co-PI). The samples displayed disease symptoms and presence of Liberibacter was verified by PCR prior to TEM imaging. Followings are the main findings. These findings are, however, still preliminary and require further investigation. 1) The plasmodesmata-pore units at the sieve element-companion cell interface are swollen. In the phloem, long distance solute transport is mediated by the sieve tube that is made of continuous linear array of sieve elements. Sieve elements are live cells but they are devoid of most organelles including the nucleus. Their survival is dependent on companion cells that are associated with sieve elements and provide materials required for sieve elements. The primary connections between sieve elements and companion cells are modified plasmodesmata, termed plasmodesmata-pore units (PPUs) and they are thought to be the principal transport pathway into and out of the sieve element, important for phloem loading and unloading. This symplastic transport apparatus comprises a wide channel at the sieve element side that is linked to multiple desmotubules at the companion cell side. The PPU sites in the cell wall are often thickened to accommodate the branching twig-shaped plasmodesmata structure. From our serial section analyses, we observed that PPU units from the sieve elements where Liberibacters inhabit are highly swollen due to insertion of non-filamentous substance around the PPU. The swelling occurs mostly in the companion cell side cell wall, widening the the companion cell side half cell wall 2~3 times thicker than the half cell wall on the sieve element side. We speculate that this alteration in the PPU structure could impede phloem loading as well as normal transport function of the phloem tissue. When compared with uninfected phloem samples and phloem samples infected with phytoplasma, the companion cell wall thickening at the PPUs was most severe in the Liberibacter-infected sieve elements. 2) Bacteria cells are seen exclusively in the sieve elements and two morphologically distinct bacteria cell types were identified. The two groups of bacteria cells were differentiated by their diameters and staining properties. The first group consists of thick (diameters: 225’92 nm) and lightly stained bacteria cells. The second group consists of thin (diameters: 103’16 nm) and darkly stained cells. To determine whether these two groups correspond to different bacterial species, molecular characterization such as immunogold labeling with a Liberibacter-specific antibody or in situ hybridization need to be carried out (Objective 2). We have prepared samples embedded in LR white resin that is compatible with immunogold labeling. We are planning to try two Liberibacter specific antibodies from Dr. Duan.
This is the first quarterly report on the Hamlin and Valencia harvests in January and April, 2009, respectively. Difference-from-control tests were run for ÒhealthyÓ juice from fruit harvested from control non-HLB symptomatic trees versus ÒHLBÓ juice from non-symptomatic (HLB-NS) fruit harvested from HLB-symptomatic trees within the same grove (15 trees/disease state/variety combination). We also ran the above juice plus juice from symptomatic fruit (small, green, lopsided) harvested from HLB-symptomatic trees (HLB-S) through a trained citrus panel. Fruit were juiced using a JBT 391 extractor and pasteurized under simulated commercial conditions (Microthermics HTST Model 25, 90 ¼C/10 s). For the Hamlin samples, the Brix ranged from 10.33 to 11.16, acid from 0.54 to 0.58 %, ratio from 19.1 to 21.1 and oil from 0.014 to 0.019 %. For the Valencia samples, the Brix ranged from 11.92 to 12.5 , acid from 0.65 to 1.18 % (HLB-S juice), ratio from 10.1 to 18.6 and oil from 0.02 to 0.037 % (HLB-S juice). So for the Hamlin harvest, there were no big differences in Brix, acid, ratio or oil content, but for the Valencia fruit, the juice from HLB-S fruit was high in acid and oil content compared to the healthy controls and the HLB-NS fruit, which were similar. Juice samples were frozen for other chemical analyses (individual sugars and acids, vitamin C, limonin, nomilin and other secondary metabolites as well as aroma volatiles) to be done in the off-season, and samples for gas chromatography-olfactometry (GC-O) work were sent to Dr. Russell Rouseff along with the transfer of $20,000 to the University of Florida for this analysis. Another batch of Valencia fruit were harvested June 25th, juiced, pasteurized and frozen, but not yet analyzed by sensory panels. For these fruit the Brix ranged from 10.65 to 12.23, acid from 0.50 to 0.58 %, ratio from 20.8 to 21.2 and oil from 0.014 to 0.028 % (HLB-S fruit). So for this harvest, the main difference was that the oil content was higher in HLB-S fruit juice, possibly due the action of the extractor on fruit of smaller size. We hope to obtain a final Hamlin sample in the fall. In addition, leaves were harvested for study from Hamlin trees (frozen for analysis), and a postdoc has been selected and is due to come on board in August, 2009 for threshold work to determine the effect of symptomatic HLB juice on flavor on normal juice. Sensory results showed that there were no differences between juices made with fruit from control or HLB-NS by the difference-from-control test for either Hamlin, harvested in January or Valencia, harvested in April. This means that the average consumer would not likely find a flavor difference between these two groups of juices for either of these varieties, much less detect a difference if this juice was blended with normal juice. Differences were found, however by the trained panel, but mostly for HLB-S fruit (small, green, lopsided, etc.) compared to healthy controls and only a few descriptors were different for HLB-NS fruit compared to controls. There were differences in Hamlin juice for orange and paint aromas as well as orange, fruity-non-citrus, fresh, sour/fermented, peppery/musty, paint, sweet, sour, salty/umami, bitter and metallic flavor descriptors between healthy controls and HLB-S juice, with the exception of the fruity-non citrus descriptor, which showed that both HLB-S and HLB-NS fruit juice to be lower in intensity compared to control juice. For the rest of the descriptors, HLB-S fruit were significantly lower in intensity for orange aroma and orange, fresh and sweet tastes; and higher in sour/fermented and paint aromas and sour/fermented, peppery/musty, paint, sour, salty/umami, bitter, metallic and tingling flavors/sensations. Results for Valencia were similar to Hamlin except that the peppery/musty and peel oil flavor showed differences (higher in HLB-NS and HLB-S juice for peppery aroma and HLB-S juice for peel oil flavor).
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.
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