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.
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.
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