The objective is to test the effect of soil applied neonicotinoid insecticides used for systemic psyllid control as inducers of SAR to determine, if possible, the direct effect of SAR on HLB disease progress in newly planted citrus trees subjected to psyllid mediated infection or budwood-inoculated infection. Hamlin trees were planted in May 2009 at the USDA-ARS, Picos Farm in Ft. Pierce FL and treated as follows: 1) untreated check, 2) foliar insecticide to control psyllids, 3) soil applied imidacloprid/thiamethoxam to induce SAR, 4) soil applied imidacloprid/thiamethoxam plus the foliar insecticide to induce SAR and control psyllids, 5) HLB bud graft-inoculated untreated check, 6) HLB bud graft-inoculated with soil applied imidacloprid/thiamethoxam to induce SAR. In 2009, the effect of SAR induction on HLB infection progress was inconclusive perhaps because the soil applied imidacloprid/thiamethoxam control of the psyllid may have caused an uncontrolled interaction with psyllid transmission. In 2010, the trial utilized soil-applied SAR inducing Actigard instead of imidacloprid/thiamethoxam in treatments 3,4 and 6. In December 2009 (6 months post-HLB inoculum challenge), 34 of 300 trees in the experiment were PCR positive (11%). In July 2010, 49 trees or 16% are PCR positive. Most of the PCR positive trees are located on the west side of the trial area which is proximal to pruned, heavily HLB infected citrus, and few infected trees on the east side bordered by citrus seed (trifoliate hybrids) and pine flatwoods. The highest number of PCR positive trees (12) occur in the untreated checks with or without graft inoculation, treatments 1 and 5, and in the graft-inoculated treated trees with soil applied SAR inducers, treatment 6; 7 positives are detected in the soil applied SAR inducer, treatment 3, 4 positives occur in the foliar insecticide,treatment 2, and 2 positives in the foliar insecticide plus soil applied SAR inducers, treatment 4. There are many new visually positive trees that cannot be confirmed as positive by PCR analysis due to the seasonally low bacterial titer in HLB affected trees throughout the state in mid summer. If the visually positive trees is are assumed to eventually be PCR positive, the effect of SAR induction on HLB infection progress remains inconclusive. Two HLB-SAR experiments of similar design are set up in Parana, Brazil. One is HLB graft-inoculated and the other is proximal to an unmanaged farm with HLB. Each of these trials utilizes soil applied Actigard for the SAR treatments. The HLB status of the Brazil trials will be assessed in September 2010 by the collaborators.
Objective 1: Survey and confirmation of HLB in seedlings from HLB-affected trees. In 2009, 500 seedlings grown from seed extracted out of mild to severely HLB-affected fruit from Pineapple orange and Murcott tangor groves in Hendry Co. were assayed for HLB detection. All seedlings were negative for HLB detection by PCR in repeated assays in July and Nov. 2009. Objective 2: Thermotherapy of HLB-affected seed for the same seed sources under Objective 1 were treated at 125F, 130F and 135F to test for the effect of heat treatment on HLB detection in seedlings. Thermal treatments were lethal to Murcott, but not Pineapple seed. The emergent seedlings at 125F (142), 130F (98) and 135F (97) were PCR tested. All seedlings were negative for HLB in repeated PCR assay in July and Nov. 2009. Objective 3: In October 2009, seed source trees in two Florida nurseries were found positive for HLB by FDACS-DPI (Nursery 1) or Southern Gardens Diagnostic Lab (Nursery 2). Discovery of infected seed source trees in two Florida citrus nurseries identifies a potential (but unconfirmed) risk of outdoor seed source trees acting as a source of inoculum for introduction into nursery propagations. In Nursery 1, seed was collected from symptomatic branches of two sources of Swingle citrumelo, four sources of ‘Kuharske’ Carrizo citrange and one source of Cleopatra mandarin. In Nursery 2, seed was collected from one source of Sekwasha mandarin. From 189 to 278 seedlings from each source provide enough leaves for PCR testing in February 2010 and June 2010. The June test yielded a single questionable PCR detection for Cleopatra mandarin. Follow-up testing of this seedling from two separate midrib DNA extractions resulted in inconsistent positive results from 16S qPCR analysis with a mean detected Las titer of 6.9 and 4.3 16S copies/reaction (76.7 and 47.8 bacteria/midrib or 3652 and 2020 bacteria/g of midrib) for the two DNA extractions. Even in this case, detection by qPCR only occurred in only 75 and 33 percent of all 16S qPCR runs for the two DNA extractions. Las was not detected using beta-operon qPCR primers in either DNA extraction even after multiple runs. False amplification at very high Ct’s, 43-50 (5 to 0.001 copies/rxn by standard curve), occurs about 33 percent of the time with the 16S primer/probe system from water and healthy controls, whereas false amplification has not been observed with the beta-operon primer/probe system. Because the positive detections are within the range of false amplifications in water and healthy DNA extracts, we are currently using nested PCR in the 16S rDNA, beta-operon gene, and OMP gene to attempt confirmation of Las presence by sequence analysis.
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apples psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. The production of greenhouse-grown citrus trees with nutrient deficiency symptoms for both zinc and boron is underway. Deficiency symptoms are expected to take a few months to develop. This is especially the case for zinc because of the high mobility of zinc within the plant allowing redistribution of existing zinc to new flush, delaying the development of symptoms. Multiplication of our stocks of HLB infected citrus is also underway to provide a constant source of symptomatic tissue for experimentation once the nutrient deficiency symptoms have developed. We are also interested to determine if strains of Las will have any effect on the attractiveness of trees to D. citri. It has been reported that Las strains have varying levels of virulence and symptomatology (Tsai et al. 2008). We have analyzed DNA samples from HLB positive trees from Polk and Highlands counties as well as the ‘Smoke Grove’ CREC greenhouse strain by PCR and sequencing. Three putative strains of Ca. Liberibacter asiaticus (Las) were found with 5 (CREC greenhouse isolate), 13, and 15 tandem repeats of DNA in the LAPGP locus described by Chen et al. 2010 and have identified sources of budwood. We are currently using the identified budwood sources to acquire greenhouse isolates of the three strains while sequencing other loci including the b-operon, OMP (outer membrane protein) gene and phage DNA polymerase to support the differentiation of the three strains as has been reported in the literature (Bastianel et al. 2005; Lin et al. 2008; Okuda et al. 2005; Tomimura et al. 2009).
The objective of this project is 1) to complete the Las genome sequence and conduct comparative genomics studies on the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. A complete circular genome of Candidatus Liberibacter asiaticus was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have obtained a complete genome sequence of Ca. L. psyllaurous with ca.1.25Mb . We have also obtained a draft genome (approximately 70%) of Ca. L. americanus using multiple displacement amplification and 454 pyrosequencing technologies. A comparative genomics of these close-related bacteria revealed useful information for understanding their pathogenesis and evolutions. The information from our genome sequence allowed us to design new primers and probes that target various regions of the bacterial genome. Using these new primers and probes, genetic diversity of Candidatus Liberibacter asiaticus (Las) samples collected from Florida, Brazil, China, Philippines, Thailand, India and Japan can be found. The relationship between the diversity and disease phenotypes were partially correlated. A putative insect-transmission determinant gene was identified and the role of this gene is under investigation. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system. This data was published in J. Bacteriol. 192:834-840, 2010. We are currently developing an antibody-based “drug” to target this protein, aimed at disrupting ATP import, which may be important for its survival. We have also characterized the individual genes of two putative zinc operons in Las, with an overall aim of interfering with the ability of Las to regulate zinc uptake. Seed transmission of Las was tested in grapefruit, sweet orange, and trifoliate orange. Relatively high titers of Las were detected from both seed coats and inner seed coats collected from HLB-affected citrus plants. A very low titer of Las was detected from the embryos and seedlings using nested PCR and real-time PCR. Most, if not all the seedlings did not show typical HLB symptoms and contained a relatively low Las bacterial titer for HLB, even in the three to four year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves, which suggested “Detection of Candidatus Liberibacter asiaticus was NOT necessarily equal to the presence of “HLB disease” in plants.” A super sensitive qPCR detection technology has been developed, which increased the sensitivity of detection by 100-2000 fold, thereby eliminating the need for DNA isolation and increasing the throughput of the detection method. The cost savings can be up to 500%. Because the detection is based on HLB bacterium-specific primers, the detection data further confirmed our results on seed transmission and HLB disease phenotypes with low bacterial titers. The role(s) of the seed-transmitted Las is under investigation.
A scFv library with activity against ‘Ca. Liberibacter asiaticus’ has been prepared at Beltsville. mRNA was purified from mouse spleens and converted into cDNA. The mice had been immunized with psyllid extracts confirmed to be carrying a high concentration of “Ca. Liberibacter asiaticus” A complete library of variable heavy chain (VH) and variable light chain (VL) genes were made by PCR amplification of the cDNA using a set of 44 primers. The (VH) and (VL) gene segments were then joined in a random combinatorial fashion by overlap extension PCR. The scFv genes were then ligated into the pKM19 phagemid vector which was used to infect Escherichia coli DH5. F’ cells with the aide of a helper phage. The resulting phage library is presently being screened to select phage clones expressing antibodies that bind to “Ca. Liberibacter asiaticus”. Our first attempts to select desired antibodies using extracts from HLB-infected rough lemon were not successful, probably because the concentration of the target bacteria in the rough lemon extracts was too low. We have therefore modified the screening procedure by incorporating magnetic microbeads. These microbeads bind to rabbit antibodies. To use them we raised standard polyclonal antisera in a rabbit against the outer membrane protein of “Ca. Liberibacter asiaticus”. These beads are added to plant and insect extracts to bind the “Ca. Liberibacter asiaticus” and then concentrated by magnetic separation. The phage libraries are then added to the “Ca. Liberibacter asiaticus” on the beads and screened in that manner. This process is ongoing. We have also cloned genes encoding several proteins with potential direct relevance for the control of HLB. Correct cloning was confirmed by DNA sequencing and these genes have been expressed in E. coli and the encoded proteins are being purified. These proteins will also be used as targets to capture scFv antibodies that bind to them. The proteins cloned thus far for this purpose include a dinucleotide polyphosphate hydolase, a pilus component protein, a type IV pilus component protein, and a polysialic acid capsule expression protein. These proteins will be biotinylated, combined with strepavidin coated magnetic beads and used to bind phage expressing antibodies that recognize these targets. Thus we will finish with uncharacterized scFv antibodies binding “Ca. Liberibacter asiaticus” as well as a set of antibodies that bind to specific pathologically relevant proteins of the pathogen.
The objective of this study is to develop a series of flexible stochastic models to predict the temporal increase and spatial spread of diseases. The models were initially characterized for citrus canker but have subsequently been extended to HLB . The models can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies. Most attention has been given to spread within plantations, including allowance for proliferation of infection along boundaries in response to vector behavior but can readily be extended to consider spread at larger scales including spread State-wide. We also considered the effects of uncertainties in the distribution of host crops for example the effects of small areas of citrus that may not be recorded but which can act as ‘bridges’ in transmitting disease. The effects of uncertainty in parameter estimates for dispersal parameters and transmission rates have also been included. Additional computer-friendly formulations of the models have also been developed to aid in education of stake-holders to illustrate the effects of uncertainty in predicting future disease spread and the effectiveness of alternative methods of control. Estimation of parameters for dispersal of HLB poses considerable statistical challenges, especially where trees may become infectious before they are symptomatic/detected. Here we use an SEIDR model (Susceptible, Exposed (latently infected but not yet infectious), Infectious but not yet symptomatic/detected, Detected and infectious and Removed trees). Using MCMC methods, and extensive data from Southern Gardens (SG) for successive snapshots of the occurrence of symptomatic detected trees in known populations of susceptible trees, we are able to estimate model parameters for the transmission rates and dispersal kernel for the disease. Current work is focused on the differential effects of host age on epidemiological parameters as well as variability across the plantation. From these it is possible to allow for uncertainty in the parameters as well as variability over time and through space. We are using Baysian methods to infer posterior densities on the model parameters. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. A front-end, a web based version of the model that users can easily access and use is under final development has now been nearly finalized. Via this web tool, both residential and commercial citrus scenarios can be tested. A wide variety of epidemiological and climate/weather variables have been included and are user selectable and changeable via sliding controls. The simulator generates epidemics that progress through time and can be visualized on the screen. Various disease control/mitigation parameters can be selected and simulations can been visualized to see the effect of these various control strategies. This web tool runs simulations one at a time and is highly instructive to growers and regulators. It is based on a more formal analytical model that can run thousands of simulations based on the same parameters and make more statistically valid predictions for regulatory intervention strategy building and regulatory/industry decision making. Currently the spread of HLB is being modeled in 10 contiguous blocks in SG, chosen to encompass both young and mature trees, since host age is likely to be an important consideration for HLB. The blocks are also relatively isolated from the surrounding outbreak. The outbreak is noticeably clustered with only a small number of isolated cases. The model continues to be improved upon to capture more and more of the true features of the data and the disease. The model is being extended to estimate spread should HLB be introduced into new areas such as TX, CA or AZ.
This project is intended to examine the potential disease control of citrus huanglongbing (HLB) by interplanting with guava. In Vietnam guava has been shown to be an effective deterrent to HLB, slowing the disease and keeping plantings alive for up to 15 years that normally succumb in 2-3 year. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring ~1 year. Both nursery and field citrus trees are assayed for HLB every 60 days, and have been assayed multiple times. Psyllid populations are also being monitored continuously every two weeks within interplanted plots to document any repulsion of the vector due to guava. Data collection continues and is currently ongoing. Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus an unprotected nursery, have were established with disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) in June 2009 and were located in the protected and unprotected plots. The guava trees were were grown to appropriate size as indicated in Vietnam prior to outplanintg. **To date no HLB+ plants have been identified in the nursery plantings after multiple assays!** Citrus/guava interplantings: 3 commercial plantings with multiple replications were established. This required considerable time (1 year per Vietnam protocols) to grow the guava trees were propagated and grown to transplant size. One trial was established in a commercial orchard with collaborators in Southern Gardens Citrus. A second trial planting was established at the USHRL Picos Farm in Fort Pierce. A third was established in a Consolidated grove in Martin county but the plantation was sold and removed before data could be collected. Severe frosts during 2008/2009 and again during 2009/2010 winters affected both the USHRL and the Southern Gardens plots. This causing a delay in the experiment. Damage has been extensive in both plots, as guava is frost intolerant. In the Southern garden plots, damage was more severe. Damaged guava trees were pruned or replaced in that the Southern Gardens plot and plots were interplanted with citrus in November 2009. Renovation of the USHRL plot was less extensive, and the guava were interplanted with the citrus as of August 2009 in the USHRL Picos Farm plot. To date, several plants have been identified in the USHRL plots after multiple assays. The majority of these positives have occurred as the result of an edge effect. That is, treatment plots closest to an existing HLB+ planting of Valencia were affected. However this data is insufficient to draw any conclusions as yet. To date no HLB+ plants have been identified in the SG plantings after multiple assays.
The goal of this project is to obtain the in vitro culture of the bacteria – Ca. Liberibacter asiaticus (LAS)- associated with the Citrus greening syndrome. The strategy consists in getting primo-cultures of the bacteria in insect cells cultures used as feeder cells. We fulfilled objectives 1 by setting up conditions of LAS inoculation in insect cell cultures. Objective 2 consisted in obtaining primo-cultures of LAS. Among various insect cell cultures in various culture media tested, two drosophila cell lines (D-mel2, DS2) and a mosquito cell line (C6/36) were selected. Those cell lines show a detection of LAS after at least 3 transfers, the minimum number of transfers after which we consider them as primo-cultures. Detection: LAS presence is detected by direct PCR. We designed new primers to avoid non specific amplification that occurred with A2/J5 (LAS .-ribosomal protein operon) and OI1/OI2c (LAS 16S rDNA), commonly used LAS specific primers. Those non-specific detections tended to occur when working with high insect cell density or when a close bacterium was present (ie: Bradyrhizobium). We had similar difficulties to quantify LAS in insect cell cultures with previously described qPCR conditions. LAS bacteria could be detected by qPCR but using the primers HLBr/HLBas/HLB probe lead to non specific signals in our cell culture conditions. New primers were designed, giving more reproductible and reliable LAS detections in our cell cultures. We are currently adjusting qPCR conditions to our material. We periodically sequence the PCR fragments obtained to validate the presence of LAS. For objective 2, We continue to test new insect cell lines. The Keyhani’s lab recently sent us Diaphorina citri cell lines. As soon as the lines are maintained and stable, we will test our inoculation protocol on D. citri cells to obtain LAS/ D. citri co-cultures. Our objectives 3 and 4 are aiming to improve culture conditions to get a higher bacterial titer and to clear the insect cells from the primo-cultures (axenization). Objective 3: Axenization. Some of our LAS/Aedes or Drosophila primo-cultures were obtained with high insect cell concentrations. In order to axenize those primo-cultures, we tried to progressively dilute the concentration of insect cells through each new passage. However, we lost LAS detection due to faster growth rate of the insect cells, meaning by diluting the insect cells, we diluted even more the bacteria cells. We are currently testing new culture medium and conditions to contain the insect cell growth itself and we are analyzing new LAS inoculations in these cell cultures. Objective 4: Medium optimization. To maintain the bacteria for a longer period of time, over more transfers and to reach higher bacterial concentrations, we started complementing the primo-cultures with various sugars, vitamins described in citrus/periwinkle phloem. In parallel we analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to define limiting factors and/or growth inhibitors. We are monitoring several LAS positive co-cultures with different additive to improve LAS concentration and decrease the insect cell ratio. Some of these cultures are positive after more than 10 transfers are we are continuously refining our culture media with new additives.
This is the first progress report for the second year of this study. No experiments have been conducted yet for the second season as this work is on fresh citrus and those experiments will begin with the fresh season in the fall (Sept. and Oct.). To summarize results from the first season: Between Oct. 5th and 30th, five experiments were conducted that included Fallglo (1 time), sunburst (2 times), red grapefruit (4 times), and navel oranges (1 time). Fruit were treated on a commercial packingline (3 experiments) or on the Indian River Research and Education Center research line (2 experiments). Treatments included 1) full wash (brush bed + high-pressure wash) + waxing (carnauba), 2) full wash, 3) brush bed only, 4) brush bed with brushes rotating half normal speed, 5) high-pressure wash (HPW) only, 6) HPW for 10 seconds, 7) HPW for 5 seconds, 8) running fruit only over PVC rollers, and 9) a control (not washed or waxed). On the commercial line, fruit remained on the brush bed for ~ 1 min. 10 seconds, and on the full HPW for ~35 seconds. Normal brush rotation speed was ~100 rpm. Fruit were also evaluated for how surface dirt obscured the ability to grade the fruit for canker and other grade defects. Fruit from all treatments were degreened under simulated commercial conditions (5 ppm ethylene, 85F, 95% RH) and color development and weight loss measured almost daily. Fruit were subsequently stored and evaluated for the development of decay and disorders during storage under ambient conditions (~70-75F). In general, all very early season Fallglo fruit were relatively clean and did not need washing for adequate grading. This changed somewhat by the end of October when grapefruit that received more extensive washing (i.e., full washing) was significantly easier to grade compared to unwashed fruit. HPW produced fruit with intermediate gradeability. However, even minimally washed fruit were sufficient for adequate grading. These experiments will be repeated this season to determine variability in initial fruit cleanliness from year to year. Washing and waxing the fruit gave the greatest inhibition of degreening, almost stopping color development completely. Compared to preliminary results in 2008, results again showed that full washing of fruit on both the brush washer and HPW, or washing on the brush bed along inhibited degreening significantly more than did washing fruit only as they passed over the HPW. Fruit that were not brushed at all, but only passed over rollers experience a slight, but significant delay in color development compared to the control, but the delay was relatively minor compared to the other washing treatments. As the season progresses, fruit exterior surfaces become more soiled with dirt and sooty mold that makes grading more difficult without washing. Experiments were conducted in July 2010 using late-season red grapefruit, harvested from a block with citrus canker to test how well the different washing methods allowed graders to detect canker and other peel blemishes. Unwashed fruit were run past commercial graders and the number of fruit with any canker or surface defects that would justify elimination based on export grade standards were counted. These fruit were then taken to a commercial packinghouse and either left un-washed (control), or washed over 1) the entire line (brush + HPW), 2) the brush line only, or 3) HPW only. Fruit were then evaluated by commercial graders again. Data is still being fully analyzed from these experiments, but it appears that commercial graders were able to detect canker lesions and other surface defects from even unwashed fruit.
The goal of this proposal is to investigate whether Ca. Las is transmitted between infected and uninfected ACP adults in a sex-related manner to better understand the mechanisms by which disease is spread in field. We carried out a series of experiments to evaluate if Ca. Las is transmitted from male to female psyllids during routine mating. Our preliminary investigations indicated that Ca. Las may be transmitted from male to female psyllids but not from females to males or among psyllids of the same sex. Pairs of Ca. Las infected male and healthy female, Ca. Las infected female and healthy male, Ca. Las infected female and healthy female, Ca. Las infected male and healthy male, Ca. Las infected female and Ca. Las infected male (positive control) healthy female and healthy male (negative control) adult psyllids were introduced separately in Petri dishes filled with agar medium. The insects were allowed to mate for 72 hrs. After 72 hrs, the insects were transferred to Ca. Las resistant Murraya koenigii plants for 12-14 days for multiplication of bacteria in recipient psyllids. DNA was prepared from each of the female and male psyllids separately and analyzed for Ca. Las presence utilizing a real time PCR assays. Our results indicated that Ca. Las was transmitted from Ca. Las infected male psyllids to roughly 15% of healthy females. Ca. Las was transmitted from Ca. Las infected males to healthy females but not from Ca. Las infected females to healthy males or among insects of the same sex. The females that acquired Ca. Las from males during mating were also able to transmit the bacteria transovarially. We were unable to detect transmission of Ca. Las when the recipient insects were analyzed for Ca. Las DNA after 72 hrs of mating. Similarly, no Ca. Las was detected in recipient insects when the recipient insects were maintained on M. koenigii plants for longer than 4 weeks. Currently, we are investigating for the presence of bacteria in genital organs of male and female psyllids with scanning and transmission electron microscopy to characterize the pathway of Ca. Las transmission from male to female psyllids. We also continue to evaluate if the Ca. Las recipient females are capable of infecting new citrus plants and to quantify the HLB transmission rate through sexual transmission.
DNA bar coding was initiated by developing and optimizing a pair of primers that amplify 1500 bp of the mitochondria COXI psyllid gene. A nested primer pair was designed to be specific to the Asian citrus COXI gene, and validated. FL field collections were obtained from cooperators that represent 2006 and onward. PCR amplification and sequencing of field collections is underway. We optimized qPCR to detect Ca. Liberibacter presence (or absence) in the psyllid colony cohorts over different AAPs, for immatures and adults (Roberts, SWFREC; Brown lab UA). Ca. Liberibacter-infected Asian citrus psyllids are now routinely maintained at SWFREC, and the potato psyllid study system is in place in AZ to explore as a more tractable system for optimizing parameters under study in Obj 3, 4 e.g. localization and time course feeding for FISH, SEM/TEM. Our FISH probes against Liberibacter support our earlier suggestion that the pathogen exists in multiple locations in the abdomen of D. citri. Distinguishing them from a potentially broader, extra-bacteriosome flora requires the bringing together of numerous visualization techniques. After fixing specimens in 2 different ways, coagulative and noncoagulative, 4′ paraffin through-sections of the same insect are mounted for three different techniques to give the best bouquet of results ‘ ISH/FISH, haematoxylin/eosin and SEM. ISH indicates areas of infection, H&E helps with anatomical interpretation, and SEM shows gross bacterial morphology as well as any possible mix of flora. These techniques are compared with SEM of exposed organs and/or extirpated organs of all life cycle stages, including the pharate adult and the teneral. These results are compared with ISH using a 10nm colloidal gold-labeled probe and TEM. TEM allows for ultrastructure of labeled bacteria. SEM of extirpated organs of infected potato psyllid has shown that one component of the flora multiplies inside and outside the gut of young adults to the extent that it overwhelms old adults and completely replaces epithelial cytoplasm. Such a scavenging species has not been found yet in uninfected psyllids. First views of the external salivary gland surface from these old adults do not show such tissue scavenging. The condition of the salivary gland internally is being investigated using SEM. The oral box, 0.06mm3 in size, delimited by the tentoria and their arms, is an extremely complex conglomerate of esophageal, pharyngeal, salivary ducal and stylet tissues that converge to allow passage of ingested, egested and secreted materials. Because of the potential ability of bacteria to proliferate in the hemocoel, and burrow through epithelia, this box has been given considerable attention, along with other, well defined, models of transmission through the gut and salivary glands, taking the study of their organization well beyond classical groundwork laid down using other model Hemiptera.
Objective: Determine if Carrizo rootstocks, either wild type or over-expressing the Arabidopsis NPR1 gene (with an enhanced, inducible defense response) have any effect on gene expression and/or the defense response of wild type (non transgenic) grapefruit scions to HLB. Some transgenic ‘Carrizo’ citrange lines (lines 854, 857, 859 and 884) transformed with the AtNPR1 were produced in Year 1 of this project. In this quarter we were able to start to propagate new transgenic lines from cuttings: 757, 761, 763, 775, 854, 857, 890, 896 and 897, all transformed with the AtNPR1 (the plants were now large enough to propagate). We have also identified sequences for several additional citrus genes that are associated with SAR, including AZI1, BLI, CHI, R13032, R20540, RAR1 and SGT1. These genes were preciously undescribed for citrus, however our microarray studies indicated that these sequences were differentially regulated by chemical and pathogen treatment. R13032 and R20540 belong to the NPR1/NPR3 family of genes in citrus and our experiments show they are all differentially expressed during SAR. Objective 1 of this project proposed to compare the response of AtNPR1 transgenic plants vs. wild type plants to the treatment of the SAR inducer salicylic acid (SA). This has been done with the first set of transgenic lines but we wish to repeat the experiment when the new plants have been propagated so we have more replications.
Objectives of this project include 1) refinement of sampling methods, 2) testing the influence of adult density and shoot infestation on precision of estimated means and distribution of population within blocks, and 3) evaluation and integration of methods for assessing psyllid density, shoot density, and infestation rates into a user friendly system accessible to consultants and managers. Accomplishments and plans for further studies are described for each objective. 1) Data from an experiment conducted at a commercial grove showed that although sticky traps caught more adults over a two week period compared to one time tap sample at the end of that period conducted at both low and high psyllid densities, they were not only expensive but consumed more time and provided data after at least a 1-week delay compared to a tap sample that provided instant data with no more supplies than a laminated sheet of letter size paper or a smooth white surface such as a clipboard, and a one-foot piece of PVC tube. We have collected more data on comparison of these methods which is being entered and analyzed. Our commercial collaborators are now using tap sampling method and shoot examination to assess ACP populations in their groves and providing us data that is being entered for analysis. Comparison of the tap sampling and sweep net sampling methods at the SWFREC orchard showed that at low density tap method was more efficient in detecting ACP than sweep net although no significant difference was observed in numbers detected and sampling time at high density. However, density averaged less than 0.5 adults per sample with either method. Therefore, we have planned to conduct this comparison at a commercial grove where ACP density is much higher and maintained at two levels (low and high) through use of insecticides and untreated control. 2) Regression analysis using bootstrapping procedure indicated that the precision targets of 0.25 and 0.01 SEM:mean were reached at high psyllid density with 8 and 66 tap-samples, respectively, compared to 12 and 75 YCRW traps and 13 and 74 ACP traps. At low psyllid density, 267 and 1929 tap-samples, 16 and 130 YCRW traps, and 43 and 219 ACP traps were needed to attain these precision ratios, respectively. At low density 100 and 571 tap-samples and 160 and 1295 sweep net samples were needed at ratios of 0.25 and 0.1, respectively, compared to 35 and 196 tap samples and 37 and 198 sweep net samples at high density. Therefore, the sweep net would take more time to provide the same data and more work and difficulty in counting at high density. As indicated above we have included additional locations to conduct these comparisons at density above the one reported here. 3) A rapid system that includes tap samples, flush inspections and natural enemy evaluations along with forms and spreadsheets is available on our website swfrec.ifas.ufl.edu/entlab/. Just during this quarter 400 tap sampling kits were distributed among growers. Five presentations and 5 workshops on psyllid monitoring were conducted at SWFREC to train 250 attendants. A survey of 27 growers in SW Florida conducted by SWFREC and IFAS extension in 2009 covered 106,148 acres of commercial citrus (80% of productive area) reported that among 96% conducting sampling, 85% use the tap method, along with shoot examination (56%), sticky traps (7%), and sweep nets (4%). We are starting another survey to collect information for 2010. We are also working on the development of a website where growers will be able to enter data on the incidence of pest and disease in order to assess the need and recommendation of appropriate treatments. Findings from the above studies were published in citrus industry and being prepared for publication in a peer reviewed journal.
Objective 1 is to conduct a field evaluation nutritional sprays for control of HLB and HLB symptom expression and yield. The field study was set up May 2010 in Southern Grove, Hendry Co., FL. Six treatments were set in 4 plots of 150 trees per treatment (interior 10 trees in each block were identified for PCR, leaf nutrition sampling, tree health and yield evaluation). Treatments were 1) non-treated check; 2) Nutri-Phite sprayed 4 times bimonthly; 3) N-Sure sprayed bimonthly; 4) Agra Sol Mn/Zn/Fe plus Nutri-Phite plus triazone urea sprayed bimonthly; 5) Keyplex 1400 DP plus Nutriphite plus triazone urea sprayed bimonthly; 6) Wettable powder nutrients (Diamond R #2) plus Nutri-Phite P+K sprayed bimonthly. The materials were applied to both sides of the tree in 125 gallons per acre with an airblast sprayer driven at 2 mph to obtain thorough coverage. Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application, Hamlin sweet orange trees have been acquired and treated with two rounds of nutritional sprays and leaf samples have been taken for baseline nutrient content of the different treatments. Graft inoculation with HLB infected budwood was performed on half of the trees in each nutritional treatment group. Monitoring of macroscopic and microscopic symptom development is underway. Monitoring of Las titer will begin when one month has passed since inoculation.
Reducing excessive vegetative growth that is produced annually by citrus trees in Florida would reduce the opportunities for Asian citrus psyllid (Diaphorina citri Kuwayama) reproduction and thereby, the spread of Huanglongbing. Excess tree growth is routinely removed through hedging and branch re-growth can be reduced after hedging in the fall season under Florida conditions because of the onset of cool temperatures. Additionally, late-summer hedging may synchronize a final late-season flush and thus, reduce new flush leaves present during the winter to support over-wintering psyllids. We determined timing effects of fall and early winter hedging of ‘Hamlin’ orange trees on vegetative growth flush and subsequent yield during a two year period (2008-2010). None of the fall/winter (Oct – Jan) hedging times tested stimulated a growth flush in either year. Yield, in terms of fresh fruit weight, was only affected by late-Nov and early-Dec hedging. This suggests that although earlier hedging times reduced the number of fruit harvested, there was sufficient time left before harvest for the remaining fruit to increase in mass, thus compensating for the pieces of fruit removed by hedging. We also tested the effects of summer hedging times (late-Aug – Sept) on yield and flush management. Similar to early fall hedging, summer hedging reduced the number of fruit harvested, but not the total weight of fruit harvested. Summer hedging times did stimulate a uniform growth flush approximately 2 weeks after hedging on which psyllid control was needed. We have concluded that hedging practices can be modified to affect flushing patterns in a way that benefits ACP control strategies. However, because of environmental effects on flushing that are poorly understood in citrus the hedging effect cannot be consistently reproduced year-to-year and is likely to be adapted by growers. These data have been published in the proceedings of the Florida State Horticultural Society (Spann et al., 2009, Proc Fla State Hort Soc 122:161-165) and are being prepared for publication in a refereed journal. We hypothesized that plant growth regulators (PGRs) could be used to reduce vegetative growth of citrus, thus reducing ACP populations. Seedlings of Volkamer lemon were treated with six commercially available PGRs (10 replications each): Embark, Sumagic, Atrimmec, Apogee, Profile and Cycocel. Untreated seedlings served as controls. Ten days after PGR application, a pair of ACP was caged on five seedlings of each treatment. The five seedlings from each treatment without ACP were used for vegetative growth measurements. All seedlings were maintained in a greenhouse at 28 C and 70% relative humidity. Over the course of fifteen days, the number of eggs laid on each plant was counted daily. Survival of ACP nymphs to adults was assessed. Adults were collected and weighed to determine adult body weight. Due to severe phytotoxicity no data were collected from Atrimmec-treated plants. Only Sumagic and Apogee significantly reduced total shoot growth. The average number of eggs laid per plant was reduced by 85% on Embark and Sumagic-treated plants, and by 65% on Apogee and Profile-treated plants. In contrast, the number of eggs laid increased by almost 40% on Cycocel-treated plants. Survival of nymphs to adults was also significantly affected by PGR treatment. Survival was lowest for plants treated with Profile (7%), followed by Sumagic (36%), untreated control plants (45%) and Apogee (47%). The highest survival rates were observed on Cycocel (72%) and Embark (68%) treated plants. ACP adults that matured on seedlings treated with Sumagic weighed significantly less than those from Embark, Apogee, Profile and control plants, but were similar in weight to those from Cycocel-treated plants. These data indicate that the PGRs tested significantly effected the overall fitness of the ACP; however, contrary to our hypothesis, these effects were observed even in the absence of significant growth reduction. This indicates that the PGRs are affecting ACP fitness through plant biochemical changes, direct toxicity or some other undetermined mechanism. Results were presented at the Plant Growth Regulation Society of America (Aug 2009), the Entomological Society of America (Dec 2009) and the Western Plant Growth Regulator Society (Jan 2010). A manuscript based on the results is being prepared for submission to a scientific journal. Continuing research on this project will investigate the plant biochemical changes induced by PGR applications that are affecting psyllid fitness, field trials to replicate greenhouse results with native ACP populations, and duration of efficacy studies in both greenhouse and field trials.
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