Under Objective 1 (Survey and confirmation of HLB in seedlings from HLB-affected trees) 200 seeds extracted from mild to severely affected fruit from HLB-affected Pineapple orange and Murcott tangor groves were assayed for HLB detection in emergent seedlings. All seedlings were negative for HLB by PCR assay. Assay of the same seedlings will be repeated. Under Objective 2 (Thermotherapy of HLB-affected seed) 100 seeds of the same seed sources were treated at 125, 130 and 135F to test for the effect of heat treatment on detection of HLB in seedlings. Thermal treatments were lethal to Murcott seed, but not Pineapple seed. The emergent seedlings were PCR tested and all were found to be negative. Under Objective 3 (Follow-up on HLB-positive trees from DPI annual survey), DPI survey of seed source trees in Florida nurseries is conducted every other year (R. Gaskalla, DPI Director, personal communication), so samples from the survey, if any, will not be available for testing until the second year of the project.
Since the last update, we focused on characterizing callose accumulation in the Liberibacter-infected phloem tissues. First, we localized callose in the infected phloem tissues by immunogold labeling. The main purpose of the experiment was to verify whether callose over-accumulation is responsible for the swelling of the plasmodesmata pore units (PPUs). We utilized a monoclonal callose-specific antibody from Dr. Bacic. This antibody has been the favorite callose antibody in the plant cell wall community during the last two decades thanks to its specificity and low background (Meikle et al., 1991). The swollen PPUs were intensely labeled by callose-specific immunogold particles indicating an excessive callose buildup. In addition to the PPUs, immunogold particles were associated with plasmodesmata between the sieve elements and with sieve pores. An interesting observation is that the inner surface of the sieve elements was covered with callose. In uninfected samples, callose was detected only in the plasmodesmata and sieve plates where callose is naturally deposited. However, numbers of associated gold particles were far smaller (less than 30%) than those in the infected samples. Callose accumulation was not seen in non-phloem cells such as mesophyll sponge cells and parenchyma cells in both infected and uninfected samples. These results indicate that sieve element cells synthesize callose in response to Liberibacter infection and the callose often swells plasmodesmata of the cells. Recently it was reported that sieve plate pores are plugged by callose in Liberibacter infected sieve elements (Kim et al., 2009). The sieve pore plugging is the first wound reaction against phloem damages, so that plants do not lose their precious photosynthetate (Ehlers et al., 2000). We examined sieve plates from infected and uninfected mid-vein samples by scanning electron microscopy (Mullendore and Knoblauch., 2009). The advantage of this microscopy technique over TEM is that cell wall structures including sieve plates, plasmodesmata, and callose are seen in three-dimension, facilitating comparison sieve pore size and callose accumulation in the phloem. In the face-on views of sieve plates, diameters of sieve pores in the infected samples were smaller than those of uninfected sieve plate pores. However, we have not found any sieve pores that are completely blocked by callose in the infected phloem samples. The pore sizes in the infected sieve plates were still larger than the width of regular Liberibacter cells. These data suggest that callose synthesis in the sieve plates is not efficient enough for completely sequestering Liberibacters to their infection sites. For the next quarter, we will investigate callose distribution by fluorescence microscopy to combine with what we have learned from high resolution electron microscopy imaging. We will also finish 3D reconstruction of Liberibacter cells and plasmodesmata deformed by callose from serial sections of citrus sieve element samples. (We have collected 3 serial sections from citrus sieve element cells.) Ehlers, K., Knoblauch, M., and van Bel, A.J.E. (2000). Ultrastructural features of well-preserved and injured sieve elements: minute clamps keep the phloem transport conduits free for mass flow. Protoplasma 214, 80-92. Kim, J.S., Sagaram, U.S., Burns, J.K., Li, J.L., and Wang, N. (2009). Response of sweet orange (Citrus sinensis) to ‘Candidatus Liberibacter asiaticus’ infection: microscopy and microarray analyses. Phytopathology 99, 50-57. Meikle, P.J., Bonig, I., Hoogenraad, N.J., Clarke, A.E., and Stone, B.A. (1991). The Location of (1-]3)-Beta-Glucans in the Walls of Pollen Tubes of Nicotiana-Alata Using a (1-]3)-Beta-Glucan-Specific Monoclonal-Antibody. Planta 185, 1-8. Mullendore, D. L. and Knoblauch, M. (2009). A new method to investigate the cell wall of living cells by high-resolution scanning electron microscopy. Annual Meeting of American Society of Plant Biologists Abstract number: P14001.
The goal of this study is to introduce broad resistance in citrus against HLB and other diseases via manipulating salicylic acid (SA)-mediated defense signaling. Specifically, we proposed to identify SA-related genes in citrus and manipulate expression of these genes genetically to elevate SA levels and/or signaling in citrus and subsequently test the transgenic citrus plants for resistance against HLB. Towards this goal, we have three specific objectives and have made the following progresses since last report. Objective 1: Identify genes positively regulating SA-mediated defense in citrus In last report, we described the identification of citrus SA homologs via bioinformatics analysis and the cloning of the citrus NPR1 (ctNPR1) gene. Now we have cloned three additional full-length citrus SA genes, ctEDS5, ctNDR1 and ctPAD4, using combination of RT-PCR and 5Õ and 3Õ RACE approaches. These three new SA genes were cloned in the pGEM vector. To provide a more detailed analysis of expression of citrus SA regulators, we infected eight-month-old trees with ÔValenciaÕ orange (Citrus sinensis (L.) Osbeck) scion on Cleopatra mandarin (C. reticulata Blanco) rootstock with Ca. L. asiaticus and are in the process of collecting tissue for RNA extraction followed by RT-PCR. At five-week post inoculation, 14 out of 30 inoculated plants were PCR-positive for Ca. L. asiaticus but no plant showed any leaf symptoms of HLB. At 11-week post inoculation, 24 out of 30 inoculated plants were PCR-positive for Ca. L. asiaticus, among which 17 plants displayed disease symptoms. Control plants were always PCR-negative and did not display any leaf symptoms. Additional time point, about 14-week post inoculation, will be used to collect leaf tissue from these plants. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues We cloned ctNPR1 in the binary vector pBINplus/ARS and already transformed Arabidopsis mutant npr1-1 and the wild type control Columbia with the ctNPR1/pBINplus/ARS construct. The T0 seeds will be harvested in about a month followed by selecting transgenic plants and possibly also testing disease resistance in the next generation. In addition, we are currently in the process of cloning ctEDS5, ctNDR1 and ctPAD4 into pBINplus/ARS for a complementation test of Arabidopsis eds5-1, ndr1-1, and pad4-1 mutants, respectively. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in citrus While we are waiting for the result of a complementation test for ctNPR1 in Arabidopsis, we have also placed the ctNPR1/pBINplus/ARS construct in the pipeline of transforming citrus.
The new spectrophotometer, a Nanodrop 8000, has just arrived. The software will be mastered and the task of adjusting the nucleic acid content of samples prior to running qPCR will begin. This is very important to the testing component of providing clean Shoot-tip grafts of new cultivars. Samples vary in nucleic acid content. Too much nucleic acids can cause inhibition, whereas too little can reduce the chance of detecting the pathogen. The greatest differences are in dry field trees compared to young tender greenhouse tissue. The first of the two microscopes, a Leica teaching microscope arrived, but during installation, it was discovered that the light source had a European plug, the light source did not fit on one of the objectives and neither objective could fit the body of the microscope (which was a newer model than the objectives). These errors should be rectified within two weeks. Meanwhile, we now are sure of the dimensions of this large microscope and plans are under way to purchase a hood to use the microscope in instead of just using it on a table. Teaching will take place in a sterile environment and the shoot-tip grafts (STGs) produced will be kept and evaluated so that the success rate of the work done can be determined. With an additional microscope and hood space, the STGing capability could also be increased when needed. During this quarter, the number of selections being cleaned up is down to 93. Four new selections have been added to the list and 22 selections were released. Four hundred seventy six STGs were set up. These represented 12 varieties including two rootstocks, two breederÕs selections, two private growersÕ selections, 2 difficult varieties, two with mild isolates of Citrus tristeza virus (CTV), and two with severe isolates of CTV. During this same time period, 36 successful STGs were grafted onto rootstocks in the greenhouse. These represented 16 varieties (which included two rootstocks, four breederÕs selections, three private growersÕ selections, two difficult varieties, two Florida Citrus Arboretum varieties, one which had a mild isolate of CTV and two which had severe isolates of CTV). For testing, over 58 STGs and parents samples were extracted and 252 real-time PCR tests were performed on STGs that grew to sufficient size in the greenhouse. One hundred forty trees were budded for increase from tested original STGs to either be planted in the Citrus Budwood Foundation at Chiefland and/or to be given back to the breeder/owner. Eighty-nine propagations were planted at Chiefland, 85 of these were breedersÕ selections. The Citrus tatter leaf virus SYBR green real-time PCR assay has been found to be robust and has been incorporated into the comprehensive qPCR testing for STGs and parents.
The two primary objectives of the current proposal are: (1) determine and compare the metagenomes of healthy and infected citrus phloem, and (2) continue to develop and apply AthenaBio’s cultivation technologies towards producing a pure culture of Ca. Liberibacter. Objective 1 Discussion. Laser cutting microdissection (LCM) will be developed to purify phloem-containing sieve cells from both healthy and infected citrus samples. DNA extracted from the purified cells will be analyzed by 16S rRNA sequencing to evaluate the contained microbial diversity. The most diverse DNA samples will be selected for large-scale metagenomic analysis. All DNA extraction and sequencing work will be performed by Dr. Ravel’s group (U. of Maryland) Objective 2 Discussion. AthenaBio will continue to develop and apply its cultivation technologies to isolate a pure cultures of Ca. L. species. In addition to Ca. Liberibacter species, we aim to cultivate other phloem endophytes to provide reference genomes for metagenomic analysis. Two approaches will be employed to disrupt quorum sensing (QS) and thereby enhance growth: (1) physical methods (i.e. a ‘flow cell’) will be used to wash QS signals away from cells, and (2) enzymes will be used to inactivate QS signals through chemical modification. The advantage of the flow cell approach is that knowledge of the chemical structure of the QS signal is not required, and this approach is therefore expected to work on a broad variety of QS systems. Both approaches (flow cell and enzyme) will be combined with various media formulations and incubation conditions. Before work could begin, a research agreement was formulated between the FCPRAC and Athena Biotechnologies, Inc. This agreement was finalized in May 2009 and work has begun to develop a method for LCM of citrus phloem cells. Healthy leaf tissue was dissected into small pieces (~5mm), immediately placed in tissue freezing medium and placed on dry ice. Sample sections were achieved using a Leica CM3050 S Cryostat and sections were cut using a Laser Capture Microdissection Microscope (LCM). We are currently optimizing section thickness and staining procedures to obtain ideal conditions for LCM processing. Once an optimum protocol has been developed, we will begin processing samples for DNA analysis.
Differential mobility spectrometer sensor for HLB-biomarker detection: Sampling protocol to collect volatile organic compounds (VOCs) from citrus leaves in open-air environment (field conditions) and analysis protocol (for GC-MS and differential mobility spectrometer-GC/MS and GC/DMS) have been developed jointly in Dr. Davis’s lab, UC, Davis and Dr. Rouseff’s lab ,UF, CREC . Sampling and analytical protocols have been exchanged between the two labs. A special positioning devise has been developed to hold the solid phase microextraction fiber for field volatile sampling. Optimal sampling time of day has been determined. Two different fiber types have been evaluated and major leaf volatiles identified. The major leaf volatiles have been identified as terpenes, aldehydes and alcohols which have been previously reported in citrus. In one sample set, the GC/MS chromatograms showed certain distinguishable patterns between healthy and HLB samples. An auto-regression model with an order of 100 was applied to extract features (auto-regression coefficients) from the chromatogram in Dr. Davis’s lab. The algorithm was able to classify the healthy from HLB-infected samples, and asymptomatic-HLB from symptomatic-HLB infected samples. In addition to these tools, other sophisticated machine learning and feature extraction (biomarker detection) approaches such as wavelet analysis, genetic algorithms, and ant colony algorithms have been developed. Portable fluorescence spectrometry for HLB detection: A laser-induced fluorescence spectrometer system was developed by Embrapa Agricultural Instrumentation for diagnosis of Brazilian citrus disease. Some preliminary experiments were conducted using the biophotonic system in the laboratory to classify healthy citrus leaves (from Valencia grafted on Swingle rootstock) from that of HLB infected leaves. The study was carried-out to calibrate the instrument. Preliminary analysis showed that the system was able to classify HLB-infected leaves with 90% classification accuracy, while healthy leaves were classified with an accuracy of 70%. The results were promising. A partnership has been set-up with Fisher Group (one of largest Brazilian producers of oranges and juice) for data collection in Citricola Farm. Hundred trees in two blocks of 1000 trees (one with low and high HLB incidence each) will be monitored using the biophotonic system each month, with data collection on weather, geo-reference, and PCR results. Data collection is expected to start on November, lasting upto a year. Hyperspectral sensors for HLB-infected trees detection: Different imaging sites having various degrees of HLB infection have been evaluated for HLB detection through aerial hyperspectral imaging. The sites selected were from citrus groves in Southweat Florida Research and Education Center (SWFREC) in Immokalee, Consolidates Citrus, and Evans Property. The hyperspectral imagery will be collected from November, 2009 with the help from Dr. Yang and imaging group at US Department of Agriculture-Agricultural Research Service, Weslaco, Texas. A photographic camera with high resolution, a multispectral sensor, and hyperspectral sensor will be used to collect the data. Greenhouse experiments are being conducted at Citrus Research and Education Center, Lake Alfred, FL to evaluate the performances of the spectroradiometer and hyperspectral imaging based sensors for their ability to detect HLB infected trees in their preliminary stages. The data collection is ongoing since last year. Analysis protocols are being developed to evaluate the classification accuracies of the sensors. Preliminary results indicated the requirement of large dataset to classify HLB-infected trees with high accuracies. Further data will be collected to evaluate the applicability of the sensor techniques.
The objectives of this project were to evaluate the effects on flush management strategies on the population densities of the Asian citrus psyllid (ACP; Diaphorina citri Kuwayama). Two hedging dates (one early in mid February and one late in mid April) and two nitrogen fertilization regimes (one application of 100 lb/acre in February and two applications of 50 lb/ac each in mid February and in mid June) were tested in a factorial design in a mature ÔMarrsÕ sweet orange block near Weslaco, South Texas. Non-hedged and non-fertilized plots served as controls for each treatment factor. Weekly counts of new flush shoot growth and ACP densities were made. Both hedging dates and application of nitrogen significantly affected ACP infestation and densities on citrus flushes. Hedging significantly altered the phenology and intensity of new flush shoot production on trees. In the non-hedged blocks, the spring flush was only followed by a very light flush shoot production in early June. In contrast, both the early and late hedging dates of trees stimulated profuse flush shoot production in June. In addition, in the late hedging treatment of April, new flush growth started in early May and lasted until late June. This alteration in flush shoot availability also impacted the population dynamics of ACP. Significantly higher ACP infestation levels and densities were recorded in the late hedging date compared to the other treatments. Although more new flush shoots were produced in the early hedging treatment relative to the non-hedged treatment, ACP populations were comparable in these two treatments. These results clearly demonstrate that early hedging should be encouraged as it prevents severe outbreaks of ACP populations, while providing the intended physiological benefit of the practice. By contrast, late pruning in spring i.e., during the active ACP growth period, will likely lead to ACP outbreaks in citrus orchards. Application of nitrogen also affected the abundance of new flush growth. Although no alteration of flush cycles resulted from N application, densities of new flush growth were higher in fertilized plots than in the non-fertilized control blocks. The effect was more dramatic in blocks where N was applied in a single dose (100 lb N/ac) compared to blocks receiving split N applications. In the one-time N application treatments, significantly more new flush shoots were produced which resulted in higher densities of ACP eggs and nymphs for most of the sampling dates. ACP densities in the split application and non-fertilized control were similar throughout the sampling period. The interaction of N fertilization and hedging dates was significant for ACP immatures, but not for ACP adults. This indicates that changes in flush shoot production can directly affect the densities of ACP eggs and nymphs. Late pruning, combined with the one-time N application treatment resulted in some of the highest ACP population counts recorded. In summary, N management, and in particular, split N fertilization and early hedging/pruning of trees were associated with lower ACP population densities on sweet orange trees. These preliminary findings from ongoing studies have provided valuable information that orchard managers can use to implement strategies to lower the risk of ACP outbreaks in citrus orchards.
Below is the abstract of a paper recently accepted for publication in Molecular Plant-Microbe Interactions. Title: “Confirmation of the sequence of “Candidatus Liberibacter asiaticus” in citrus and assessment of microbial diversity in Huanglongbing-infected citrus phloem using a PCR-independent, metagenomic approach”. Authors: Heather L. Tyler, Luiz F.W. Roesch, Siddarame Gowda, William O. Dawson, and Eric W. Triplett The citrus disease, Huanglongbing (HLB), is highly destructive in many citrus-growing regions of the world. The putative causal agent of this disease, Candidatus Liberibacter asiaticus, is difficult to culture and Koch’s postulates have not yet been fulfilled. As a result, efforts have focused on obtaining the genome sequence of Ca. L. asiaticus in order to give insight on the physiology of this organism. In this work, three next generation high-throughput sequencing platforms, 454, Solexa, and SOLiD, were used to obtain metagenomic DNA sequences from phloem tissue of Florida citrus trees infected with HLB. A culture independent, PCR-independent analysis of 16S rRNA sequences showed the only bacterium present within the phloem metagenome was Ca L. asiaticus. No viral or viroid sequences were identified within the metagenome. By reference assembly, the phloem metagenome contained sequences that provided 26-fold coverage of the Ca L. asiaticus contigs in GenBank. By the same approach, phloem metagenomic data yielded less than 0.2-fold coverage of five other alphaproteobacterial genomes. Thus, phloem metagenomic DNA provided a PCR-independent means of verifying the presence of Ca. L. asiaticus in infected tissue and strongly suggest no other disease agent was present in phloem. Analysis of these metagenomic data suggest that this approach has a detection limit of one Liberibacter cell for every 52 phloem cells. The phloem sample sequenced here is estimated to have contained 1.7 Liberibacter cells per phloem cell. In addition to the manuscript abstract above, we have begun the PCMAT work described our proposal. We have extracted protein from infected and uninfected phloem tissue for the purpose of making antibodies. We expect to have the antibodies delivered to us in a few weeks. We are also training a new student in the nanosensor approach described in the proposal.
Trials have been established in mature citrus groves located south of Lake Placid and east of Arcadia to evaluate the impact that delaying the treatment of stumps with Remedy, where trees were removed due to greening, will have on sprout formation. Remedy, an approved herbicide for citrus stump treatment, is being applied at 25% or 50% v/v mixed with diesel fuel and applied to the stump at 4 different time intervals. Each treatment will be evaluated and compared to the untreated control as to how effective the treatments are on controlling sprouts from citrus stumps. Each trial consists of 9 treatments replicated 7 times. Treatments are: 1) untreated control; 2) treatment at time of clipping with a solution of 25% Remedy and 75% diesel fuel; 3) treatment at time of clipping with a solution of 50% Remedy and 50% diesel fuel; 4) delaying the application by 24 hours with a solution of 25% Remedy and 75% diesel fuel; 5) delaying the application by 24 hours with a solution of 50% Remedy and 50% diesel fuel; 6) delaying the application by 48 hours with a solution of 25% Remedy and 75% diesel fuel; 7) delaying the application by 48 hours with a solution of 50% Remedy and 50% diesel fuel; 8) delaying the application by 72 hours with a solution of 25% Remedy and 75% diesel fuel; 9) delaying the application by 72 hours with a solution of 50% Remedy and 50% diesel fuel. Each trial will be evaluated at 30-day intervals for sprout formation from citrus stumps that remain after tree removal by clipping the tree off above the soil line. All removed trees were surveyed by greening scouts prior to removal and determined to visually have greening. Once sprouts are observed, the entire stump will be placed in an enclosure and allowed to continue to grow. The enclosed structure will be made of the same material that is currently approved for citrus nursery greenhouses. At approximately 120-days after treatment, citrus sprouts from stumps will be tested for the presence of HLB by PCR testing methods.
During the second quarter of 2009, numerous activities have been conducted by the citrus extension agents to enhance grower knowledge of greening management practices via a number of venues. In April, the Greening Symposium was conducted in Bartow whereby 354 individuals registered to participate in the day-long educational event. The program discussed multiple aspects of greening and its management. All oral presentations from the event are currently available as well as most presentations being in a printed version and available at: http://citrusagents.ifas.ufl.edu/events/Citrus_Greening_Symposium_2009/Videos/CitrusGreeningSymposium2009.htm . Since the April symposium, over 5,350 hits have been received relating to the symposium presentations. This agents’ web site also has posted the presentations from the Greening Summit in April 2008 for viewing. These presentations are a valuable resource to aid in greening education. The extension agents’ web site posts numerous other resources, including citrus agents’ newsletters and links to enhance knowledge and resource availability. During the quarter: 38,613 down loads were for newsletters and over 96,000 hits for the site to obtain information. The agents have been active in conducting and presenting data obtained via various field trials. Agents made three presentations at Florida State Horticultural Society Annual Meeting (June 2009) and are as follows: Determining Greening Infection Levels Using Multiple Survey Methods in Florida Citrus; Detection of Greening in Sprouts from Citrus Tree Stumps; and The Effect of K-phite and Salicylic Acid on Container Grown Citrus Graft Inoculated with HLB Budwood. Information from these studies will be presented at other grower meetings as well as in various formats in the near future. Grove visits have been conducted to assist growers with greening management in all parts of the state and exceeded 70 during this quarter. As a result of the visits, agents have submitted 24 samples to various labs for analysis. Current trials and studies are also being conducted and include: a) assessment of psyllid numbers using sticky traps; b) methods of plant growth regulation; c) HLB impact on fruit size; and d) various aspects of low volume applications on control of psyllids. Additional ‘county’ or local meetings have been conducted to enhance grower knowledge and have included: HLB/Canker training; scouting for greening; and low volume application. These program activities have involved more than 370 individuals. A citrus grower tour has been conducted whereby growers were provided the opportunity to visit Brazil. This tour was conducted in May 2009. These annual events now represent the management of citrus companies exceeding 40% of the industry as well as representatives from industry publication and trade groups.
The Ca. Liberiacter asiaticus is the most prevalent species of Liberibacter associated to HLB in the American continent. In the current project we aim to develop serological and molecular techniques for diagnosis of Lib. asiaticus. The serological approach is based on heterologue expression of Outer Membrane Protein (OMP) gene in E. coli, its purification, and injection into rabbit for antibody production followed by purification. The molecular approach is the development of nested-PCR based on sequences of OMP of Lib. asiaticus. All OMP sequences deposited on GenBank were aligned and the consensus used for primers designing. The external primers (OMP1F-tgtaattcggcgtgaacttg and OMP1R-cacgcggacctataccctta) amplified a 884 pb fragment and the internal ones (OMP2F-ggcgtagaagggcatattga and OMP2R-acgtggcacaattgggttat) a 411 pb fragment. From 600 psyllids analysed, which fed on HLB-infected citrus plants, 40% of the positive samples were detected by nested-PCR whereas 42% were detected by real-time PCR. However a good relationship between both methodologies was obtained. The next step will be the establishment of one-step nested PCR to avoid cross-reaction among samples and false positiveS, which is normal in the nested-two step PCR. At the present date the grants was not provided for us due the bureaucracy of Brazilian Bank, although it was sold by the FCPARC. The reagents for the immunological procedures including purchase of animals, culture media and culture flasks for growing the hybridome cells are expensive as well as the procedure is time demanded. The delay for release the money by the Brazilian Bank was impeditive to buy the necessary reagents for antibody production as well as to have a student working full time in this project. As soon we get the grants we will start to work in the antibody production.
The purposes of this project have been: 1) to compare the temporal progress of HLB caused by C. L. asiaticus (CLas) and by C. L. americanus (CLam), both naturally transmitted by D. citri, having infected Citrus or Murraya paniculata as primary inoculum sources; 2) to compare the acquisition efficiency of both Ca. Liberibacter species by D. citri in Citrus and M. paniculata; 3) to determine incubation and latency periods of HLB after transmission of CLas or CLam by D. citri; 4) to determine when citrus plants infected by CLas or CLam become an effective source of inoculum for D. citri; 5) to quantify and compare the HLB symptoms progress in sweet orange cultivars infected with CLas or CLam by D. citri at different ages. To achieve these objectives, 3 experiments have been carried out under insect-proof conditions to guarantee the source of inoculum and the time and site of inoculation. Under objectives 1 and 2, one experiment began in April/08, having two compartments of the screenhouse (replication) with 4 sweet orange plants each, 2 inoculated with CLam and other 2 inoculated with CLas, as primary source of inoculum. Other compartment had 2 sweet oranges inoculated with CLam and other compartment had 2 M. paniculata also inoculated with CLam (Unfortunately no M. paniculata with CLas was available to make a complete comparison between CLam and CLas in M. paniculata, but we are still working to get M. paniculata with CLas for the next round of experiment next year). Monthly, one hundred adult psyllids have been confined in the source of inoculum plants and after 7 days released in each compartment. Leaf samples of all the plants were collected with the objective to detect the presence and to identify the bacterial species in each plant by real time PCR in July/08, October/08, January/09 and May/09. The first diseased plants appeared in samples of October/08 for CLas from citrus source. Real time PCR of the samples collected in January/09 indicated that the psyllids had transmitted CLam from M. paniculata and from citrus inoculated with CLam to other citrus plants (it was the first time that the transmission of CLam from M. paniculata to citrus by psyllid was observed). Also, it was observed the predominance of diseased plants with positive results for CLas than for CLam when the inoculum sources were infected citrus plants, even the symptoms in citrus plants with CLam are more severe. Real time PCR and conventional PCR confirmed the acquisition of CLas and CLam from the diseased plants by D. citri. All samples (leaf and psyllid) from other assessment dates are being processed. Under objectives 3 and 4, the experiment did not begin yet because the inoculum source plants for this experiment are being prepared (To get budwoods of citrus with CLam in the field has been difficult as to get Murraya plants infected with CLas). Under objective 5, 6 insect-proof screenhouses were built in a commercial citrus farm in Dec/08 in anticipation of funding. Each screenhouse covered 15 trees. The following combinations of sweet orange cultivar grafted over Rangpur lime/planting year were selected: Hamlin/99, Hamlin/04, Hamlin/06, Valencia/99, Valencia/04, and Valencia/06. Five monthly assessments looking for the presence of HLB associated bacteria in each protected tree by real time PCR were done, and unfortunately in each screenhouse the CLas were detected in few trees, that were immediately removed. At the beginning of July/09, one single young shoot of each tree received previously infected psyllid with CLas or CLam. Afterthat, monthly assessments for symtom severity are ongoing. The funding for this project was sent in May/09, and we are waiting the grant convertion to Reais by Brazilian Federal Bank to start building 3 more insect-proof screenhouses to protected sweet orange cultivar Pera (midseason) at 3 different ages in the same farm.
Experiment 1 has two objectives: to compare the temporal progress of HLB caused by C. L. asiaticus (CLas) and by C. L. americanus (CLam), both naturally transmitted by D. citri, having infected Citrus or Murraya paniculata as primary inoculum sources; and to compare the acquisition efficiency of both Ca. Liberibacter species by D. citri in Citrus and M. paniculata. This experiment began in April/08. Leaf samples of all the plants were periodically collected with the objective to detect the presence and to identify the bacterial species in each plant. Psyllids were also periodically collected to evaluate their infectivity. The samples were submitted to conventional PCR and real-time PCR. Preliminary results show a predominance of diseased plants with positive conventional PCR for CLas in the two compartments where citrus plants served as primary source of inoculum of CLas and CLam. Although bacterial source plants infected with CLam showed more severe symptoms than plants infected with CLas, real-time PCR detected 13 and 6 plants with CLas in compartment 1 and 2, respectively, while no CLam infected plant was detected in any compartment yet. There were 8 plants with CLam (infected and symptomatic) in compartment 4, where M. paniculata served as primary source of inoculum and no plant with positive PCR results for CLam in compartment 3, where citrus plants served as primary source of inoculum. Also, the number of psyllids (adults and nymphs) was higher in compartment 4. Psyllids samples are being processed for infectivity. The note ‘First Report of ‘Candidatus Liberibacter americanus’ Transmission from Murraya paniculata (L.) Jack to Sweet Orange (Citrus sinensis Osbeck) by Asian Citrus Psyllid (Diaphorina citri Kuwayama)’ was submitted for publication. Experiment 2 has two objectives: to determine incubation and latency periods of HLB after transmission of CLas or CLam by D. citri; and to determine when citrus plants infected by CLas or CLam become effective sources of inoculum for D. citri. This experiment did not begin yet because there are not enough inoculum source plants for CLam. The idea is to start this experiment only with CLas next December. Experiment 3 wants to quantify and compare the HLB symptoms progress in sweet orange cultivars infected with CLas or CLam by D. citri at different ages. Three new insect-proof screenhouses were built in a commercial citrus farm to protect Pera sweet orange on Rangpur lime planting in 1999, 2004 and 2006 adding to other 6 insect-proof screenhouses for cv. Hamlin and Valencia. Plants from these first six screenhouses were inoculated at the beginning of July/09, and monthly assessments for symptom severity are ongoing. No symptoms were observed yet. Plants from cv. Pera are being prepared to be inoculated soon. We continue trying to get good sources of M. paniculata infected with CLas and Citrus infected with CLam, but it is getting very difficult. In Sao Paulo State, at field conditions, there is a predominance of M. paniculata infected with CLam and Citrus infected with CLas.
The purposes of this project have been: (i) to study the effect of different frequencies of removal of symptomatic trees on HLB progress; ii) to study the effect of vector control on HLB progress; iii) to establish the most efficient and economic combination of frequency of inoculum reduction and vector control to manage HLB; iv) to verify the possibility of growing an economically feasible planting using such strategies in an region with high external pressure of inoculum; and v) statistically compare our results with those elucidated by Gottwald et al. form similar research in their proposal (HLB Epidemiology and Disease Control). In May/06, a new grove of Valencia sweet orange grafted on Rangpur lime was planted in a HLB epidemic region of Sao Paulo State, Brazil, and 8 treatments including 4 different frequencies of symptomatic tree removal and 2 vector control programs (with and without vector control) divided in 24 plots were applied. The first HLB-symptomatic tree was found 13 months after planting in May/07, but for most of plots the first symptomatic trees appeared after November/07. In December/08 the average HLB incidences in plots with and without vector control plots were 1.6 and 3.2%, respectively. In the last 6 months, the disease incidence increased very fast, achieving 15.4 and 34.1% in plots with and without vector control, respectively. Until now, there was no significant relationship between the frequency of local inoculum reduction and all assessed variables. The short period of application of eradication treatments associated to the long incubation period of the disease could contribute for that. The local vector control program was able to significantly reduce adult psyllid population and also to avoid psyllid breeding in treated trees. In a preliminary temporal analysis, despite no significant difference observed on the delay of the beginning of epidemics and on disease progress rates, psyllid control significantly reduced the cumulative number of HLB-symptomatic trees. However, the tested vector control program did not completely avoid HLB contamination from external source of inoculum (15.4% of HLB-symptomatic trees 37 months after planting) and raises the question if the efficiency of local vector control is enough to guarantee the profitability of citrus groves where HLB is endemic (data for economic analysis have been collected). Preliminary spatial analysis showed that at the beginning of HLB epidemic in each plot (HLB incidence = 1%), the spatial pattern of symptomatic trees were at random originated from long distance movement of infected psyllid from external source of inoculum. In a higher disease incidence (5%), for plots without vector control, aggregation patterns were detected in 8 of 12 plots, indicating that secondary infections at short distances also occurred. For plots with vector control program, non-aggregation of HLB-symptomatic trees was significant detected in 9 of 12 plots, indicating that vector control has an effect in reducing the local transmission that operated over short distances within the plots even in plots where HLB-symptomatic trees are exposed to adult psyllid for some months. The preliminary results point to a need for regional control strategy and that for small plantings that can not control neighbors will be really hard to profitability manage the disease. The assessments on this experiment will continue to allow more detailed temporal, spatial and economical analysis for better conclusions.
Callose was identified by immuno-gold labeled callose antibody in transmission electron microscopy (TEM) as the amorphous plugging material in HLB affected tree phloem sieve elements. An antibody specific to phloem protein 2 (PP2) was obtained and this coupled with gold labeling showed that the filamentous plugging material was a lectin produced from PP2 . Neither plugging material cross labeled with the antibody for the other material. A manuscript reporting these findings is now being prepared. These plugging materials are responsible for the upset of normal phloem translocation of carbohydrates to other parts of the plant and ultimately the root system is deficient in carbohydrates and starves (Etxeberria et al, 2009, accepted). This may be the main reason for tree decline in response to HLB infection. Field samples are being collected and prepared to determine the relative amounts of amorphous versus filamentous plugs in order to evaluate if one type is more important than the other in disrupting phloem sap flow. Additionally, work is underway to understand the mechanism by which the bacterial infection leads to this phloem plugging. Insufficient bacteria are present in the phloem to directly plug the phloem. Work is underway to determine how the causal bacteria elicits the over expression of phloem plugging materials. Currently, one Agilent microarray has been designed based on the genome sequence of Candidatus Liberibacter asiaticus. The gene expression analysis of the HLB pathogen is underway. More than 50 genes containing signal peptides for secretion and other virulence factors have been selected for further analysis of their roles in pathogenicity. Multiple genes for callose formation have been incorporated into grapefruit resulting in potential over-expression of this material. Shoots of this material have been propagated on other citrus plants which will be challenged with the HLB bacteria by using infected buds as soon as the shoots start to grow. The PP2 gene has been cloned into a protoplast transformation vector and new sweet orange suspensions for protoplast transformation have been started. Transformation experiments will begin this quarter. The amount of phloem plugging will be monitored in this material when it becomes PCR positive. Rootstocks that do not show strong symptoms when challenged with the HLB bacteria in the greenhouse will be infected in an isolated field location and evaluated for phloem plugging development as they become PCR positive.
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