This first section of the report covers the entire period that Huanglongbing Diagnostic Laboratory has been in service since February 2008 because one of the objectives for the funding was to continue to provide continuous, seamless service for as long as the growers deemed it necessary. 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. In addition to providing diagnostic services to the growers, the lab continued to develop and apply techniques, protocols and efficiency. Samples The lab has been in operation for nearly 2 years, and has received to date more than 16,000 grower samples, approximately 5,700 research samples, and 2,100 screen house samples from the Budwood facility located on the SWFREC property. Approximately 2600 of the sample total were submitted between October through December 2009. Additionally, of the 5,700 total samples have been processed for research, of which nearly 700 were within that same time frame. These totals represent a several-fold increase in number of sample submissions when compared to the year’s previous two quarters, reflecting seasonal fluctuation in sample volume. 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 The new DNA isolation kit implemented last quarter into routine operation of the lab 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 are using liquid nitrogen to grind all samples as this provides the most effective maceration. We continue to use the magnetic particle based system, which has proved both reliable and fast. 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 have attempt to culture the organism Candidatus Liberibacter asiaticus either in co-culture or isolate it in pure culture using reports cited in literature and will use the pure culture to calibrate the sensitivity of our lab protocols to determine the minimum number of cells of Liberibacter needed for detection.
Irrigation and fertilizer practices in the Advanced Citrus Production System flatwoods site at SWFREC indicate potential for improved plant nutrition of three year old trees. Hamlin and Valencia trees were planted in 2006 at the flatwoods site at 151, 198 and 545 trees per acre on rootstocks Cleopatra mandarin (Hamlin) or Volkamer lemon (Valencia), Swingle citrumelo, and Flying Dragon; respectively. Irrigation/fertigation practices were daily drip fertigation (Drip OHS), daily irrigation and weekly fertigation with a strip shaped microsprinkler (Microsprinkler OHS) and a Grower Microsprinkler control with periodic irrigation based on soil depletion and monthly fertigation. Adequate tree nutrient status, root density in the irrigated zone and water conservation were three factors investigated in 2009. Leaf N , P and K content were similar for all treatments at the beginning of 2009 (January and March), however samples taken in June and September indicate that the Drip OHS and Microsprinkler OHS maintained leaf concentrations greater that those for the Grower Microsprinkler irrigation treatment. The exception to this is P, where no significant differences were found among treatment at any sample date. All treatments maintained average leaf nutrient concentrations in the optimum or high range. Root length density (RLD, root diameter <1mm) varied as a function of irrigation/fertilizer treatment (p<0.05), decreasing with depth (p<0.0001) and distance from the tree (p<0.0001). RLD was consistently higher for Drip OHS than the other two treatments. Prior to the start of the summer rainy season, Drip OHS significantly increased RLD by 13-90% for roots <3mm in diameter in both the irrigated and non-irrigated zones when compared with the conventional practice. After the rainy season, RLD in the Drip OHS plots were 19% and 112% higher than conventional practice in the non-irrigated and irrigated zones, respectively. These data indicate that RLD in the drip irrigation zone may not be greatly influenced by the rainy season as first speculated. Thus, irrigation methods such as drip which apply water and fertilizer frequently and in small pulses within a limited root zone offer a viable option for increasing root water and nutrient uptake efficiency compared with the microsprinkler based systems when the trees are small. Total water used was greatest for the Grower Microsprinkler treatments plots and increased with planting density ranging between 10 and 18 inches. Water use was lowest for Drip OHS irrigation treatments at low (5 in yr-1) and moderate (6 in yr-1) densities compared with the Grower Microsprinkler treatments at the same densities (10 and 14 in yr-1, respectively). Thus, overall water use efficiency for the Drip OHS and Grower Microsprinkler treatments were 0.03 and 0.07 inches per tree, respectively. However, the highest water user was the high density planting with drip irrigation (19 in.). The reason water use increased about 3 to 4 fold for the drip treatment at high tree density is that each tree had the same number of drippers and thus water use was a function of tree density (i.e. 545 trees at high density, 151 and 198 at low and moderate densities). The relationship of increased water use with increased tree density existed for the microsprinkler treatments but not at the same ratio as tree density because the emitter output and pattern sizes were selected to give similar application rates on a gal. per area basis with little overlap.
QUARTERLY RESEARCH REPORT (Jan. 08, 2010) In the past 3 months (from Oct. 2009 to Dec. 2009), the research was focused on the on-going evaluation of the new candidate compounds for HLB control using the optimized regeneration system in periwinkle, the effectiveness of the compounds that killed Liberacter in periwinkle to control HLB in citrus and the effects of PS on microorganism diversity in rhizosphere soil. RESULTS 1. On-going evaluation of the new compounds MDL, OPP and SA were selected and evaluated against the Las bacteria and for their phytotoxiticy using the optimized regeneration system in periwinkle. MDL is an antibiotic drug used against anaerobic bacteria. The results showed MDL is effective in eliminating Las bacteria in the Las-infected periwinkle cuttings. The Las can’t be detected in the treated, regenerated plants. MDL is currently being tested in the citrus grove at the Pico Farm, USDA. OPP and SA were not effective. High concentrations of OPP and SA inhibited regeneration rates in Las-infected periwinkle. The regenerated plants from OPP- or SA-treated cuttings kept high titers of Las bacteria. The average Ct value in the regenerated plants was less than 30. 2. Effects of the screened compounds on HLB-affected citrus HLB-affected container-grown citrus was treated with PS, one of the screened effective compounds against Las bacteria. The HLB-affected citrus seedlings were soaked in PS three times. Citrus samples were taken once each month for Liberibacter analysis. The Las bacteria could not be detected in the container-grown citrus two months post treatment, by Q-PCR. The PS-treated citrus grew better than citrus receiving control treatments. When PS combined with other PGRs was injected into HLB-affected, container-grown citrus, PS can also eliminate the Las bacteria in the greenhouse. Thus, PS can control Las in container-grown citrus when applied either as a root drench or by injection. In August 2009, PS at three different rates (2.5g. 5g and 10g per tree) was injected into HLB-affected field citrus trees with 10~15 cm in diameters. Leaf samples were randomly taken and tested in Oct and Dec, 2009. The average Ct value in the PS-treated citrus grove increased from 26 in the pre-treatment to 32 in the post-treatment. All treatment rates were effective. Some other compounds, such as MDL and KESU are also being evaluated in the field. 3. Effect of PS on microorganism diversity in rhizosphere soil Rhizosphere soil samples were taken from the PS-treated citrus pot. Soil DNA was extracted using a Soil DNA Extraction Kit (Mio-Bio). Diversity of microorganisms in the soil was evaluated by RFLP. The results showed that treatment of field citrus with PS had no effect on the diversity of soil microorganisms. The residues of PS in the citrus fruit will be tested later. CONCLUSIONS 1. PS was effective against Las bacteria in periwinkle or in citrus. 2. MDL is also effective compound against Las, but MDL is not easily dissolved in water. Improving the water solubility of MDL and would be was necessary for MDL application in HLB control. 3. OPP or SA was not effective in control of Las bacteria.
Experiment 1 – Since the last report, no new symptomatic plant were observed in any 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 in November/09 (total of 1268 samples collected since the beginning of the experiment in April/08). Psyllids were collected in November and December/09 to evaluate their infectivity. The samples were submitted to conventional PCR and real-time PCR. These analyses are in progress. In December/09 we stopped to release new ACP and killed all psyllids present in the compartments. All plants were moved to other insect-proof screenhouse and will be there for symptoms observation and leaf sampling for PCR analysis. New inoculum source Citrus and Murraya plants are being prepared to repeat this experiment after March/10. At this time we want to include sources of M. paniculata infected with CLas. Experiment 2 – The delay to start this experiment was caused because took so long to obtain inoculum source plants for CLam, but this problem was already solved. During January/10, ACP will be reared on symptomatic inoculum sources for CLam and CLas. The emerging adults from nymphs reared on such inoculum sources will be used for inoculation in February/10. After the inoculation, adults of ACP free of liberibacters will be periodically feed on these inoculated plants to detect the moment when they can acquire the bacteria from inoculated plants. Experiment 3 – Nine insect-proof screenhouses were built in a commercial citrus farm to protect Hamlin, Pera and Valencia sweet oranges with three different age. Plants from Hamlin and Valencia were already inoculated at the beginning of July/09 with infected adults of ACP and plants from Pera will be inoculated in January/10. Monthly assessments for symptom severity have been done, but no HLB-symptoms were observed yet. Also, leaf samples have been collected to detect the presence of Liberibacter species on inoculated shoot. PCR analyses are in progress, no result yet.
Data still have been collected from this field experiment. The longer time of exposition of symptomatic trees in plots with longer frequencies of local inoculum reduction treatments did not result in significant differences on HLB progress rate and HLB incidence 42 months after planting. The tested program of ACP has been efficient to reduce the number of adult psyllids captured on yellow stick traps in 78.6% and to reduce the number of eggs and nymphs observed on new shoots in 94.6%. The disease progress rate (estimated by Gompertz model) in plots with ACP control program was significantly reduced in 21% compared to the disease progress rate in plots without ACP control, reducing the incidence of HLB-symptomatic trees in 53.2% during these 42 months. No significant difference was observed on the delay of the beginning of epidemics. From October/09 to December/09 the disease incidence increased from 20.3 to 28.9% in plots with ACP control program and from 44.9 to 61.8% in plots without vector control. Like observed in past years, the seasonality of HLB symptoms was repeated this year. The incidence of symptomatic trees in each assessment started to increase after February (end of summer) reaching high values in April to June (autumn), and started to decrease after July, reaching low values in September (beginning of spring). However, in 2009 there was a second increase of symptomatic trees detection from October to December probably associated to abnormal amount of rain during the winter. All psyllids captured on yellow stick traps since the winter/07 were tested for the presence of Candidatus Liberibacter spp. by conventional PCR. Higher frequencies of PCR-positive ACP have been observed during spring and summer. Until the first harvest in September/09, US$ 7.37 per plant was spent on psyllid vector control program. The cumulative costs for scouting symptomatic trees were respectively US$ 1.56 and 0.12 per tree for 14 and 182 days of inoculum reduction frequencies. US$ 0.79 was the average cost to remove and eliminate each symptomatic tree. Considering the average cost of HLB management, the cost of eliminated trees and the income by first fruit harvest (US$ 4.12per box paid to citrus growers in Sao Paulo in 2009), no treatment was economically sustainable so far. Additional data for economic analysis have been collected. Annual maps of HLB-symptomatic trees were prepared for spatial analysis using stochastic models (MCMC) to verify the effects of each treatment on primary and secondary spread of HLB. This analysis will be done at USDA lab in Fort Pierce by Gottwald’s team. The assessments on this experiment will continue at least for one more year to allow more detailed temporal and spatial analysis and get better conclusions. According to the estimated disease progress rates, the HLB incidence will be 62.8 and 83.4% respectively for plot with and without vector control at the end of 2010. These results will be presented in the next Florida Citrus Show (January 27th).
The objective of this project is 1) to complete Las genome sequence and to conduct comparative genomics of 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 has been obtained using metagenomics approach, and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin in USDA-ARS, Parlier, California, we have obtained approximate 1.2Mb, a nearly complete genome of Ca. L. psyllaurous with less than 20 contigs, which has ca. 34X coverage . We have also obtained the draft genome (approximately 70%) of Ca. L. americanus using multiple displacement amplification and 454 pyrosequencing technologies. We are currently confirming the sequence of these contigs both in the psyllids and host plants. Preliminary comparison revealed significant difference between Ca. L. asiaticus and Ca. L. americanus. 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, we revealed the genetic diversity of Candidatus Liberibacter asiaticus (Las) collected from Florida, Brazil, China, Philippines, Thailand and Japan. The relationship between the diversity and disease phenotypes were partially correlated. A putative insect transmission determinant gene was identified. The role of this gene is being investigated. We have characterized the ATP/ADP translocase of Las, and proved its function in the heterologous E. coli system, and published in J. Bacteriol. doi:10.1128/JB.01279-09. We are currently developing a antibody-based “drug” to target this protein, aiming to disrupt the life cycle of the Las bacterium. The seed transmission of Las is tested in grapefruit, sweet orange and trifoliate orange. Relative high titer of Las detected from seed coat and inner seed coat of the seeds collected from HLB-affected citrus plants. Very low titer of Las was detected from the seedlings, ranging from 3 to 42% using nested PCR. Most, if not all the seedlings did not have typical HLB symptoms and the threshold of the bacterial titer for HLB, even in the three year old seedlings. The results indicated that the seed-transmitted Las could not cause HLB by themselves. The role(s) of these seed-transmitted Las is under investigation. A super sensitive qPCR detection technology has been developed, which increased the sensitivity at least by 100 fold and thereby eliminated the need of DNA isolation and increased the throughput of detection. The cost savings can be up to 200%. Because of the detection is based on HLB bacteria-specific primers, the detection data confirmed our previous results on seed transmission and the HLB disease phenotypes with low bacterial titer. A BAC library (61,440 clones with insert size 30Kb to 200Kb) of Las-infected psyllids was constructed. BAC clones of Las bacterium are being used for multiple genomics projects.
We proposed to identify and evaluate potential genes for RNAi-induced lethality of sap-sucking Hemipteran insects using both in vitro and in planta dsRNA feeding assays. Since our last report, we have the nearly completed the first objective of our proposal: to evaluate possible candidate genes for their negative effects on Diaphorina citri and our model organism, Myzus persicae, using artificial feeding assays. To date, we have cloned sequences from nine homologous D. citri and M. persicae transcripts. To test the ability of these sequences to instigate RNAi-induced effects on sap-sucking insects, we generated large quantities of double-stranded RNA (dsRNA) derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 and constitutively expressed S4e ribosomal protein from M. persicae using in vitro transcription. The sequences tested were chosen based on their different expression patterns to allow the examination of possible effects resulting from tissue-specificity on RNAi efficiency. Artificial feeding assays were carried out on M. persicae using feeding media that included 750 ng/’l of each dsRNA, respectively. Control assays utilizing feeding media without dsRNA and water, respectively, were also conducted. Three replicate assays were performed in each case. Our initial results indicate that there were significant reductions in survival rates, as well as the number of offspring generated, in the assays bearing dsRNA. We have also begun to make excellent progress on Objective 2: to evaluate the RNAi strategy in vivo using the model plant (Arabidopsis thaliana) and the model tree (Prunus domestica) for its effects against M. persicae. This strategy may prove to be of crucial importance in this study since new evidence suggests that RNAi in sap-sucking insects may function more effectively in planta than in vitro. This research will necessitate the use of Gateway-based vectors that will express our chosen insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We previously cloned both the AtSUS1 and AtSUC2 promoters from A. thaliana, as well as the rolc promoter from Agrobacterium rhizogenes, and have since confirmed their phloem-specificity in transgenic plants in which each promoter was fused to the .-glucuronidase (GUS) reporter gene. Expression driven by the AtSUS1 promoter appeared to be more specific, but less robust, than that induced by the AtSUC2 and rolc promoters. As reported earlier, we have also cloned two separate alleles of the CsSUS1 promoter, as well as the CsSUT1 promoter (which appears to be homologous to AtSUC2) from Citrus sinensis cv. valencia. To test their phloem-specificity in a range of plant species, we have fused all three citrus sequences to the GUS coding region and transformed the resulting vectors into A. thaliana, as well as the woody species P. domestica (plum) and Malus domestica (apple). Transformed plants are currently being grown and GUS assays will be carried out on leaf tissues in the coming weeks. We have also begun the process of replacing the 35S promoter contained within our original Gateway vector with a multiple cloning site that will allow the introduction of the most efficient phloem-specific promoter for use in driving the expression of insect-derived dsRNA for our in planta RNAi assays. In summary, we have successfully cloned a number of transcripts from both D. citri and our model organism, M. persicae, and have analyzed a subset of derived dsRNAs for their lethality to M. persicae using in vitro assays (objective 1). We have also cloned several potential phloem-specific promoters from various organisms, including Citrus sinensis, and are now in the process of evaluating their expression patterns in a number of plant systems (objective 2). Finally, we are currently developing new Gateway-derived vectors bearing a constitutive promoter and phloem-specific promoters, respectively, for use in RNAi against sap-sucking insects in planta (objective 2).
The overall goal of this project is to characterize the virulence mechanisms of Candidatus Liberibacter asiaticus, the citrus Huanglongbing (HLB) pathogen, thus to come up with new management strategies by genome sequencing and functional genomics approaches. The original goal of the proposed research is to further complete the genome sequencing of Candidatus Liberibacter asiaticus, for which a draft sequence is available. The goal was modified to meet the current progress in genome sequencing of Ca. L. asiaticus with the advice and permission from program manager of FCPRAC. The tile has been changed to the following to better suit the goal: Understand the virulence mechanism of Ca. L. asiaticus by genome sequencing and functional genomics approaches. Comprehensive metabolic reconstruction is being used to further understand the biology of Ca. L. asiaticus and identify potential genes for targeting using small molecules and other chemicals. Some novel insights were acquired regarding its respiration, amino acids synthesis, Co-factors and nutrient transportation. Those information provide hints to cultivation and have been shared to groups to culture Ca. L. asiaticus. In addition, genome comparison of Ca. L. asiaticus against Mycoplasma genitalium, one of the smallest bacterial genomes, has identified more than 200 homologs. Those shared genes might be close to the minimal set required for viability of bacteria and good candidates for screening antimicrobial small molecules. Several potential targets including SecA and GalU are being used to screen potential antimicrobial small molecules to control HLB. Bioinformatics analysis was performed to identify potential virulence factors. The SignalP v3.0 program was used to predict the presence of signal peptide within the proteins. The secretomeP 2.0 program was used to predict the non-classical secretion proteins without signal peptide. ORF containing transmembrane domains was predicted by TMHMM2.0 program. Smart and other programs were used to further mine the genome sequence of Candidatus Liberibacter asiaticus. Totally 28 potential virulence genes were cloned into TMV30bGFP viral vector for transient expression on Nicotiana benthamiana plants. Out of the 28 genes cloned, 13 were successfully assayed on tobacco plants for symptom expression. Three out of the 13 showed interesting symptoms, and hence were selected for further characterization. The following symptoms were observed: LasA1 showed vein clearing after about a week after inoculation, and subsequent wilting and death of the whole plant within 2-3 weeks. LasA2 showed phyllody, stunting and very clear growth defects. LasA3 showed very severe yellowing. The symptoms were significantly different from the infection using the empty vector (TMV 30BGFP). The expression of the genes in planta were checked using RT-PCR. Microscopy analysis is being conducted to understand how those virulence factors cause symptoms. The leaf, petiole and roots of tobacco plants infected with the viral vector containing LasA1, LasA2, and LasA3 were collected after ~3weeks for observation under the light microscope. To further confirm the result acquired using the TMV30bGFP vector, two binary vectors were also chosen to clone the genes of interest, pTLAB31 and pGR106. All three genes LasA1, LasA2, and LasA3 were cloned into pTLAB31 and confirmed by sequencing. The constructs, with the genes present in pTLAB31 were then introduced into two strains of Agrobacterium, AGL1 (for tobacco plants) and EHA105 (for citrus). The constructs in AGL1 were then used for making transgenic tobacco plants, and also for transient expression of the genes in tobacco plants. The constructs in EHA105 will be used for making transgenic citrus plants. Understand the virulence mechanism of Ca. L. asiaticus is critical for management of HLB.
The overall goal of this project is to characterize the virulence mechanisms of Candidatus Liberibacter asiaticus, the citrus Huanglongbing (HLB) pathogen, thus to come up with new management strategies by genome sequencing and functional genomics approaches. The original goal of the proposed research is to further complete the genome sequencing of Candidatus Liberibacter asiaticus, for which a draft sequence is available. The goal was modified to meet the current progress in genome sequencing of Ca. L. asiaticus with the advice and permission from program manager of FCPRAC. The tile has been changed to the following to better suit the goal: Understand the virulence mechanism of Ca. L. asiaticus by genome sequencing and functional genomics approaches. Comprehensive metabolic reconstruction is being used to further understand the biology of Ca. L. asiaticus and identify potential genes for targeting using small molecules and other chemicals. Some novel insights were acquired regarding its respiration, amino acids synthesis, Co-factors and nutrient transportation. Those information provide hints to cultivation and have been shared to groups to culture Ca. L. asiaticus. In addition, genome comparison of Ca. L. asiaticus against Mycoplasma genitalium, one of the smallest bacterial genomes, has identified more than 200 homologs. Those shared genes might be close to the minimal set required for viability of bacteria and good candidates for screening antimicrobial small molecules. Several potential targets including SecA and GalU are being used to screen potential antimicrobial small molecules to control HLB. Bioinformatics analysis was performed to identify potential virulence factors. The SignalP v3.0 program was used to predict the presence of signal peptide within the proteins. The secretomeP 2.0 program was used to predict the non-classical secretion proteins without signal peptide. ORF containing transmembrane domains was predicted by TMHMM2.0 program. Smart and other programs were used to further mine the genome sequence of Candidatus Liberibacter asiaticus. Totally 28 potential virulence genes were cloned into TMV30bGFP viral vector for transient expression on Nicotiana benthamiana plants. Out of the 28 genes cloned, 13 were successfully assayed on tobacco plants for symptom expression. Three out of the 13 showed interesting symptoms, and hence were selected for further characterization. The following symptoms were observed: LasA1 showed vein clearing after about a week after inoculation, and subsequent wilting and death of the whole plant within 2-3 weeks. LasA2 showed phyllody, stunting and very clear growth defects. LasA3 showed very severe yellowing. The symptoms were significantly different from the infection using the empty vector (TMV 30BGFP). The expression of the genes in planta were checked using RT-PCR. Microscopy analysis is being conducted to understand how those virulence factors cause symptoms. The leaf, petiole and roots of tobacco plants infected with the viral vector containing LasA1, LasA2, and LasA3 were collected after ~3weeks for observation under the light microscope. To further confirm the result acquired using the TMV30bGFP vector, two binary vectors were also chosen to clone the genes of interest, pTLAB31 and pGR106. All three genes LasA1, LasA2, and LasA3 were cloned into pTLAB31 and confirmed by sequencing. The constructs, with the genes present in pTLAB31 were then introduced into two strains of Agrobacterium, AGL1 (for tobacco plants) and EHA105 (for citrus). The constructs in AGL1 were then used for making transgenic tobacco plants, and also for transient expression of the genes in tobacco plants. The constructs in EHA105 will be used for making transgenic citrus plants. Understand the virulence mechanism of Ca. L. asiaticus is critical for management of HLB.
The new spectrophotometer, a Nanodrop 8000, is used to quantify nucleic acid (NA) content of all samples. An excel spreadsheet with formulas is used to determine what quantity of water needs to be added, if any, to standardize the NA content. Each sample then has the requisite amount of water added to dilute it to the pre-determined concentration in the master plate. Plates for all assays are drawn from this master plate so that the precise amount of NA determined to be optimal for that set of primers is used. The Leica teaching microscope is now fully functional and has been used both to teach and also to evaluate dissection technique. We have a new employee starting next week and this teaching microscope will be instrumental in teaching. We have also found it very useful for examination of developing shoot-tip grafts (STGs). One person handles the tube, the other takes the notes and with two opinions where needed, STGs can be more successfully examined with the result of fewer root sprouts being sent to the greenhouse. Also by careful examination of which STGs are successful, dissection technique can be improved. The second microscope, a Leica dissecting microscope was received in good working order. The old hood the new Leica dissecting microscope is used in has a single body construction and the vibrations are too much to be able to use the microscope in the hood. Several avenues are being explored to solve this problem. During this quarter, the number of selections being cleaned up is up to 99. Seventeen selections have been added to the list and 22 selections were released, several others had their status changed. Five hundred fiftyeight STGs were set up. These represented 13 varieties. During this same time period, 34 successful STGs were grafted onto rootstocks in the greenhouse. These represented 14 varieties. For testing, 38 STG and 7 parent samples were extracted and 84 real-time PCR tests were performed on parents and STGs that grew to sufficient size in the greenhouse. Seventynine 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. Thirty propagations from STGs were planted at Chiefland, five represented a breedersÕ selection. Testing of all selections at the Chiefland Foundation for the 2009 testing season has begun using samples adjusted to the proper titer for each set of primers. This represents the first time testing will be done based on data. Whether or not samples even have adequate nucleic acids present will be known. Citrus tatter leaf virus SYBR green real-time PCR assay will be used on the foundation trees for the first time.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. The specific objectives of this proposal are: transcriptional and microscopic analyses of citrus varieties which are either susceptible or tolerant to Ca. L. asiaticus infection at different infection stages in greenhouse and citrus grove; and transcriptional and microscopic analyses of host response to Ca. L. asiaticus infection with a model system tobacco. Microarray analysis and/or suppressive subtractive hybridization libraries approaches will be used to study the host response to the HLB pathogen infection followed by confirmation with Northern blot or quantitative reverse transcriptional PCR. Anatomical study will be performed with light microscopy using different staining methods. Major achievements: One refereed journal article was published. Kim et al. 2009 Response of sweet orange (Citrus sinensis) to ‘Candidatus Liberibacter asiaticus’ infection: microscopy and microarray analyses. Phytopathology. 99:50-7. Investigation of the host response was examined with citrus microarray. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes related to plant pathogenesis/stress, anthocyanin biosynthesis, cell wall metabolism, cell division, detoxification, lipid metabolism, metabolite transport, metal transport, nucleotide metabolism,phenylpropanoid / flavonoid / terpenoid metabolism, phytohormones, protein kinase, protein metabolism, protein-protein interaction, signal transduction, sugar metabolism, transcription/translation factors and unknown/hypothetical genes. The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since ‘Ca. Liberibacter asiaticus’ does not form aggregate in citrus. The upregulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants. The cDNA sequence of PP2 has been requested by four different research groups to use as the target to suppress the HLB symptom development. To further expand our current understanding of Ca. L. asiaticus-host interaction, we are currently comparing two susceptible and two resistant/tolerant cultivars grown in greenhouse. To understand the genetic mechanism of resistance/tolerance will help the breeding program in the long run. Suppression Subtractive Hybridization is being used to study grapefruit since microarray is not available for grapefruit. Currently, subtractive cDNA libraries were constructed. We are collecting samples We are collecting samples from the resistant/tolerant cultivars due to initial problems of inoculation to those plants. We are further investigating host response of sweet orange (Valencia) in the field conditions. Both leaf and root samples have been collected. Microarray data is available from one experiment (three biological repeats) using the leaf samples. More comparisons have been planned and are being conducted. Finding key genes involved in HLB symptom development will reveal potential management strategy and lead to innovative research to control HLB.
Comparison of the microbiomes associated with HLB pathogen positive and negative citrus will illuminate the causal agent of citrus greening. Potential beneficial endophytic microorganisms could be identified from escape plants which survived in heavily infected citrus groove with HLB. Beneficial microorganisms have been shown in previous studies to have the capacity to control plant diseases by accelerating seedling emergence, promoting plant growth and development, and preventing the invasion of plant pathogens. The investigation of the microbiomes associated with different hosts will help understand the transmission of microorganisms between different hosts. Major achievements: This research has resulted in three referred publications (two published, one submitted). 1. A comprehensive study of the bacterial diversity associated with healthy and HLB diseased citrus indicated that Candidatus Liberibacter asiaticus as the pathogen responsible for HLB disease in Florida. Phytoplasma was not found in any of the samples collected from Florida. Publication: Sagaram et al. 2009 Bacterial diversity analysis of Huanglongbing pathogen-infected citrus using PhyloChips and 16S rDNA clone library sequencing. Applied and Environmental Microbiology (AEM). 2. We characterized the effect of HLB on the bacterial community associated with citrus roots. This research has been summarized in the following manuscript ‘Huanglongbing, a systemic disease, restructures the bacterial community associated with citrus roots’ which has been submitted to AEM for publication. In this study, 16S rDNA clone library analysis of the citrus roots revealed shifts in the microbial diversity in response to pathogen infection. The clone library of the uninfected root samples have a majority of phylotypes showing similarity to well known plant growth promoting bacteria including Burkholderia, Pseudomonas, Stenotrophomonas, Bacillus and Paenibacillus. Infection by Ca. L. asiaticus restructured the native microbial community associated with citrus roots and led to the loss of detection of most phylotypes while promoting the growth of bacteria such as Methylobacterium and Sphingobacterium. The relative proportions of different groups of bacteria changed significantly after pathogen infection. These data indicate that infection of citrus by Ca. L. asiaticus has a profound effect on the structure and composition of the bacterial community associated with citrus roots. 3.A method was developed to screen antagonistic bacteria against uncultured HLB pathogen. The method uses the discrimination of live-dead cells by EMA and speed and sensitivity of QPCR. This finding is published on European Journal of Plant Pathology which has attracted tremendous interest. 4. Isolation of plant growth promoting bacteria from potential escape citrus. Isolation of bacteria with the potential of plant growth promoting and biological control potential might reveal innovative ways controlling the HLB disease. Fifty-four morphologically distinct isolates were obtained from surface sterilized roots of symptomatic and asymptomatic (potential escape trees) citrus plants from a citrus grove with a HLB infection rate of more than 60% and an infection history of approximate five years. Qualitative screening showed that for all of these criteria, asymptomatic plants harbor a significant greater diversity of potentially beneficial bacterial strains. Six different bacteria have been shown to be able to reduce the population of Ca. L. asiaticus when tested in vitro. We are further characterizing the potential mechanism to utilize it to control HLB. Furthermore, we have also located tress which have survived HLB for more than 15 years in China. We plan to investigate whether beneficial bacteria could be isolated from those trees to inhibit the population of the HLB bacteria in citrus. 5.Samples (leaf and root) from different varieties including grapefruit, Murcott, and Hamlin were collected from Florida. Samples (leaf and psyllid) from three different locations were collected. The bacterial diversity of those samples are being studied.
In this third quarter, we have primarily focused on the first two objectives of our proposal as planned in our timeline. Objective 1: Identify genes positively regulating SA-mediated defense in citrus So far, we cloned four citrus SA homologues, ctNPR1, ctEDS5, ctNDR1 and ctPAD4 via the RT-PCR approach. We are currently working on cloning the fifth gene, ctEDS1. We have also finished collecting citrus tissues infected with Ca. L. asiaticus in a time course. Samples will be used to study the expression profile of salicylic acid (SA) homologues and SA analysis. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues We cloned all four citrus homologues, ctNPR1, ctEDS5, ctNDR1 and ctPAD4 into the binary vector pBINplusARS and already transformed Arabidopsis with each construct. We obtained T0 seeds for all these constructs and selected some T0 seeds for T1 transgenic plants. Our transgenic lines and expected outcomes are summarized below: 1. CtNPR1/pBINplusARS to Col or the npr1-1 mutant: we have obtained T1 seeds and will test defense phenotypes in the next generation (T2). If the ctNPR1 gene is functional in Col, we expect that the transgene confers enhanced disease resistance in Col background and complement the npr1-1 mutant. 2. CtNDR1/pBINplusARS to Col or the ndr1-1 mutant: we have obtained T0 seeds and will select T1 seeds on Kanamycin containing plates and test T2 transgenic plants for defense responses in both Col and ndr1-1 background. 3. CtEDS5/pBINplusARS to Col or the eds5-1 mutant: progress is similar to the ctNDR1 construct. 4. CtPAD4/pBINplusARS to Col or the pad4-1 mutant: progress is similar to the ctNDR1 construct. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in Citrus A ctNPR1/pBINplus/ARS construct has been in the pipeline of transforming citrus.
We have examined callose accumulation in the Liberibacter-infected citrus phloem tissues by epifluorescence microscopy since the last quarterly report. Although it is a low-resolution technique, epifluorescence microscopy has an advantage over transmission electron microscopy (TEM) in that it enables callose imaging across larger leaf areas, facilitating extensive and thorough callose measurement. In healthy leaves, no callose was detected in the phloem. In contrast, callose deposition in sieve pores and plasmodesmata pore units (PPUs) was observed in huanglongbin (HLB) symptomatic leaf phloem cells. It was shown that callose accumulates in the phloem cells of senescing citrus leaves (1), raising a question as to whether the callose deposition is a secondary phenomenon following leaf deterioration by Liberibacter infection. To address this question, we examined callose deposition in citrus leaves that are positive for Liberibacter infection (assayed by PCR) but display no HLB signs yet. Callose was detected in the sieve pores and PPUs of the asymptomatic leaves almost as much as those in the symptomatic leaves. This demonstrates that callose synthesis in the Liberibacter-infected phloem precedes development of HLP symptoms. Callose deposition in the plasmodesmata is a natural plant defense reaction but it reduces solute transport efficiency through the plasmodesmata (2). This led us to hypothesize that the callose synthesis in the PPU accompanying Liberibacter infection may affect phloem loading in the citrus leaves. To test the possibility, we injected carboxyfluorescein into intercellular spaces of healthy leaves, Liberibacter-infected asymptomatic leaves, and Liberibacter-infected symptomatic leaves. When we imaged carboxyfluorescein at 24 hours after injection, the dye was seen to concentrate in the vein and spread through the vein in healthy leaves. However, dye remained at the injection site in the Liberibacter-infected leaves, indicating that phloem loading is inhibited in the infected leaves. The massive starch accumulation in the parenchyma cells of HLB symptomatic leaves suggests a blockage in photosynthetate export and inhibition in phloem loading of the infected leaves provides an explanation for the blockage. Our results also suggest that cell death in the HLB citrus trees is caused by inappropriate plant response against Liberibacter rather than damages actively done by the bacterial cells, which is consistent with the fact that this phloem-limited bacterium devastates the entire tree at a very low titer. As stated in the second report, we have identified phloem cells containing bacterial cells in HLB symptomatic leaves. We examined their bacterial cell shapes by serial section 3D reconstruction. (An image from our 3D models is shown at http://news.ifas.ufl.edu/2009/12/09/uf-researchers-find-lone-culprit-behind-greening/) We found two morphologically distinct bacteria cells in periwinkle leaf phloem cells. (Please see the first progress report.) But all the bacterial cells in the citrus leaf phloem cells displayed uniform morphological features including diameter, length, cytoplamic staining and cell wall staining, and so on. This observation suggests that Liberibacter vastly outnumbers other bacteria, if there is any, in the HLB-infected citrus phloem tissue and agrees with a recent metagenomic sequencing result by (3). 1. M. J. Jaffe, R. Goren, Physiologia Plantarum 72, 329 (Feb, 1988). 2. Y. L. Ruan, S. M. Xu, R. White, R. T. Furbank, Plant Physiol 136, 4104 (Dec, 2004). 3. H. L. Tyler, L. F. W. Roesch, S. Gowda, W. O. Dawson, E. W. Triplett, MPMI 22, 1624 (Dec, 2009).
We have examined callose accumulation in the Liberibacter-infected citrus phloem tissues by epifluorescence microscopy since the last quarterly report. Although it is a low-resolution technique, epifluorescence microscopy has an advantage over transmission electron microscopy (TEM) in that it enables callose imaging across larger leaf areas, facilitating extensive and thorough callose measurement. In healthy leaves, no callose was detected in the phloem. In contrast, callose deposition in sieve pores and plasmodesmata pore units (PPUs) was observed in huanglongbin (HLB) symptomatic leaf phloem cells. It was shown that callose accumulates in the phloem cells of senescing citrus leaves (1), raising a question as to whether the callose deposition is a secondary phenomenon following leaf deterioration by Liberibacter infection. To address this question, we examined callose deposition in citrus leaves that are positive for Liberibacter infection (assayed by PCR) but display no HLB signs yet. Callose was detected in the sieve pores and PPUs of the asymptomatic leaves almost as much as those in the symptomatic leaves. This demonstrates that callose synthesis in the Liberibacter-infected phloem precedes development of HLP symptoms. Callose deposition in the plasmodesmata is a natural plant defense reaction but it reduces solute transport efficiency through the plasmodesmata (2). This led us to hypothesize that the callose synthesis in the PPU accompanying Liberibacter infection may affect phloem loading in the citrus leaves. To test the possibility, we injected carboxyfluorescein into intercellular spaces of healthy leaves, Liberibacter-infected asymptomatic leaves, and Liberibacter-infected symptomatic leaves. When we imaged carboxyfluorescein at 24 hours after injection, the dye was seen to concentrate in the vein and spread through the vein in healthy leaves. However, dye remained at the injection site in the Liberibacter-infected leaves, indicating that phloem loading is inhibited in the infected leaves. The massive starch accumulation in the parenchyma cells of HLB symptomatic leaves suggests a blockage in photosynthetate export and inhibition in phloem loading of the infected leaves provides an explanation for the blockage. Our results also suggest that cell death in the HLB citrus trees is caused by inappropriate plant response against Liberibacter rather than damages actively done by the bacterial cells, which is consistent with the fact that this phloem-limited bacterium devastates the entire tree at a very low titer. As stated in the second report, we have identified phloem cells containing bacterial cells in HLB symptomatic leaves. We examined their bacterial cell shapes by serial section 3D reconstruction. (An image from our 3D models is shown at http://news.ifas.ufl.edu/2009/12/09/uf-researchers-find-lone-culprit-behind-greening/) We found two morphologically distinct bacteria cells in periwinkle leaf phloem cells. (Please see the first progress report.) But all the bacterial cells in the citrus leaf phloem cells displayed uniform morphological features including diameter, length, cytoplamic staining and cell wall staining, and so on. This observation suggests that Liberibacter vastly outnumbers other bacteria, if there is any, in the HLB-infected citrus phloem tissue and agrees with a recent metagenomic sequencing result by (3). 1. M. J. Jaffe, R. Goren, Physiologia Plantarum 72, 329 (Feb, 1988). 2. Y. L. Ruan, S. M. Xu, R. White, R. T. Furbank, Plant Physiol 136, 4104 (Dec, 2004). 3. H. L. Tyler, L. F. W. Roesch, S. Gowda, W. O. Dawson, E. W. Triplett, MPMI 22, 1624 (Dec, 2009).
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