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.
The main focus of our experiments on the time-course of movement of Ca. Liberibacter through the psyllid anatomy is dependent upon establishing a quarantined, high-titer plant/psyllids rearing system so that ÐqPCR psyllids can be introduced and tracked by dissections and microscopy through their ontogeny. This rearing system is under construction and excellent progress has been made and some adult and immature as well as plant samples have been collected for qPCR analysis (in progress). At the same time, we have studied the insect/plant/bacteria epidemiology in the local orchards, and from these trees have collected and preserved the first specimens for electron microscopy and FISH technique optimization. As a consequence of our pilot studies and communications with other laboratories, we have affected a paradigm shift from the earlier understanding that flush was the source of greatest infectivity for the psyllids, to a new understanding that older leaves contain the highest titer. Psyllids oviposit preferentially on the flush, and it appears that they accumulate HLB by maintaining their position on the growing leaves during their ontogeny. Evidence is accumulating that HLB may reproduce in the psyllid body also. These new concepts and other newly arriving information are constantly steering and revising our ongoing methodology. As we continue to build our rearing system to generate large numbers of specimens guaranteed to have baseline high titers, several specimen lines were collected and processed in different ways to yield different sets of information at this early stage in our study: 1) We have adapted a procedure for rapid, gross, localization of whitefly endosymbionts to the psyllid so that we can quickly and roughly determine degree of infectivity in the general areas we found organs of transmission to occur. +qPCR and -qPCR specimens were preserved for this technique. The results thus far (May collections) tindicated high variability of signal present in free orchard psyllids vis-ˆ-vis a baseline from non-infective psyllids from quarantine colonies. The qPCR reports (Ct values) for 25 psyllids sampled randomly from orchard (left) and quarantine ÐqPCR colonies (lowest value of 20 indicates high HLB titer, highest value of 40 indicates no HLB titer. Mean = 32.965, SD = 5.835). 2) The same lines of specimens were also processed for high quality ultrastructural detail using the electron microscope. These lines will be compared to determine the morphology of the pathogen and the tissues that harbor it. Refereed Publication: Cicero, J., Brown, J.K., Roberts, P. D., and Stansly, P.A. 2009. The digestive system of Diaphorina citri (Kuwayama) and Bactericera cockerelli (Sulc) (Homoptera: Psyllidae). Ann. Entomol. Soc. Am. 102:650-665.
Funds were received in April of 2009. Since that time citrus and other host plants of Candidatus Liberibacter asiaticus were established, propagated and infected with Liberibacter asiaticus at Beltsville for use in this project. A half time student worker was hired to assist in greenhouse duties. Plants were confirmed to be positive for the pathogen by real time PCR assays. Colonies of the insect vector of the pathogen were also established and maintained at Lake Alfred, Florida and Ft Detrick, Maryland. Test shipments of insects were sent to Beltsville to confirm shipping procedures and to perform initial extracts for testing. Many administrative tasks were completed, including transfer of funds to subcontractors at the University of Florida, USDA ARS, Ft Detrick, MD and Agdia, Elkhardt, IN. Material transfer agreements were negotiated with Sigma-tau Pharmaceutical Company, Rome, Italy to acquire the specialized vector for phage display technologies and with Agdia, Elkhardt IN for exchange of materials. Visiting scientists were recruited internationally, identified in China and Italy and arrangements were made for their study visits in Beltsville, to begin August 10 and October 20, 2009. Funding arrangements were made with the USDA Foreign Agriculture Service to support these visitors on this projectÕs funds.
A procedure of shallow cuts in the tree trunk sprayed with some effective herbicides usually killed HLB infected trees in trials in commercials groves. The most effective herbicides found so far contain Imazapyr. As little as 0.33 oz. of a 1 to 4 diluted product was effective at all times except during the spring flush, when apparently a one to one dilution was needed. A vehicle attachment to cut the trunk and spray apply the herbicide is now being developed. This will be mounted on a 4 wheel drive utility vehicle and tested under commercial conditions. Further tests of herbicide dilution, other products and the possible need for higher concentrations in the spring-summer period are planned also. Some chemicals still to be tried include: Velpar (hexazinone) and Ally (metsulfuron) and Clearstand (imazapyr+metsulfuron). Tree killing using Midas (Iodomethane) continue with another trial scheduled for July. This trial will evaluate deeper placement in the soil and a surface spray on barrier to retard volatile loss of the chemical. Trials of aerial applications of herbicides to kill abandoned citrus trees are being monitored. These trials were applied the week of 6 July by the South Florida Water Management District (SFWMD) in areas near Ft. Pierce, FL. About 600 acres were sprayed for kill of an area to be reverted to watershed. Another area was divided into 6 acre plots to test chemicals and rate. All applications were made using a helicopter. It is planned to make the first evaluation of the plots the second week of August. The results of that test which included Arsenal and Clearstand will be in the next report. Other chemicals will be tested for tree kill under abandonment conditions using a simulated aerial spray. A Curtec or Proptec sprayer will be set up to spray from the upper register only, droplet size will be adjusted and volume will match aerial applications of 5 to 10 gal/acre. Materials to be tested will include Velpar (hexazinone) and Ally (metsulfuron) and Clearstand (imazapyr+metsulfuron). The Imazapyr products will be the standards to compare other products to. The effectiveness of the Imazapyr products in the SFWMD tests will provide a guide for how well the simulation test did compared to the aerial test.
Primary: Cultural Practices to prolong productive Life of HLB Infected Trees The objective of duplicating the positive results obtained by citrus grower Maury Boyd with his nutrient/SAR foliar spray program appears to be well on the way to achievement. The foliar spray mixture was broken into component groups of micronutrients (Mg, Zn, Mn, Mo) SARs (Salicylic acid, Serenade Max WP), Phosphorus acid, Hydrogen peroxide, and other (macronutrients, citrus spray oil). Three trial sites, randomized & replicated, of 6 to 7-yr-old Valencia orange on Swingle rootstock were established; 1) two acres 100% infected at IFAS Immokalee, 2) six acres 50-60% infected commercial grove, and 30 acres 15-20% infected commercial grove. The trials were started at the beginning of 2008 with all combinations of the component groups being applied to the 3 flushes in 2008 and spring flush 2009. In 2008 trees were all symptomatic of HLB and confirmed positive with PCR. Trees had dieback and poor growth. Results at the end of 2008 showed treated trees varied in their response, most decreased in tree rating and the full treatment maintained trees in many plots as compared to controls and untreated trees. Winter 2008 and spring 2009 saw mild freeze damage followed by extreme drought stress. Tree yield was to low to harvest at sites 1 and 2. A dramatic recovery was apparent in many of the treatment group with the 2009 spring flush. Trees grew vigorous shoots with normal to long internodes and full size leaves. Many trees flowered and set a heavy crop load for young trees. Most treatments improved but the best results are being seen with the complete mixture. Monitoring BoydÕs grove has recorded a fourth year of fruit set and vigorous growth. Canopy density, shoot length, and leaf size is normal. HLB symptoms remain evident on foliage and new vigorous shoots initiate from infected stems. Yields the past three years have maintained or increased. Fruit drop was observed on HLB infected trees at fruit maturity. Juice quality is high and equal to uninfected trees. Root samples have shown no root deterioration or sloughing. Primary cost estimates of HLB Foliar Spray program are that net production costs increase by less than $200 per acre. The threshold for removing HLB infected trees could by less than 2% per year if nutritional and SAR treatments preserve production of infected trees for at least 10 years. Subcontract: Evaluation of Systemic Acquired Resistance Inducers Combined with Psyllid Control to Manage Greening in Infected groves The trial began on march 19, 2008 and occupies 12 acres of ÔValenciaÕ orange on ÔSwingleÕ citrumelo, divided into 16 plots in an RCB design with 4 replicates and 4 treatments: ± a micronutrient SAR cocktail and ± insecticide applications as needed to control psyllids. We have been able to maintain low populations in the sprayed plots 0.7 ± 0.2 adults per 10 tap samples compared with 9.9 ± 1.4 adults per 10 tap in neighboring non-treated plots. Trees receiving the cocktail are visibly in better condition but differences in production after 1 year were not significant although trends were favorable: (1.30±0.13 for treated plots vs 0.98±0.1 boxes for untreated). The treatments are not yet having a measurable effect on the incidence of greening. Instead, a geostatistical analysis of the distribution of infected plants indicates edge effects.
The objectives of the research during this period is to (1) perform genome-wide sequence analysis to identify Simple Sequence Repeat (SSR) loci from genomic sequences of “Candidatus Liberibacter asiaticus” (Las). (2) Design and develop PCR-based SSR molecular markers for detection and genetic analysis. We carried out research experiments and successfully met milestone of the objectives. Followings are the summary of research activities and accomplishments 1. Genome wide sequencing analysis to identify Simple Sequence Repeat loci We conducted genome sequence analysis to identify simple sequence repeat (SSR) loci. A complete ÒCandidatus LiberibacterÓ asiaticusÓ (CLas) genome sequences has been obtained by pyrosequence technology using the DNA extracted from a single Las-infected psyllid (Duan, et al., 2009). The bacterial has genome size 1.23 M base pairs represented in a circular chromosome with average 36.5% GC content. The software, ÒTandem Repeat FinderÓ was used to search SSR loci genome wide. These searches included mono, di-, tri-, tetra-, penta-, hex- type of perfects, in-perfect and compound types of simple repeats. The criteria to identify SSR loci for designing primer were as followings: 1) for each type of repeat, we selected loci containing at least 5 or more of SSR motifs. 2) each locus has one copy per genome. These researches led to the identification of 123 loci that met the criteria and were potentially useful to design SSR markers. 2.In silico CLas sequence validation. To ensure that sequence loci selected for SSR marker development are unique to CLas, in-silico sequencing analyses were carried out using BLAST to compare the 123 SSR candidate loci with all the available microbe sequences in the NCBI databases. Next, selected SSR sequences were aligned by CAP3 program to remove any duplicate loci. Ten of SSR loci were found to be duplicate and therefore were removed from the list. About 200 bp up and down stream of flanking sequences of each selected loci were then selected for SSR primer designs. 3. SSR primer design. ÒMolecular BeaconÓ software (V. 7.0) was used for primer design. As much as possible, the same parameters were applied for all SSR primer designed, which included, ~50% GC, Tm=55oC, primer length Å 20bp, self dimer/cross dimer .G= -5 kcal/mol., and amplicon sizes ranging from 150 to 350 bp (Lin et al., 2005). Under these criteria, all designed primers will work in the same PCR conditions thereby facilitating SSR primer validation. Of 113 SSR selected loci, 45 of them passed the requirements for SSR primer design. To further evaluation multiplex detection, computational algorithms were used to search possible multiplex primer sets that had the least formation of self and cross primer dimmer under multiplex conditions. Using above criteria, 24 SSR primers had been designed. 4. PCR validation. To evaluate the usage of SSR markers, PCR assays were conducted using multiple CLas strains collected from Florida, Brazil, China and India. Specificity assays also performed to check if these SSR primers interact other citrus related pathogens such as Xylella fastidiosa 9a5c, Xanthomonas axonopodis pv. Citri, Citrus tristeza virus, ÒCa Liberibacter africanus, ÒCa Liberibacter americanusÓ and ÒCa Liberibacter solanacearumÓ. PCR products were then resolved by a 5% polyacrylamide gel and visualized by the silver staining. Of 24 primers we designed, 20 SSR primers showed clean and expected fragment sizes. Seven SSR primers are capable of differentiating CLas strains among various collections. (1)Duan et al., 2009. Complete genome sequence of citrus huanglongbing bacterium, ÒCandidatus Liberibacter asiaticusÓ obtained through metagenomics. Molecular Plant-Microbe Interaction. 22:1011-1020. (2) Lin, et al., . 2005. Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of Xylella fastidiosa. Applied and Environmental Microbiology 71:4888-4892.
The objectives of the research during this period is to (1) perform genome-wide sequence analysis to identify Simple Sequence Repeat (SSR) loci from genomic sequences of “Candidatus Liberibacter asiaticus” (Las). (2) Design and develop PCR-based SSR molecular markers for detection and genetic analysis. We carried out research experiments and successfully met milestone of the objectives. Followings are the summary of research activities and accomplishments 1. Genome wide sequencing analysis to identify Simple Sequence Repeat loci We conducted genome sequence analysis to identify simple sequence repeat (SSR) loci. A complete ÒCandidatus LiberibacterÓ asiaticusÓ (CLas) genome sequences has been obtained by pyrosequence technology using the DNA extracted from a single Las-infected psyllid (Duan, et al., 2009). The bacterial has genome size 1.23 M base pairs represented in a circular chromosome with average 36.5% GC content. The software, ÒTandem Repeat FinderÓ was used to search SSR loci genome wide. These searches included mono, di-, tri-, tetra-, penta-, hex- type of perfects, in-perfect and compound types of simple repeats. The criteria to identify SSR loci for designing primer were as followings: 1) for each type of repeat, we selected loci containing at least 5 or more of SSR motifs. 2) each locus has one copy per genome. These researches led to the identification of 123 loci that met the criteria and were potentially useful to design SSR markers. 2.In silico CLas sequence validation. To ensure that sequence loci selected for SSR marker development are unique to CLas, in-silico sequencing analyses were carried out using BLAST to compare the 123 SSR candidate loci with all the available microbe sequences in the NCBI databases. Next, selected SSR sequences were aligned by CAP3 program to remove any duplicate loci. Ten of SSR loci were found to be duplicate and therefore were removed from the list. About 200 bp up and down stream of flanking sequences of each selected loci were then selected for SSR primer designs. 3. SSR primer design. ÒMolecular BeaconÓ software (V. 7.0) was used for primer design. As much as possible, the same parameters were applied for all SSR primer designed, which included, ~50% GC, Tm=55oC, primer length Å 20bp, self dimer/cross dimer .G= -5 kcal/mol., and amplicon sizes ranging from 150 to 350 bp (Lin et al., 2005). Under these criteria, all designed primers will work in the same PCR conditions thereby facilitating SSR primer validation. Of 113 SSR selected loci, 45 of them passed the requirements for SSR primer design. To further evaluation multiplex detection, computational algorithms were used to search possible multiplex primer sets that had the least formation of self and cross primer dimmer under multiplex conditions. Using above criteria, 24 SSR primers had been designed. 4. PCR validation. To evaluate the usage of SSR markers, PCR assays were conducted using multiple CLas strains collected from Florida, Brazil, China and India. Specificity assays also performed to check if these SSR primers interact other citrus related pathogens such as Xylella fastidiosa 9a5c, Xanthomonas axonopodis pv. Citri, Citrus tristeza virus, ÒCa Liberibacter africanus, ÒCa Liberibacter americanusÓ and ÒCa Liberibacter solanacearumÓ. PCR products were then resolved by a 5% polyacrylamide gel and visualized by the silver staining. Of 24 primers we designed, 20 SSR primers showed clean and expected fragment sizes. Seven SSR primers are capable of differentiating CLas strains among various collections. (1)Duan et al., 2009. Complete genome sequence of citrus huanglongbing bacterium, ÒCandidatus Liberibacter asiaticusÓ obtained through metagenomics. Molecular Plant-Microbe Interaction. 22:1011-1020. (2) Lin, et al., . 2005. Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of Xylella fastidiosa. Applied and Environmental Microbiology 71:4888-4892.
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 multiple displacement amplification and 454 pyrosequencing technologies from a single Las-borne Asian citrus psyllid (Diaphorina citri). Annotation of the 1.23 Mb genome sequence uncovered 1136 predicted coding sequences (CDS), and all but 300 of these CDS could be assigned to one of the defined Clusters of Orthologous Groups of proteins (COGs). Phylogenetic analysis of the full genome sequence has shown that within the Rhizobizles, Las is closely associated with members of the Rhizobiaceae family. Completion of this genome has also revealed that Las has a limited capacity for aerobic respiration, is likely auxotrophic for at least five amino acids, lacks Type III and IV secretion systems although the Type I secretion machinery appears to be present and 40 ATP-binding cassettes (ABCs). With such a large number of ABCs in relation to the small size of the Las genome and the nature of ABCs, it is quite possible that some of these transporters affect virulence, host range and/ or symptom elicitation. A more extensive discussion can be found in our paper entitled ÒComplete genome sequence of the citrus huanglongbing bacterium, ÔCandidatus Liberibacter asiaticusÕ obtained through metagenomics, which is published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin in USDA-ARS, Parlier, California, we have obtained a nearly complete genome of Ca. L. psyllaurous with 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. 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 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. 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.
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