Although Year 2 of this project officially began on July 1, 2010, funds did not reach any of the researchers until 9/9/10, and at this time, only the UF PI (Moore) and the UF subcontractors (Grosser, Gmitter) received funding. The USDA subcontractors did not get their funds until late in September due to procedures at UF and USDA. Therefore little new research was started in this quarter. Other funds were found to maintain materials and personnel.
Objective: Determine if Carrizo rootstocks, either wild type or over-expressing the Arabidopsis NPR1 gene (with an enhanced, inducible defense response) have any effect on gene expression and/or the defense response of wild type (non transgenic) grapefruit scions to HLB. We recently started to propagate new lines from cuttings of 9 individually transformed plants: lines 757, 761, 763, 775, 854, 857, 890, 896 and 897, all transformed with the AtNPR1 (We had to wait until the plants were large enough to withstand the taking of multiple cuttings). However, we have found that propagation by cuttings is difficult with certain lines and, even when it is possible, it may take several months for new growth on the cuttings. This process is still underway. Concurrently, we have standardized more probes and primers for the detection of SAR-associated citrus genes. Making these primers and probes requires knowledge of nucleotide sequence of the genes. Then the primers and probes must be tested and conditions optimized before experiments can be done. The list of genes we can test now includes: AZI1, BLI, CHI, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, PR1, R13032, R20540, RAR1 and SGT1, in addition to our controls 18S and COX. We chose these 15 genes because they are either important in the early induction and regulation of SAR (AZI1, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, R13032, R20540, RAR1 and SGT1) or are targets of the regulatory SAR pathway (BLI, CHI and PR1). In Objective 1 of this project, we propose to compare the response of AtNPR1 transgenic plants vs. wild type plants to the treatment of the SAR inducer salicylic acid (SA), by testing for expression of the above listed genes. This has been accomplished for the first set of lines. We intend to repeat this experiment with the increased number of transgenic lines once they are ready, with the increased number of genes we have identified, and using the commercial version of SA (Actigard, Syngenta Corporation).
Candidatus Liberibacter asiaticus (CLas), the causative agent of Citrus Huanglongbing (HLB) also known as citrus greening, contains1.23 Mb genome which is smaller compared to other members of the family. The genome encodes large amount of hypothetical proteins, which constitutes nearly 26% of the genome. It is possible that the important genes responsible for pathogenicity might be buried in this large amount of gene pool encoding hypothetical proteins . The proposed research concerns the expression of putative effector genes, part of the gene pool encoding hypothetical proteins, in citrus. Using bioinformatics tools, we have identified a number of putative effector genes based on the genome sequence of CLas. By the use of citrus tristeza virus (CTV) vector, we could express putative effectors (pathogenicity and virulence genes) of the CLas bacterium directly inside the phloem of citrus. At present twenty different genes encoding putative effector proteins from Las infected citrus plants have been amplified and cloned behind heterologous beet yellows virus CP subgenomic RNA controller element engineered between the CPm and CP genes in the CTV vector and transferred into Nicotiana benthamina. The CTV virions purified from Nicotiana benthamiana containing different HLB effectors will be used to inoculate citrus plants by bark-flap inoculations. The resulting systemic spread and expression of the putative effectors throughout citrus trees will enable us to understand the role of the putative effectors in disease induction.
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. Major achievements: 1. Microarray analysis of host response of sweet orange to Las infection in greenhouse. The results have been published in the following paper: Kim, J., Sagaram, U.S., Burns, J. K., and Wang N*. 2009 Response of sweet orange (Citrus sinensis) to Candidatus Liberibacter asiaticus infection: microscopy and microarray analyses. Phytopathology 2009 99:50-7. 2. We are currently assessing citrus genes modulated by Las infection in 1) citrus stems and roots, 2) citrus grove, 3) citrus varieties that show tolerance, and 4) at different infection stages. Using Affymetrix microarray analysis, we detected a total of 2,795 and 1142 probe sets with significantly (p< 0.05) altered expression levels in stems and roots of Valencia sweet orange (Citrus sinensis Osbeck), respectively. At a cutoff point of 1x log fold change (logFC), a total of 580 transcripts were significantly up-regulated and 350 down-regulated in stems. A relatively lower number was up-regulated (58) and down-regulated (58) in roots. Different sets of plant genes including those related to response to biotic or abiotic stress, transcriptional factors, transport, cell wall re-modeling and biogenesis were represented in both sets. Highly up-regulated genes (>3x logFC) in stems included 2OG-Fe(II) oxygenase family protein, WAK-like kinase, Lectin-related protein precursor, Cu-Zn superoxide dismutase, Zinc transporter protein ZIP1, many of which are involved in oxidative stress that produce highly toxic reactive oxygen species (ROS). Homologs of nucleotide binding and Leucine-rich repeat (NB-LRR) domain containing proteins involved in gene-for-gene resistance were down regulated in both tissues with some such as TIR-NBS-LRR proteins being re-pressed in stems only. Suppression subtractive hybridization analysis of RNA from Las-infected Mandarin line (Citrus x limonia Osbeck) showed up-regulation of pathogenesis/resistance, biotic stress related, cell wall re-modeling gene groups and transcriptional factors and down regulation of NB-LRR domain containing proteins. Zinc is a cofactor in many redox reactions and zinc deficiency-like in plants have been attributed to damage by ROS. This suggests that the zinc-pattern-deficiency symptoms associated with HLB is caused by ROS generated by citrus plants in response to Candidatus Liberibacter infection. Currently, QRT-PCR assays are being used to further confirm the results of microarray analysis. In addition, SSH is being used to compare the several varieties different in susceptibility and tolerance.
Citrus canker is a serious disease of most commercial citrus cultivars in Florida. The goal of the proposed research is to identify and characterize novel and critical genes involved in pathogenicity and copper resistance present in Xanthomonas axonopodis pv. citri (Xac) and related strains. Identification of critical virulence factors is a crucial step toward a comprehensive understanding of bacterial pathogenesis, host-species specificity, and invasion of different tissues thus to design new management strategies for long term control. Treatment of citrus with copper-based bactericides is one of the most common practices used for control. However, there is potential for horizontal gene transfer of copper resistance genes from other closely and distantly related bacterial strains, which will drastically reduce the efficacy of copper bactericides. Currently, copper resistant strains of other xanthomonads, including X. a. pv. citrumelo, the citrus bacterial spot pathogen, have been isolated from fields in Florida. Understanding the potential mechanisms of copper resistance in Xac and potential horizontal gene transfer of this resistance to Xac is also important for the long-term management of citrus canker. Major achievements: Currently, five Xac related strains are being sequenced, which includes Xac Aw and A* strains which have restricted host range compared to the A type strain, X. axonopodis pv. citrumelo strains (copper resistant and non-copper resistant), and Argentinian strain (copper resistant). Both 454 Titanium and Illumina (solexa) methods were used. The genome sequence of Xac Aw strain is completed. Aw strain: Complete De novo genome sequencing of Xanthomonas axonopodis pv. citri strain Aw was done. 454 Titanium paired end reads were used for making contigs and scaffolds using Newbler with 24X coverage. It was then assembled and closed by using contigs made from Illumina 75bp paired end reads using CLC Bio with about 300X coverage. Finishing was done using Opgen MapSolver. Plasmids for the strain were obtained by reference sequencing using only the Illumina 75bp paired end reads. The size for genome was 5.34 Mb and for plasmids pAw1 and pAw2 was 29 and 60 Kb approximately. 200 clones of the two plasmids have been sent out for sequencing to close the gap. Similarly, the genome sequence of X. axonopodis pv. citrumelo is completed. Its relationship with Xac is more distant but sufficiently close that they share nucleotide sequence identity over 80% in most genomic regions. Comparative analysis of X. axonopodis pv. citrumelo and X. axonopodis pv. citri is underway.
This project has been revised extensively to take into consideration of suggestions from reviewers and CRDF council. The title has been revised as: Control of citrus Huanglongbing by screening small molecules which are antimicrobial against Candidatus Liberibacter asiaticus. Specifically, the target has been suggested to be SecA for the first year. Protein secretion in bacteria is a critical and complex process. SecA is the protein translocase ATPase subunit and a superfamily 2 RNA helicase, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. Identification of small molecule inhibitors that intrude the function of SecA could lead to potential antimicrobial agent. In order to find the novel inhibitory structures we followed the below steps in the current design. 1) Identification/Build the 3D protein structure & optimization 2) Pharmacophore design based on ATP binding site 3) Virtual screening of commercial databases 4) Generation of a small subset of structures 5) Molecular docking studies & filtration of the structures 6) Selection of best candidates by scoring functions & chemical intuition 7) Biological activity studies against SecA Several 3D structures of SecA protein have been reported in Protein Data Bank (PDB), nevertheless we used the best resolution (<2.0Ao) and ATP bound model 2FSG.pdb in our study. Maestro module of Schrodinger software was used to add hydrogen's, electro static potential charges to the protein and optimized the structure with molecular minimization by using AMBER force field. Then identified the pharmacophores at ATP binding site and subjected these to virtual screening with the Lead Like structures from commercially available ZINC databases. Further with the best hits from database structures we build a small set of ~5000 structures as subset and all these structures were docked at ATP binding site to evaluate the docking scores and their molecular poses at the active region. Based on virtual screening and followed by dock we identified ~4500 structures from millions of commercially available database structures. Then those structures were again docked with xtra-precession by using glide6 program and chosen 2% of top scored structures based on the scoring functions, physicochemical properties and our chemical intuition. Further these structures were energy minimized by molecular minimization studies and evaluated their binding energies to pick the best twenty structures. The selected compounds were used to perform the biological activity studies against SecA. Among these twenty compounds three have shown less than 10'm activity. The high active structures will be utilized to design a potential inhibitor compound against SecA to lead a pesticide drug. All the molecular modeling studies have been performed on HP ProLiant Linux system by using Schrodinger suite7 programs and ATPase assay kit and purified SecA enzyme was used for biological testing.
Five field studies are underway to evaluate the effects of various foliar nutrient applications on the expression of HLB in infected trees by evaluating tree nutrient status, growth, yield and visual tree appearance through photographic documentation. The first trial is a survey-type trial to monitor the health and yield of trees in Maury Boyd’s grove in Felda. We have harvested the same Hamlin and Valencia trees for two seasons and will begin the third harvest on these trees as fruit reach maturity. It is clear from this observational study that HLB infection does not kill well managed citrus trees, and that trees can be maintained with economically profitable yields of quality fruit for at least five years after known infection. The second of these trials is in a heavily infected mature Hamlin grove in south Florida. Since the initiation of the project the trees in this study have received seven foliar applications of nine different treatments. Untreated trees serve as controls. The trees were harvested in December 2009. This was the first harvest since the beginning of the trial and did not reveal any significant differences among treatments; however, that is not unexpected following only 1 year of treatment. That trial will likely be harvested in December 2010 for the second time. The second study is in a young (3-5 years old) commercial Valencia grove in Haines City. Treatments in this study have been underway for approximately 12 months and include fertigation in addition to foliar nutrient sprays. Our initial efforts at this site have been to demonstrate the ability to raise the levels of specific nutrients involved in plant defenses within trees. Since treatments began, B levels have been successfully raised to near toxic levels within infected and healthy trees using both foliar and fertigation applied B. This has demonstrated that good nutrient uptake can be achieved through the treatment methods in a relatively short period of time. Unfortunately, the grower-cooperator began applying a foliar nutrient program to this grove, including our treatment plots, so the future of this trial is now in question. Analysis of the large quantity of data these studies have generated is still being analyzed as of the writing of this report. As soon as the analyses are complete information will be passed along to the Florida citrus community. Two additional field studies were begun during 2010 in research blocks at the CREC. One of these trials involves the application of a commercially available foliar nutrient product with and without the application of SAR inducing compounds. Three treatment applications have been made in 2010 and the trees (Valencia) will be harvested in spring 2011. The second of these trials involves standard and high application rates of foliar nutrients in combination with standard and elevated ACP control. Trees in this 10 acre block are Valencia and Grapefruit and will be harvested when mature. Both blocks continue to be scouted for HLB occurrence and are also being photographed to document visual changes in tree appearance. A hydroponics system has been constructed in an HLB approved greenhouse at the CREC. Trees are growing well in the system and samples are being taken periodically to assess tree nutrient status. Trees are also being monitored for the development of HLB symptoms and will be confirmed by PCR when suspects are detected. Once trees are known to be infected data collection of how the disease develops in trees under different nutrient deficiencies will be collected. This experiment will allow us to begin to separate nutrient and HLB effects on plant growth and development.
A major objective of this project is to develop an understanding of how the HLB bacterium (Las) interacts with citrus genotypes to cause disease. After finding that different citrus genotypes respond differently to Las from extremely sensitive (sweet orange and grapefruit) to tolerance with minor symptoms, we have focused on the one citrus genotype that is most resistant to citrus. Las is restricted to very low levels in Poncirus trifoliata. Most plants remain PCR negative, but a few have barely detectable levels of Las. We have found that under some conditions Las appears not to be able to move through poncirus. We have plants with lower living inoculum that is highly infected with Las, but sensitive sweet orange shoots grafted on top of the poncirus plants have not become infected. We are examining the value of using Poncirus rootstocks and interstocks to reduce or prevent spread of the disease in sweet orange or grapefruit. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las for those plants can be as little as 6 weeks. We are examining this process in more detail now. Second, when we allowed the infected psyllids a choice of different citrus genotypes, there was a large difference in the time and number of plants that were inoculated by the psyllids: (Citrus macrophylla >> Swingle citrumelo >> Volkamer lemon = Duncan grapefruit > Madam Vinous sweet orange >> Carrizo citrange). Most of the Citrus macrophylla plants became infected with only 2 months of exposure in the epidemic room, whereas only a few of the sweet orange and grapefruit became infected after 4 months. Since there was such a clear preference, we are now investigating its cause ‘ whether the preference is related to genotype, growth habit, flushing, or other possible differences. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. We have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or anti-psyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Another large experiment is underway. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and Kirsten Pelz-Stelinski’s lab for psyllid transmission experiments. Among the plants being screened for resistance or tolerance to HLB for other labs are: 1) a series of elite lines for the citrus improvement group; 2) a series of transgenic plants designed to examine the relationship of pectin production to disease development for Jude Grosser, Gene Albrigo, and Nian Wang; 3) we are testing a series of transgenic plants that we made in collaboration with Zhonglin Mou to have increased disease resistance. The trees, which have high resistance to citrus canker, so far do not look like they have resistance to HLB.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 50 different antibacterial constructs have been tested in trees. We have found two peptides that appear to effectively protect sweet orange trees from HLB. However, we and other labs continue screening for better genes that more effectively control HLB and can be approved for use in a food crop. In the California lab, we developed methods to rapidly screen anti-bacterial peptides against Ca. L. psyllaurous in tobacco plants. Tobacco plants were either inoculated with Ca. L. psyllaurous by using the tomato psyllid (Bactericerca cockerelli) and challenged one week later with recombinant Tobacco mosaic virus (TMV) expressing the specific peptides, or the plants first were inoculated with recombinant TMV, followed one week later by using B. cockerelli to inoculate Ca. L. psyllaurous. These assays are being analyzed presently. We also are improving the CTV-based vector to be able to produce multiple genes at the same time. This could allow expression of genes against HLB and canker or multiple of genes against HLB. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. After some delays, we have received permission for USDA APHIS and are now establishing the field test.
Recently we identified several sulfur chemicals from guava that repel Asian citrus psyllid (ACP) in the laboratory, but are difficult to formulate into controlled release devices for field use because of their high volatility. As we continue to work on formulating these sulfur compounds into devices that will have practical application, we have also investigated several potential “of-the-shelf” essential oils for their repellency against ACP. These were chosen based on their known repellency to many insects and based on their perceived similarity to guava in chemistry. ACP generally rely on olfaction and vision for detection of host cues. Certain plant volatiles and plant-derived essential oil products are known to repel several insect species and are considered minimum-risk pesticides. We examined the effect of five essential oils previously reported to have activity against various insect species on ACP behavior in a two-port divided T-olfactometer in the laboratory in an effort to identify an effective natural repellent and/or insecticide for ACP. Volatiles from essential oils of coriander, lavender, rose, thyme, tea tree oil and 2-undecanone, a major constituent of rue oil repelled ACP adults compared with clean air. Also, coriander, lavender, rose and thyme oil inhibited the response of ACP when co-presented with citrus leaves. Volatiles from eugenol, eucalyptol, carvacrol, .-caryophyllene, .-pinene, .-gurjunene and linalool did not repel ACP adults compared with clean air. Chemical analysis of the headspace components of coriander and lavender oil by gas chromatography-mass spectrometry revealed that .-pinene and linalool were the primary volatiles present in coriander oil while linalool and linalyl acetate were the primary volatiles present in lavender oil. Coriander, lavender and garlic chive oils were also highly toxic to ACP when evaluated as contact action insecticides using a topical application technique. The LC50 values for these 3 oils ranged between 0.16 to 0.25 ‘g/ACP adult while LC50 values for rose and thyme oil ranged between 2.45 to 17.26 ‘g/insect. Our current efforts are focusing on quantifying the airborne concentrations of these essential oils found to have behavioral activity against ACP that are required to induce the effect. Our current results suggest that garlic chive, lavender, and coriander essential oils should be further investigated as possible repellents or insecticides against ACP. Also, these repellents may be useful in organic citrus production, which currently has few available tools for management of ACP. We have also developed a method with which to sample and quantify the airborne concentrations of sulfur violates directly in the field. We are perfecting this method so as to be able to directly quantify the airborne concentration of DMDS in the field that is associated with our SPLAT treatments. We believe this will help us understand why certain applications of DMDS show effectiveness in suppressing ACP populations while others do not. Our field results with DMDS released from SPLAT have been mixed. While some trials appeared to show reductions of ACP populations, others did not. We have almost completed a large investigation of four new SPLAT formulations of DMDS and will have that information compiled soon.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. The rationale is that by constitutive expression of an R protein, the plant innate immunity response will be at a high state of alert and will be able to mount a robust defense against infection by phloem pathogens. Overexpression of R proteins often results in lethality or in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: We sequenced all of the constructs introduced into Arabidopsis which consisted of the AtSUC2 promoter (940 upstream from ATG) driving expression of the mutated (constitutive) and wild type forms of SSI4 and SNC1 (R proteins). For SSI4, which is derived from the Nossen cultivar of arabidopsis, two closely related genes (MUF8.3 and 8.2) are present in the Columbia cultivar. We cloned both, and the original from the Nossen cultivar, and cloned them behind the AtSUC2 promoter. These wild type versions of SSI4 will be used as controls for non-active forms of the R protein (pathogen activated). The four AtSUC2/R protein constructs (mutant and wild type of each of the two R proteins) were transferred from the pCAMBIA1305.1 vector, which confers hygromycin resistance, into pCAMBIA2301 with kanamycin resistance since the former is detrimental to transformation into citrus. The four constructs were submitted to the UF Citrus Research Facility at Lake Alfred for transformation into citrus. Transformation of these constructs into the Duncan variety of grapefruit is currently in progress at the Lake Alfred Citrus Research and Education Center (Dr. Vladimir Orbovic). Conclusions: Our hypothesis was that phloem-restricted expression of the R protein constitutive mutants would limit potential negative impacts on growth. The Arabidopsis transgenic plants expressing R protein mutants did not seem to be significantly affected in the majority of cases. Approximately 8% of the snc1 transgenics exhibited a stunted phenotype, very similar to the snc1 mutant expressed from its native promoter (not phloem specific). The first series of ssi4 transgenics (construct 5-2) had a point mutation (C>T) in the coding region that generated a premature stop codon and shortened the protein by 78 aa in the C-terminal region, past the leucine rich repeat (LLR). Five percent of these truncated ssi4 transgenics showed phenotypic differences mostly in the rosette appearance and lighter green, splotchy coloring. However, this effect will be investigated again in the new full-length ssi4 transgenic plants.
This report covers the period April 1 through June 30, 2010. This project was funded July 1, 2009. A no-cost extension of this project was applied for in June 2010 and was granted during this reporting period. Two coordinating meetings were held during this period. The field personnel, in particular, closely coordinated their activities with the personnel analyzing data. Coordination with the aerial application program through Southeastern Air and the Indian River Citrus league continued as did the mapping of data for subsequent time and space analyses of Asian citrus psyllid populations. Due to the massive amount of data being collected in the field, four Garmin GPS units and five laptop computers were purchased during this reporting period. The Garmin units were delivered on June 25, 2010.. This program increased its monitoring activities by increasing the number of tap samples. There were 730 tap sample sites: Indian River 265, St. Lucie 315, and Martin 150. 1. 4040 traps were set and retrieved during this reporting period: Indian River 1,325, St. Lucie 1,575 and Martin 750. 2. Trapping locations by county: Indian River 205; St. Lucie, 225; Martin 75. 3. There were 9,845 psyllids caught in traps duirng this reporting period: Indian River 5,433, St. Lucie3,672 and Martin 740. Over 12,000 miles were driven by field personnel during this period. The data have been analyzed by time and space by Drs. Hall and Gottwald(USDA) and hot spots identified. In our previous report we stated that the data analyses from the first aerial application were inconclusive. The data from the second application in Feb-Mar 2010 also led to an inconclusive conclusion. The second application was made over a period of one month and the lengthy application period appears to be the reason for this result. The one month application period was the result of adverse weather and interference with harvesting.
1- The physical construction/renovation of the growth room did not start yet. A draft of the final layout of the lamps and benches was presented. Several scenarios about air filtration and distribution were discussed as well as safety, security and the irrigation system. Regarding irrigation, it seems like there are concerns about the amount of run-off water leaving the growth room. A holding tank was proposed as a way to contain the water that is coming out of the facility, however the existence of a retention pond south of the building might be the best solution. No decision has been reached at this point regarding the disposal of the water. We make clear to everyone that we need hose bibs in the growth room and there will approximately 2000 gallons/week of run off water coming from the growth room, this is inclusive of watering the plants and maintaining cleanliness. 2 – The first material to establish mother plants from Hamlin 1-4-1 was released from Dr. Peggy Sieburth lab. The clean shoot tips are approximately 4 weeks old and they will need to be grafted in approximately 4 more weeks on rootstocks that are currently growing inside the lab. These rootstocks are being maintained inside the lab for 6 months under laboratory conditions and they needed to be transferred to bigger pots 2 months ago. Under these conditions the grafting will be delay until the growth room is available. Since these rootstocks are already suffering, a new batch was started as a backup. We hope that the shoot tips can hold a few months more until the grafting can be performed. We will need to transfer them several times which in normal conditions is not required. Maintaining shoot tips for a long time on in vitro conditions might also induce juvenility, which we will want to avoid. 3 – Construction of the growth room will start on October 25th according to information provided by the CREC maintenance supervisor. 4 – A grower was selected and he will be at CREC in a few weeks.
In this quarter, the research was focused on the efficacy of the screened chemical compounds against citrus HLB in the field. A completely randomized design with three replicates was conducted at the USHRL farm in Fort Pierce. Seventy-two HLB-affected citrus trees were treated with PS (penicillin and streptomycin) by trunk-injection. Water was used as a control. The applications were repeated once after two months, and samples were taken for PCR assay. Potential phytotoxic effects of PS were also determined in the field. The primary results showed PS may eliminate the Las-bacteria in the treated citrus. When HLB-affected citrus were injected in the field with different dosages of PS, the resulting Ct values increased from 25.7 at pre-treatment up to 34.2 at 2 months after treatment with PS-5 (5.0 g penicillin+ 0.5g streptomycin in 100 ml water solution), which is an approximate 500-fold decrease in the Las bacterial population in the treated citrus plants. At 4 months after the second treatments, the Ct value was ‘ 40.0 (undetectable) indicating elimination of the Las bacterial population in the treated plants. Similar results were found with PS-10 (10.0 g penicillin+ 1.0 g streptomycin in 100 ml water solution). However, phytotoxic effects on citrus were found with PS-10. Penicillin residues in the fruit from the treated citrus were positive (more than 5 ppb) at one week after treatment, but negative (less than 5 ppb) after one month. A grafting system using HLB-affected citrus scions was optimized and used to screen the chemical compounds. HLB-affected citrus (Lime) cuttings (three to four buds) were collected and soaked overnight in solutions of MDL, PS, Kasu, AG, DBNPA, Chaptin, Oxy and KO, respectively, and grafted into Las-free grapefruit. Scions without any treatment were used as controls. A randomized block design was conducted with three replicates and 45 scions per treatment. New growth from the scions was observed in 1 to 2 weeks. Leaf samples were collected for PCR assay 3 month post-grafting. The results suggested that the scion treatment system may be used for screening anti-Las bacteria compounds to control citrus HLB. HLB-affected trees characteristically have a damaged root system and nutritional deficiencies because of interrupted transport of photosynthetic products from shoots to the roots and mineral nutrients from roots to the shoots. Greenhouse and field experiments were conducted to develop an integrated approach for keeping groves with HLB productive by means of disease-curing antibiotics combined with nutritional priming and root restoration. Key nutrients, plant growth regulators, and transporters were screened and several chemicals, called growth-priming substances, have been identified to be useful in keeping the HLB-affected citrus growing and promoting root restoration. Soil application once at two-week intervals with the growth-priming substances in combination with antibiotics was found to reduce the accumulation of starch in HLB-affected citrus leaves and enhance the emergence of new flushes from the severely diseased young trees. These growth-priming substance and antibiotic combinations are being evaluated in field trials.
Obj. 1,2. Construct cDNA libraries from (a) adult/immature psyllids, dissected gut, salivary glands and accessory salivary glands; Sequence random cDNA clones, assemble ESTs, and select unigene sets; Obj.3. Produce RNA-seq libraries and compare transcript levels in psyllids and digestive organs at key acquisition access periods (AAP) to determine the relative abundance of insect mRNAs affected by bacterial infection. We have received from NCGR the Illumina assembled sequences from the Diaphorina citri psyllid libraries: (1) adult gut liberi-infected, (2) immature whole body liberi-infected, (3) adult whole body, and (4) immature whole body. Each library resulted in over 24M sequences and the total set of ~100M sequences assembled into 121444 contigs. The distribution of lengths is very similar to that of our 454 assembled sequences. We have also downloaded the Hunter labs’ Sanger sequences from GenBank, which will be assembled with the 454 and Illumina consensus sequences for further analysis. The website software has been enhanced based on feedback from Dr. Brown. A web domain has been purchased for this project, where the results will be made public once analysis is complete. The first four libraries (above) have been assembled, organized in PAVE, and predictive annotation is nearly complete with links to the GenBank database. Data-mining, qPCR analysis of transcripts, FISH probe design and optimization using our epifluorescence microscope equipped with a filter for Cy5-labeled probes will commence Oct 1 with the hiring of a post-doc. The last two of six adult D. citri (from our P. Stansly/Roberts collaborators) EST libraries have been constructed from, and are undergoing preparation for, Illumina sequencing to improve coverage over initial 454 runs. Space and energy, devoted to rearing potato psyllids, have been ramped up to facilitate transmission studies, dissections, FISH, and SEM/TEM. Use of potato psyllids has accelerated this study because it is a far more tractable system. With it, we can dissect a detailed time-course study to produce data points for extrapolation to the more difficult-to-study D. citri-asiaticus system. Toward B. cockerelli library preparation, two of three collections of whole adults and immatures (all stages) have been made, as well as 1000 dissected, liberi-infected, adults guts. 1000 liberi-uninfected guts will be in hand in less than two weeks. RNA isolation and cDNA synthesis will proceed for these libraries. Transmission studies to determine % efficiency transmission by single B. cockerelli psyllids to tomato plants have been initiated using liberi-infected and -uninfected colonies and a range of AAPs. Two replicated experiments have been completed and a third is underway (0-24 hrs). Next, the more time consuming studies using Liberi-uninfected psyllids will be initiated to determine AAPs and IAPs over time-course, and to be correlated with FISH, qPCR, and TEM (project-34),and quantitative gene expression analysis (RNA-seq). Key time points will be identified and results investigated by RNA-seq analysis and/or qPCR to validate transcript involvement for the D. citri-Liberibacter system.
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