Since the recent release of the citrus genome sequences, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory. In some genes, however, we observed single nucleotide polymorphisms in our cloned genes, compared with the ones published in the citrus sequence database. We think that it might be due to the difference in the plants used in our lab from that used in the citrus genome sequencing. Nevertheless, we found that most published defense genes have full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes. The cloning of these genes is at various stages, some have been amplified from the cDNA library and cloned into pGEM vector while others have already been moved to the binary vector pBinARSplus. The genes newly cloned into the binary vector pBinARSplus include ctNHL1, ctMOD1, and ctJAR1. Since previously cloned ctEDS5 had many single nucleotide polymorphisms (SNP) from the published sequence, we recloned the ctEDS5, which now has fewer SNPs, into pBINARSplus. We have planted the corresponding Arabidopsis mutants plants and transformation will be conducted with the constructs, ctEDS5, ctNHL1, ctMOD1, and ctJAR1 soon. For ctNDR1 + ndr1-1 plants, we are continuously characterizing the defense phenotypes of the homozygous lines. So far we observed a range of defense phenotypes displayed by different individual plants, which is normal with independently transformed lines from one construct. Some plants with stronger disease resistance are also associated with minor cell death phenotypes. For ctPAD4 + pad4-1, ctEDS1 + eds1-2, and ctEDS5 + eds5-1 plants, we are trying to get more transgenic plants, obtain homozygous lines, and/or perform initial defense tests.
We are continuing to examine the interactions between the psyllid, the plant, and the greening bacterium. We are examining the disease epidemic under confined conditions. 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 is much shorter that we expected. 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 also 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. Trees are not being examined in the field that first were maintained under heavy inoculation pressure by infected psyllids for several months. Other peptide protected plants are being prepared for field testing. 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. We routinely screen citrus genotypes or transgenic citrus for other labs for tolerance or resistance to greening or psyllids.
The main objective of this project is to examine how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development and how it correlates with the distribution of the bacterium within infected trees. During this period of funding we established a psyllid colony for the use in our experiments. One portion of healthy psyllids is maintained on healthy Murraya paniculata plants in special insect cages and the other portion is exposed to HLB-infected citrus plants to generate HLB-infected psyllids that can be further used for inoculation of new citrus plants. Psyllid transmission experiments were initiated using already available HLB-infected plants. Additionally, we inoculated new sweet orange and grapefruit plants using the infected psyllids and graft-inoculation with HLB-containing buds to generate more plant material that can be used in our further experiments. The newly inoculated plants will be monitored for development of HLB infection and then used for transmission experiments. To examine acquisition of the bacterium from different types of flushes, healthy psyllids (as nymphs and adults) were placed on either a young growing flush or an older symptomatic flush of an infected tree. Psyllids were secured on those flushes by using small traps made up of mesh material. After 21 days traps were removed and psyllids were analyzed by PCR with HLB-specific primers. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. However, TEM observations of the material extracted from psyllids demonstrated that psyllids fed on different types of flushes contained different forms of Las: psyllids fed on the young flushes contained mostly rod shaped bacteria, while bacteria extracted from psyllids that fed on the old symptomatic flushes were mainly present in a round form. We are now conducting experiments that will determine the viability of the bacteria acquired during psyllid feeding on different tissue sources. Another interesting observation was made during these experiments. Psyllids fed on the old symptomatic tissue exhibited phenotypical abnormalities. Our next step is to assess whether these developmental abnormalities are due to psyllid feeding on the symptomatic diseased tissue or similar phenotype can be obtained from incubation of psyllids on old flushes of healthy plants. During this period we also began experiments in which we are examining what types of flushes are more susceptible to psyllid inoculation. In these experiments infected psyllids are placed on different types of flushes of healthy plants. Plants are grouped in two sets: first set in which only young flushes are exposed to psyllids and second set in which psyllids are placed on old flushes. Psyllids are kept for a period of 21 days on those plants. The experiments are in progress. Percentage of plants that become infected will be analyzed.
The initial indexed mature material that was maintained in vitro for a long time did not adapt properly after planting in soil. The plants have already 3 months and they have very long leaves and are growing stunted, most of them are not behaving like normal plants. We are going to continue monitoring them the next couple of months to see if they recover and come out of this stage. The new batches of indexed mature material introduced from May to July 2011 are being grafted on Swingle citrumelo, Macrophylla and C. Volkameriana that were growing originally inside the laboratory. This material will also be used to establish mother plants and to establish the first batch of plants that will give us the material for mature transformation experiments. The rootstocks that we started planting when the growth room was finished are not ready for grafting yet. Only a few plants coming from this material were available to graft and the whole group will be ready in October 2011. The Growth Room is still under “modification”. It took almost 5 months to be able to have completely access to the computer program, however it is still an issue to get access for users that come and go which is the case with the personnel we currently have. The Citrus Research and Education Center does not have enough IT help to assist in a timely fashion with the several needs required for this project. The IT people usually respond within weeks instead of hours to any problem we may have. The humidifier in the small room is still not working properly. The company responsible for the job was not able to coordinate the different subcontractors to finalize the job. The warranty in this case will not work and we will need to pay for them to finish this task. Another problem that we are currently facing is related with filtrations among the different areas where the floor meets the walls but also window sealing. The water is moving between growth rooms and between the growth room and the office. The caulking applied to close the gaps and to provide a seal between the concrete floor and the panel walls is not working. The subcontractor came and applied a new coat of caulking but it was not enough to stop the filtration. It is still happening as of today. Water leaking through the window has not been addressed yet. We also experience problems with tripped breakers and bad electrical connections of some lamps. We are still monitoring the situation.
The causal agent of greening, Candidatus Liberibacter asiaticus ( L asiaticus) is a fastidious phloem-inhabiting gram-negative bacterium. It is transmitted by Asian citrus psyllid (ACP) during feeding. Transmission mechanisms of Las have been intensely investigated recently, yet key information gaps still existed. We investigated whether Las is transmitted between infected and uninfected ACP adults during courtship. Our results indicate that Las was sexually transmitted from Las-infected male ACP to uninfected females at a low rate (< 4%) during mating. Sexual transmission was not observed following mating of infected females and uninfected males or among adult pairs of the same sex. Las was detected in genitalia of both sexes and also in eggs of infected females. A latent period of seven days or more was required to detect the bacterium in recipient females. In addition, Las was detected in the unlaid eggs and offspring of infected females. Rod shaped as well as spherical structures resembling Las were observed in ovaries of Las-infected females with transmission electron microscopy but were absent in ovaries from uninfected ACP females. The size of the rod shaped structures varied from 0.39 to 0.67 'm in length and 0.19 to 0.39 'm in width. The spherical structures measured from 0.61 to 0.80 'm in diameter. Although, the overall percentage of sexual transmission of Las from males to females appears small, it could be a highly significant contributing factor to pathogen spread given that thousands of psyllids colonize individual trees. Collectively our results demonstrate that Las is transmitted from male to female ACP at a low rate during mating. These results suggest that the presumed causal agent of HLB may spread within populations of the vector even in absence of infected host plants. These findings provide an alternative sexually horizontal mechanism for the spread of Las within populations of ACP, even in the absence of infected host trees. Detection of Las in female psyllids reared on Las-free citrus plants after mating with infected males strongly suggests that this plant pathogenic bacterium was transferred sexually from male to female insects during mating. This was also confirmed with lack of transmission from females to males, or between same sex pairs. While this phenomenon has been demonstrated experimentally for endosymbiotic bacteria of insects, this is the first report of sexual transmission of plant pathogenic bacteria among insect vectors. The distribution of Las appears to be ubiquitous throughout the hemolymph and organs of infected psyllids, although the bacterial titer is highest in the alimentary canal and salivary glands. We observed a low bacterial titer (Cq values 37 to 39) in 2.3% and 8.9% of individual male and female genital samples, respectively, suggesting a low bacterial titer in the reproductive organs of ACP. Cq values could also be high because of the small amount of tissue that is processed during DNA extraction even though there may be a high bacterial load relative to the size of the organ examined. If the overall titer of bacteria in the infected psyllid donor is low, the likelihood of bacteria occurring in the reproductive organs should also be correspondingly low. Detection of Las in the unlaid eggs and offspring of infected females is congruent with previously reported transovarial transmission of Las by ACP. Sexual transmission of Las provides additional, albeit indirect, evidence that the relationship between the pathogen and ACP could be mutualistic rather than pathogenic.
A scFv library with activity against ‘Ca. Liberibacter asiaticus’ has been prepared at Beltsville. mRNA was purified from mouse spleens and converted into cDNA. The mice had been immunized with psyllid extracts confirmed to be carrying a high concentration of “Ca. Liberibacter asiaticus” A complete library of variable heavy chain (VH) and variable light chain (VL) genes were made by PCR amplification of the cDNA using a set of 44 primers. The (VH) and (VL) gene segments were then joined in a random combinatorial fashion by overlap extension PCR. The scFv genes were then ligated into the pKM19 phagemid vector which was used to infect Escherichia coli DH5. F’ cells with the aide of a helper phage. The resulting phage library is presently being screened to select phage clones expressing antibodies that bind to “Ca. Liberibacter asiaticus”. Our library is estimated to contain 2.1 x 10 7th primary, unique antibody clones. Because our antigen was individual psyllids from Florida infected with high concentrations of ‘Ca. Liberibacter asiaticus’, the library contains antibodies for both the pathogen and the vector insect. Our first attempts to select desired antibodies using extracts from HLB-infected rough lemon were not successful, probably because the concentration of the target bacteria in the rough lemon extracts was too low. These approaches are described in previous reports. We have now obtained a unique set of scFv antibodies including ‘B665, B705, B717, B734 and B743’ that bind to a portion of the major outer membrane protein, OmpA; B900, B905, B913, B932, B1037, B1071, B1072, B1075 and B1083 that bind to flagellar protein FlgL; B947, B968, B969, B982, B984, B1096, B1114, B and B1115, B1119 and B1128 that bind to flagellar protein FlhA; B435, B466, B467, B468, B475, B500, B547, B556, and B557 that bind to a pilus protein; B442, B443, B479, B494, B520, B1191, B1194, B1199, B1202 and B1212 that recognize a protein that polymerizes cell surface polysaccharides.We have also isolated a number of scFv that recognize the TolC protein of ‘Ca. Liberibacter asiaticus. We isolated these scFv by using genomic sequence data to clone and purify specific proteins to beused as capture antigens. Portions of these proteins predicted by software to be exposed on the cell the surface were cloned. Correct cloning was confirmed by DNA sequencing and these genes have been expressed in E. coli and the encoded proteins have been purified. Emphasis is on recovering the scFvproteins in native, soluble form, which was difficult, but we have now been able to accomplish it. Thus we have developed and demonstrated a protocol that will allow us to isolate scFv antibodies that, in principle, will recognize any proteins from “Ca. Liberibacter asiaticus” or the insect vector, Diaphorina citri. Several of these proteins have also been successfully used in Das-ELISA assays and in dot blot assays of plant and insect extracts containing ‘Ca. Liberibacter asiaticus’ from Florida. Premature termination of scFv proteins has been encountered in the soluble expression system. This is because ‘stop’ codons accumulate in the phage selection system because the host E. coli is an amber suppressing strain. But these ‘stop’ codons become a problem for production of soluble scFv. Therefore we are checking all clones by sequence analysis to be sure premature stop codons are not present. This approach allows production of soluble scFv. Several of these antibodies detect antigen in plant but not in insect extracts. scFv antibodies may be useful as labels for ultrastructural studies of infected plants and insects, advanced detection assays, and possible even for HLB control through a ‘plantibody-based’ approach.
The main objective of this project is to manipulate calcium signals by over-expressing calcium signal modifier genes (CSM) from citrus to develop broad spectrum disease resistance. During the fiscal year 2010-2011 we produced 17 transgenic C-22 rootstocks, three Valencia and six Hamlin oranges over-expressing the CSM-1 gene. Previous grapefruit plants produced with this gene Were tested for disease resistance and shown to be resistant to citrus canker, Phytophthora nicotianae and Alternaria alternate and to the toxin tentoxin. Furthermore we engineer a new Agrobacterium binary vector with a citrus lectin gene to be used for genetic transformation during the fiscal year 2011-2012. As part of the transformation procedure, we are trying to accomplish genetic transformation without the use of antibiotic, and using only citrus genes. We were able to find a substance that increase the regeneration capacity of citrus stem segments used for genetic transformation and at the same time have the potential to be used for selecting transgenic plants and replace the antibiotic selection system. We are currently optimizing the system. We performed several crosses of Rio Red X Hirado Buntan pummelo and Rio Red X Wilking tangor. Hybrids will be recovered in November 2011.
In our initial schedule, the mature transformation facility (lab and greenhouse) at the CREC in Lake Alfred had to be implemented during the first year of the project. An existing laboratory was modified to fulfill the requirements of a tissue culture facility. The laboratory is now fully operative. Regarding the greenhouse, it became impossible to accommodate the budget to our plans for constructing the outstanding facility we requested. Alternatively, a growth room was designed profiting an existing structure at the CREC. The growth room construction was initiated in October 22nd 2010. The projected date of completion was February 11th 2011. Technically it was finalized by mid-April, however there were several technical/operational problems that came out during the following 4-5 months, especially regarding the refrigeration system (environmental conditions were not estable; some air handlers were not producing the air the manufacturer claims, in some cases the thermal expansion valve was changed because it was defective, air filters were not the ones we requested and they were changed, the humidifier in the small room is not located in the appropriate place for working properly), computer program (growth room technician still doesn’t have access to the program though this is currently being solved), water elimination after irrigation is defective, soil sterilizer (it needs a special accommodation to work ‘safely’). A generator should be purchased; without it, any prolonged electricity cut could jeopardize the whole project. The manager from Florida (Dr. Cecilia Zapata) completed her training in Spain during the first year, moved to Florida and has been working hard to set up the mature transformation facility at the CREC during the whole second year. Two part time OP technicians were hired to work on tissue culture and on plant preparation and a third OP technician was hired to work care at the growth room, under the supervision of the manager (and the PI at the initial stage). Another technician has been recently hired to help in the growth room and the lab because one of the OP technicians is leaving soon due to personal reasons. The Spanish lab has been monitoring the progress of the Florida facility. The PI and his manager at the IVIA greenhouses traveled to Florida last March 2011 to supervise the growth room construction and to set up healthy citrus germplasm bank establishment. The PI and his greenhouse manager will travel again to Florida next October 2011. An IVIA scientist with experience in mature citrus transformation will travel to Florida to help setting up the facility tentatively next November 2011. It is programmed that the IVIA scientist will spend three months in Florida (November 2011-February 2012). In Spain, mature tissues from the three sweet orange types (Hamlin, Pineapple and Valencia) plus Carrizo citrange were readily transformed. For our second objective, improving citrus tree management, we proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. We have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange over-expressing either CsFT or CsAP1 flowering-time genes already established in the greenhouse. We have characterized these transformants at the molecular level and continue characterizing them phenotypically in detail in the greenhouse. Moreover, for generating a dwarf-dwarfing rootstock, we have incorporated a construct aimed to induce RNA interference to downregulate the expression of a crucial gene in gibberellin biosynthesis, CcGA20ox1, in mature Carrizo citrange.
Objective 1: Comparative methodology was initiated to compare phloem plugging and collapse with different staining techniques. Phloem samples were collected and prepared from healthy and HLB affected trees. Preliminary results suggest that a rapid survey of plugging and starch accumulation is possible for general surveys of functional and non-functional phloem. Objective 2: Transformation experiments to produce plants that over-express the citrus ‘-1,3-glucanase gene: Experiments to validate the stability of the ‘-1,3-glucanase gene in transgenic greenhouse plants were initiated. PCR analysis on 9 of 10 plants showed the ‘-1,3-glucanase gene band, indicating stable transformation. All recovered transgenic clones (mostly Valencia, Duncan and Carrizo) were micropropagated, producing 4-5 replicates of each clone for further testing, including HLB challenge. Embryogenic callus transformation with the ‘-1,3-glucanase gene: More than 200 GFP+ somatic embryos were recovered from sweet orange OLL#20, and > 100 transgenic shoots were transferred to rooting medium. About 50 GFP+ embryos and shoots from Jin Cheng sweet orange and 20 GFP+ embryos and shoots from Valencia were also recovered ‘ which should result in stable transgenic plants in these important cultivars. Objective 3:To identify the putative genes involved in HLB disease development and psyllid transmission, quantitative reverse-transcriptional PCR (QRT-PCR) assays using total RNA isolated from infected plants and psyllids were conducted. Gene specific primers were used to check the expression of 506 genes in Ca. L. asiaticus. The genes showing a differential expression of two fold or more in either the plant or psyllid were categorized into Clusters of Orthologous Groups of proteins functional categories. Potential virulence related genes including hypothetical genes, which were overexpressed in planta, were selected. Differential expression of these selected genes were also evaluated in susceptible and tolerant varieties of greening infected citrus. Several ABC transporter genes and genes involved in protein export, along with many genes involved in porphyrin and chlorophyll metabolism, were upregulated in the plant. Most of the genes that were overexpressed in the psyllid included those involved in cell motility. The genes overexpressed in planta identified in this study will also be screened by transient assays on Nicotiana benthamiana plants. The results from this study will be useful in identifying the potential virulence genes involved in symptom expression of this pathogen in planta.
For the construct containing the ctEDS1 gene in the binary vector pBINplusARS, we selected the T0 seeds and obtained so far 15 independent T1 transgenic plants. The T2 plants will be planted to select for homozygous lines and also for an initial disease resistance test. For ctNDR1 transformation of the ndr1-1 mutant, we currently obtained 11 homozygous lines. We reported earlier that some of the lines showed enhanced disease resistance. Now we are at a stage to systematically analyze the defense phenotypes of the ctNDR1 overexpressing transgenic plants, using the homozygous lines that we have. For ctPAD4 + pad4-1 and ctEDS5 + eds5-1 plants, we have obtained over 10 and 3 independent T1 transformants, respectively. We have planted the T2 seeds and will test the segregating T2 plants for disease resistance and harvest seeds from multiple individual plants for selection of homozygous lines. For ctEDS5 + eds5-1, we are also selecting more T0 seeds in order obtain additional transformants. Additional newly cloned genes include ctSID2, encoding the major biosynthetic enzyme for salicylic acid biosynthesis, and ctNHL1, which is a homolog of NDR1. These two genes were obtained from RACE followed by RT-PCR. We have moved the ctNHL1 cDNA fragment from the pGEM T-easy vector to the binary vector pBINplusARS for plant transformation. For ctSID2, we only obtained the cDNA clone in the pGEM T-easy vector. However, we have had some trouble in moving this fragment into pBINplusARS. We are currently trying a few different approaches to address this problem. Since the recent release of the Citrus sinensis (sweet orange) and clementine genome sequence, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory with this support. In addition, we found that most published defense genes are present in citrus with full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes. The cloning of some of these genes is underway.
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: The transgenic plants containing AtSUC2/snc1 and AtSUC2/ssi4 mutants, as well transgenic control plants are growing in the laboratory of Dr. Orbovic at the UF Citrus Research Facility (Lake Alfred) until they are ready for the next level experiments. Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. By further restricting expression of the R protein to a single cell that is pierced by the insect stylet, we anticipate that a defense can be mounted without a manifestation of a dwarf phenotype. Results: The vast majority of T1 and T2 transgenic Arabidopsis plants expressing snc1 and ssi4 mutant coding sequences under the control of the AtSUC2-940 promoter have wild type phenotypes. Although the AtSUC2 promoter has been reported to be phloem-specific, we have found that it often does not maintain this tissue-specific pattern of expression in transformed Arabidopsis. However, despite the likelihood of expression in tissues other than phloem, only a few transformants showed any negative developmental or growth abnormalities. This lack of a negative phenotype in Arabidopsis provides a basis for optimism for similar results in transformed citrus. Our working hypothesis is that expression of the constitutive R proteins (mutants) in the phloem will active components of the innate immunity response to provide enhanced protection from Liberibacter infection in phloem cells. In order to monitor the activation state on the innate immunity system, we will cross the R protein transformants with transformed Arabidopsis lines containing pathogen-inducible promoters driving GUS reporter genes. We cloned the PR2 (also known as BGL2), and PR5 pathogen-inducible promoters in front of the GUSplus gene in pCAMBIA 2301. They were sequenced, transformed via electroporation into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis (strain GV3101) through the floral dip protocol in order to generate stable transgenic lines. We currently await the T-1 seeds from these transformations. In parallel, we acquired BGL2-GUS (in pBI101 vector; from Dr. Xinnian Dong from the Duke University) stable transgenic line to use as an alternative donor. The introduction of our R protein constructs into reporter lines by crosspollination will be faster and more efficient than transformation by agrobacterium. Being able to monitor constitutive activation of the innate immunity system by GUS will provide a test of the hypothesis that our constructs will activate pathogen-inducible promoters and will allow us to select lines that have strict phloem-specific expression for further study.
Research on LAS in this quarter has focused on monitoring viable LAS concentrations over time in different culture treatments. Replicate culture treatments were created and inoculated with a LAS suspension obtained from seed of infected pomelo fruits. Monitoring has been conducted with two methods: 1) quantitative polymerase chain reaction (qPCR) of cells treated with ethidium monoazide (EMA) and 2) microfluidic chamber observations. Several LAS experiments have been conducted using three different culture treatments The three different culture treatments are created in duplicate culture flasks. One contains only King’s B (KB) media at a 1/3 diluted concentration. The other two treatments contain the same media with 25% or 50% juice solution. The juice solution is obtained by grinding the pulp from the infected fruit with deionized water and filter sterilizing the resulting solution. Each culture medium is then inoculated with a bacterial suspension created from infected seeds. At the time of inoculation, samples are collected, either treated with EMA or not treated, and frozen. Samples are then collected every two days from that point and treated in the same manner. DNA is extracted from frozen samples and analyzed by qPCR to determine the numbers of viable and non-viable LAS cells. Samples are also collected and injected into the microfluidic chambers at the time of culture inoculation, using the same media described above. The chambers are monitored daily with a microscope for visible cells and any signs of bacterial aggregation or biofilm formation. After cells are monitored for ~1 week, remaining cells are eluted from the chambers and tested with EMA-qPCR as was done for the culture flask samples. Initial results indicated that LAS cells may lose viability more slowly in media containing juice. However, these results came from ripe pomelo fruits that were collected in early summer and represented the last of last year’s fruit. Now, the fruits that can be collected are this year’s immature green fruits. These results have not been replicated with the new fruits. The new fruits have much higher initial cell titer as well as cell viability (~20%) compared to the older ripe fruits (~5%). However, the immature fruits seem to have some endophyte that grows in the cultures as a contaminant. This contamination causes the cultures to be unusable after ~3-5 days post-inoculation. We are working on a way to deal with this problem. In the microfluidic chambers, cells similar in size to that reported for LAS cells have been observed aggregating in some experiments. We eluted them to try to determine if they were LAS cells. Using EMA-qPCR, the samples sometimes have detectable LAS DNA, but no viable LAS DNA. It is unclear if this is because there are not viable cells in the chambers or if it is because they are below the limit of detection. Future work will try to clarify this.
Research on LAS in this quarter has focused on monitoring viable LAS concentrations over time in different culture treatments. Replicate culture treatments were created and inoculated with a LAS suspension obtained from seed of infected pomelo fruits. Monitoring has been conducted with two methods: 1) quantitative polymerase chain reaction (qPCR) of cells treated with ethidium monoazide (EMA) and 2) microfluidic chamber observations. Several LAS experiments have been conducted using three different culture treatments The three different culture treatments are created in duplicate culture flasks. One contains only King’s B (KB) media at a 1/3 diluted concentration. The other two treatments contain the same media with 25% or 50% juice solution. The juice solution is obtained by grinding the pulp from the infected fruit with deionized water and filter sterilizing the resulting solution. Each culture medium is then inoculated with a bacterial suspension created from infected seeds. At the time of inoculation, samples are collected, either treated with EMA or not treated, and frozen. Samples are then collected every two days from that point and treated in the same manner. DNA is extracted from frozen samples and analyzed by qPCR to determine the numbers of viable and non-viable LAS cells. Samples are also collected and injected into the microfluidic chambers at the time of culture inoculation, using the same media described above. The chambers are monitored daily with a microscope for visible cells and any signs of bacterial aggregation or biofilm formation. After cells are monitored for ~1 week, remaining cells are eluted from the chambers and tested with EMA-qPCR as was done for the culture flask samples. Initial results indicated that LAS cells may lose viability more slowly in media containing juice. However, these results came from ripe pomelo fruits that were collected in early summer and represented the last of last year’s fruit. Now, the fruits that can be collected are this year’s immature green fruits. These results have not been replicated with the new fruits. The new fruits have much higher initial cell titer as well as cell viability (~20%) compared to the older ripe fruits (~5%). However, the immature fruits seem to have some endophyte that grows in the cultures as a contaminant. This contamination causes the cultures to be unusable after ~3-5 days post-inoculation. We are working on a way to deal with this problem. In the microfluidic chambers, cells similar in size to that reported for LAS cells have been observed aggregating in some experiments. We eluted them to try to determine if they were LAS cells. Using EMA-qPCR, the samples sometimes have detectable LAS DNA, but no viable LAS DNA. It is unclear if this is because there are not viable cells in the chambers or if it is because they are below the limit of detection. Future work will try to clarify this.
After we received the notes from the Evaluation Committee on May 10, 2011, thirty-seven compounds from the listed 48 were ordered. Eleven compounds are not available, and they should be supplied by the participators. One additional compound sent by Syngenta was received for a test. Thirty-three compounds have been used to treat the HLB-affected scions (Lime) and grafted onto the healthy rootstocks (Duncan). Thirty scions were treated for each compound. All the grafted scions grew well now. More than 300 rootstock seedlings have also been purchased and planted in the greenhouse for further test.
We are continuing to examine the interactions between the psyllid, the plant, and the greening bacterium. We are examining the disease epidemic under confined conditions. 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 is much shorter that we expected. 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 also 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. Trees are not being examined in the field that first were maintained under heavy inoculation pressure by infected psyllids for several months. Other peptide protected plants are being prepared for field testing. 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. We routinely screen citrus genotypes or transgenic citrus for other labs for tolerance or resistance to greening or psyllids.
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