Objective 1 (To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface). Xanthomonas citri subsp. citri (Xcc) A strain types are causal agents of Citrus Bacterial Canker (CBC) on most Citrus sp. and close relatives. Two narrow host range strains of CBC, Aw and A*, from Florida and Southwest Asia, respectively, are only able to infect Mexican Lime. As for many phytopathogens in the early stage of infection, Xcc must ingress the leaf surface to colonize the apoplast. This process may be mediated by plant signals originating from the sites of entry, such as stomata or wounds. In this study, the chemotactic profile of wide and narrow host range strains of CBC was compared with other xanthomonads causing Citrus Bacterial Spot (CBS) and Crucifer Black Rot (CBR) and related to carbon source utilization evaluated by Biolog GN system. Differences among these species and types of xanthomonads were found for motility, chemotaxis, bacterial growth and the profile of chemicals that act as chemotaxis inducers. The diversity of chemotaxis profiles was related to the patterns of methyl-accepting chemotaxis proteins (MCPs) that act as chemotactic sensors. Cluster analysis based on chemotaxis profiles and MCPs grouped narrow host range CBC strains into the same clade. Chemotactic response of CBC and CBR strains towards leaf fractions from sweet orange, Mexican lime and Chinese cabbage were compared. Differential chemotaxis responses occurred for leaf washes and apoplastic fluids depending on the combination of xanthomonad and host plant. These results suggest that xanthomonads sense signals from the host which facilitate the location of leaf entry points for specific bacterium plant associations. Objective 2 (To investigate bifofilm formation and composition and its relationship with bacteria structures related with motility in different strains of Xcc and comparison to non-canker causing xanthomonads). Differences in biofilm formation were demonstrated among the diverse CBC strains and compared to X. campestris and X. alfalfae subsp. citrumelonis. Presently, type IV pilus from Xanthomonas citri subsp. citri strains is being purified in order to obtain antibodies to be used in a microscopy strategy to confirm such protein as a main component of the protein fraction of the biofilm matrix and determine possible differences among bacterial strains. In addition assays to detect cellulose and amyloid fiber production by CBC strains, using calcofluor and Congo red, respectively, are underway. Amyloid fiber but not cellulose has been detected in preliminary assays. Moreover, role of DNA in the biofilm matrix is being evaluated by applying DNAase at different concentrations during the biofilm formation or removal process. First results confirmed the positive effect of the DNAase and therefore the role of free DNA as a component of the biofilm matrix. More assays are underway to compare altering of DNA with that of other biofilm matrix elements. Finally, initial assays of gene expression revealed differences in the level of transcription between wide and narrow host range strains of CBC for genes related to biofilm and motility.
he objectives are 1) apply a protocol for sampling grapefruit for streptomycin resistance, 2) quantify the local systemic activity of streptomycin for control of Xcc inoculum in lesions of grapefruit; 3) evaluate the efficacy of mixing copper with streptomycin compared to streptomycin alone for reduction in risk of streptomycin resistance in Xcc. Treatments of streptomycin in three grove company locations as well as a trial with additional sprays this season will be monitored in September 2013 for incidence of resistance using a sampling protocol previously developed in our program. As soon as the results are available they will be forwarded to EPA section 18 for their information/comment.
The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. During this quarter we accomplished: ‘ The paper ‘Distribution of QoI resistance in population of tangerine-infecting Alternaria alternata in Florida’ was submitted to Plant Disease. This paper was accepted for publication. ‘ The book chapter ‘Fungicide resistance in citrus’ was submitted to be published in the book ‘Fungicide resistance in North America, 2do edition’. ‘ The tangerine non-pathogenic Alternaria populations were tested for azoxystrobin sensitivity. During this time 124 isolates were screening for sensitivity using the rezasurin-based microtiter assay. ‘ In summary: 90 isolates (72%) were resistant to azoxystrobin (EC50 values higher than 10 ‘g/ml), while 34 isolates (28%) were sensitive (EC50 values lower than 0.5 ‘g/ml). ‘ DNA of 20 tangerine non-pathogenic isolates was extracted to identify the G143 point mutation in the cytochrome b gene. ‘ In vitro fitness components of A. alternata tangerine pathotype were established using five QoI-resistant (R) and five QoI-sensitive (S) isolates. This experiment was performed twice. Fitness parameters evaluated were: mycelium growth, sporulation and conidium germination. Those isolates were added to the last 10 isolates tested previously. ‘ DNA extraction and RFLP-PCR analyses were performed using the cytochrome b gene amplification of the remaining 10 isolates (used in the In vitro fitness experiment) to identify the G143A mutation in resistant isolates. In summary: all five R isolates carried the G143A mutation. In contrast, the five S isolates did not have the G143A point mutation. ‘ The study of baseline sensitivity to boscalid was started using the rezasurin-based microtiter assay. During this trimester, 405 isolates (arbitrary selected from 2008-2010 survey) were tested. The mean Effective concentration needed to reduce the growth by 50% (EC50) was 0.57 ‘g/ml. ‘ The paper ‘Stability and fitness of Alternaria alternata tangerine pathotype’ was started.
The Mature Tissue Transformation Laboratory (MTTL) continued to increase its preparedness for the first incoming orders. Number of rootstock plants available for budding was increased significantly and supply is now at the level necessary for normal operation. In the last three months, seven co-incubation experiments were performed. In three experiments, 2212 explants of Valencia were used. Two co-incubations were done with 497 Hamlin explants. One experiment was done with 240 explants from Pineapple orange plants and one with 701 explant of Ray Ruby. The experiment with Ray Ruby was the first experiment done using the grapefruit explants. The data were analyzed from three experiment performed in the previous reporting period and from four experiments performed in this reporting period. For both binary vectors used pCAMBIA2301 and pTLAB21, transformation rate is about 3%. Because of the problems with the budding success rate, the decision was made to change provider of grafting services again. Within the last 12 months, major efforts were directed towards keeping the facility operational, employees retained, and number of rootstock plants increased to levels needed for performing 9-10 experiments per quarter. Those goals have been achieved. By doing multiple repetitions of transformation experiments with bacterial strains carrying two different binary vectors, proper estimation of transformation success rate was obtained. That rate is at satisfactory level for citrus mature tissue transformation. However, there is a possibly lingering problem that needs to be addressed. Many of the plants produced to be the source of explants in co-incubation experiments have thorns that are one of the major features of juvenility. As a result, at least a half of transgenic plants already produced in the MTTL also have thorns. Within next few months, the oldest transgenic plants in our inventory will reach the age where they should theoretically flower. If they do not flower, protocol used for production of transgenic plants will have to be re-evaluated. Also, we must make sure that the sources of our germ-free certified material for production of ‘mother’ plants are really mature trees.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. This report marks the end of the first year of the project, during which we have achieved large-scale production of CLas positive ACP. To date on this project, a technician dedicated to the project has been hired, a second career technician has been assigned part-time, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies are being used for caged infestations. A total of 3,583 transgenic plants have passed through the screening program. A total of 71,760 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects.
The goal of this experiment is twofold, first to determine the effects of plant growth regulators on addressing vascular degeneration and fruit drop, and second to determine the effects of HLB and ACPS citriculture on drought tolerance. A field experiment was installed in April 2013 to test the efficacy of the synthetic auxin 2,4-D and a micro-emulsion ‘based surfactant to reduce HLB symptom severity in a mature ‘Hamlin’ orange block. The HLB incidence in the block is currently more than 50% and consequently the fruit yield losses due to pre-harvest fruit drop from symptomatic trees were devastating in the 2012/13 season. The experimental design is a 4×4 Latin Square with four replications and four factorial foliar spray treatments consisting of 2,4-D, Eco-Agra’ surfactant, 2,4-D + Eco-Agra’, and untreated control. Each whole plot is split into two sub-plots containing Swingle and Carrizo rootstocks. A basal foliar nutrient spray treatment applied to the whole experiment consists of a comprehensive, balanced fertilization program of micronutrients, macronutrients and potassium phosphite products timed to coincide with the major leaf flushes. The basal ground-applied fertilizer program consists of a dry granular bulk-blended N-P-K +Ca +Mg + Fe + Mn +Zn +B +S product applied four times in the growing season. The automated micro-sprinkler irrigation system is used to apply water to the trees according to seasonal evapotranspiration demand as needed, up to twice per day. Measurements of the trees in this experiment will begin in the summer, and are designed to determine if the PGR and surfactant treatments can reduce the debilitating damage caused by the Liberibacter pathogen to the phloem tissue, and to the whole-tree physiology. They will include photosynthetic rates, phloem transport functions, HLB symptom severity, leaf nutrient concentrations, leaf drop and fruit drop. Preliminary hydraulics data suggests that trees grown under conventional microsprinkler irrigation systems, and watered 2-3 times per week are more resistant to drought induced declines in hydraulic conductivity compared to trees in the ACPS system. We are now sampling the trees again for hydraulics measurements to confirm the preliminary results and are also investigating the anatomical components of the resistance.
Understanding the transmission of CLas within the citrus tree remains one of the principal obstacles in the global efforts to undermine the pathogenicity of HLB (citrus greening). The movement of CLas has been assumed to follow the photoassimilate stream through the phloem. However, many observations based on our knowledge of the bacteria and general phloem anatomy have exposed inconsistencies with the accepted beliefs. The brevity of available information on the ultrastructural properties of citrus phloem sieve elements has hindered efforts to understand the spread of the disease within a tree. For example, lateral movement of CLas around an infected stem appears improbable given the size of cytoplasmic plasmodesmata connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from the roots to uninfected aerial tree parts through the phloem seems highly unlikely given the direction of phloem sap. To date we lack a thorough investigation into the ultrastructure of citrus phloem and the surrounding tissue, the potential pathways that CLas could utilize to move long distances through citrus trees, and the location of CLas habitat within different citrus tissue. Using a variety of grafting and girdling experiments, SEM, TEM, confocal, high resolution computed tomography, and PCR tissue analysis we aim to gain a better understanding of the anatomical traits that facilitate the spread of CLas through citrus. These data will allow us to develop new screening tools that breeders can use to select for resistant scion/rootstock combinations to confer resistance or tolerance to HLB. As of this progress report the Valencia/Swingle trees are in the process of being grafted together. Once the grafting heals the trees will have HLB infected buds and branches grafted onto the scion or rootstock depending on the experiment. Trees will be dissected prior to grafting on the HLB infected material, and then trees will be sampled for anatomical studies and PCR testing at regular intervals for the next year. We have hired personnel to maintain the plants in the greehouse during the duration of the experiments.
The main goal of this project is to optimally deploy the superinfecting Citrus tristeza virus (CTV)-based vector to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. We have several sets of the experiments in which we are examining how prior infection with different CTV strains affects the ability of the superinfecting CTV vector to infect and get established in the same trees as well as examining the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. Plant material that is being used in this project and CTV inoculum sources (different isolates of CTV propagated in the greenhouse as well as collected on the field) have been prepared. The experiments to assess the effect of preexisting CTV infections on multiplication of the superinfecting vector in inoculated citrus trees are ongoing. We first graft-inoculated sweet orange trees with the T36,T30 or T68 isolate of CTV, the isolates that were propagated in our greenhouse, as well as with CTV-infected material obtained from field (FS series isolates). We are using isolates that contain only single strains and isolates that contain mixtures of strains for primary inoculations. Real time PCR analysis protocol is being optimized for quantification of multiplication of CTV genotypes in the inoculated trees. Trees with developed CTV infection along with uninfected control trees were challenged by graft-inoculation with the superinfecting vector carrying a GFP gene. The latter protein is used as a marker protein in this assay, which production represents a measure of vector multiplication. The trees are now being examined to evaluate level of replication of superinfecting virus. Tissue samples from the challenged trees are observed under the fluorescence microscope to evaluate the ability of the vector to superinfect trees that were earlier infected with the other isolates of the virus. Levels of GFP fluorescence are monitored and compared between samples from trees with and without preexisting CTV infection. Additionally, real time PCR quantification is also being employed to these tests. To select rootstock/scion combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the foreign protein of interest from this vector, we are preparing trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla. The plants are used for the experiments similar to the experiments described above.
Work continues on the construction and characterization of new FT constructs using cDNA clones. The experiments are underway to compare the new FMVcDNA27 construct, which contains an FT3 cDNA insert in the pCAMBIA2201 vector with a constitutive FMV promoter, with a corresponding genomic clone, which we have been using up to this time. Transformation of Carrizo and tobacco tissue is underway in order to compare the action of these two constructs. The new construct was created as a first step towards the development of a new FT3 construct with an inducible promoter. We have arranged for the materials transfer of two inducible promoter systems from the Danforth Foundation. Both of these promoters are inducible by the chemical methoxyfenozide, a widely-available pesticide, approved for field use on citrus. However, we have not yet received the inducible promotors. One system is driven by the CsMV constitutive promoter, and the other by the RTBV vascular-specific promoter. Once we have verified that the smaller and more manageable cDNA is as effective as the original genomic version of the FT3 gene, we hope to begin development of the inducible promoter constructs. Experiments to determine the behavior of the three genomic clones from citrus when overexpressed in tobacco have been completed and a manuscript is being written. Expression of the genes in mature nonstransgenic citrus plants is being recorded monthly.
Two naturally occurring terpenoid essential oils have been selected as the key components for the pesticide formulations. They are known to have antimicrobial properties which allow us to believe that they will be potent in combating the targeted bacteria. Agriculturally approved surfactants of selected HLB values have produced formulations that permit for encapsulation of the oil. Both essential oils have successfully formed stable micro-emulsions. Large changes in temperature cause some instability with higher oil loading. However, at room temperature, system is re-equilibrated. Essential oil A requires additional input of energy (sonication), where as essential oil B is more readily encapsulated. With the addition of co-surfactants, the oil loading percentages greatly increase in the respective microemulsions. For essential oil A, loading is increased from 1% to 7% (w/w). Essential oil B increases from 8% to 14% (w/w). Particle sizing measurements have been conducted and show mean particle sizes by number distribution of ~3 nm for essential oil A and ~7 nm for essential oil B. The droplet size of Essential oil B microemulsion was increased to about ~30 nm with increase in oil loading. These results have been compared using two particle-sizing instruments, Microtrac Nanotrac and Malvern Zetasizer Nano ZS. Further investigations are being carried out to determine the correlation between the particle size and the amount of oil in the system. Currently, surfactant weight percentages are ~ 20% for both Essential oil microemulsion systems. Additional experiments are being performed to increase the Essential oil loading percentages in both microemulsion systems.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/16th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. Dr. Grosser removed the unsuccessful trees from the first planting and planted additional transgenics among the promising trees still under trial. Additional plantings are welcome from the research community.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants with citGRP1 were transferred to soil. Nineteen of them were test by PCR and ten of them are transgenic plants with citGRP1 insertion. They will soon be ready for RNA isolation and RT-PCR to check gene expression. More than thirty kan resistant shoots were obtained from citGRP1 transformed Hamilin. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting. Seven transgenic Hamlin with MADS6 were planted in soil. In addition, numerous putative transformants are present on the selective media transformed with different constructs. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many kan resistant transformants were generated on the selective media. About twenty kan resistant shoot are rooted in rooting medium and one of Hamlin transformatn was planted in soil. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. The construct pBinARSplus:nbFLS2 was used to transform Hamlin and Carrizo. Many putative transformants were generated on the selective media. About forty resistant shoots were rooted in rooting medium and ten Hamlin transformats were plant in soil. Other targets identified in genomic analyses are also being pursued. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
Our recent progress towards proposed research goals: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. A] Mutagenesis of EFR to produce elf18-CLas responsiveness: Our initial approach of random mutagenesis and screening in tomato was unsuccessful, indicating the necessity to generate multiple mutations for elf18-CLas recognition. Presently we are evaluating phage display for this purpose. To this end, we have defined suitable conditions for specific binding of ectodomain fragments of EFR to biotinylated elf24. Elf24 has been used for these experiments to allow linkage of the biotin group to Lysine 24; this peptide is fully functional in the elicitation of ROS. We are in the process of evaluating binding of biotinylated elf24-CLas to different regions of EFR. Once optimal regions are determined, mutagenesis will be performed on these regions and cloned into phage display vectors for screening. B] Screening for natural variants of EFR: A small selection of Brassicae has been screened for elf18-CLas response, however none of these were positive. Pending the outcome of mutagenic approaches, the screen for elf18-CLas response will be expanded to a large number of Brassicae. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Assessing XA21 function in dicots: Transgenic XA21-EFR, XA21 and EFR lines have been generated in Arabidopsis and are now ready to assess their effectiveness in pathogen defence. We plan to test these lines against Xanthomonas, Pseudomonas and Argobacterium. In addition, we have generated transgenic tomato lines expressing XA21. These plants will be crossed with EFR tomato lines to determine the pathogen resistance conferred by these two genes in a heterologous system. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. We will initiate the construction of appropriate expression vectors of genes for citrus transformation and expression.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the LasA1 gene, the N-terminal region (LasA1-A), two version for the repeat region with different number of the repeat sequences (LasA1-B0 and LasA1-B1), the C-terminal region (LasA1-C), and the full LasA1 gene. We are analyzing these constructs for their transient expression in Nicotiana benthamana and stable expression in transgenic Arabidopsis thaliana. The transgenic lines were obtained by floral-dip transformation of Arabidopsis Col-0 plants and we are currently verifying the gene insertion and mRNA expression level on T2 Arabidopsis. Three transgenic T3 lines expressing the gene are selected for analyzing phenotypes and protein localization using GFP pGWB2 vector. We are testing the expression level of the gene constructs that were transiently expressed in N. benthamiana with 35S, PFLAG and GFP pGWB2, 6 and 12 vectors. Localization of different constructs of LasA1 and LasA2 proteins using GFP vectors and pull-downs for protein-protein interactions using the PFLAG vector are in progress.
Cytoplasmic (CiLV-C and CiLV-C2) and nuclear (CiLV-N) citrus leprosis virus cause citrus leprosis disease in North and South American. All types of the CiLV are reportedly transmitted by Brevipalpus mite species. We continued mite transmission experiments at the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD utilizing endemic healthy Brevivalpus yothersii (syn. phoenicis) mites from Florida. We completed mite transmission experiments after receiving the citrus leprosis affected samples from Mexico (CiLV-N) & Colombia (CiLV-C2). Six to seven weeks after completion of the transmission experiments none of the citrus seedlings showed any leprosis symptoms. For confirmation of the negative test results asymptomatic leaf tissue from the experiments was analyzed by reverse transcription polymerase chain reaction (RT-PCR) using CiLV type-specific primers but all plant samples were negative. Recently, nuclear CiLV was reported from Mexico but no prior sequence information was available. In work on another funded project we have successfully determined the entire genome sequence of nuclear CiLV and deposited the sequence in the NCBI Genbank. A manuscript also has been accepted for publication in the journal Genome Announcement (‘Genome assembly of citrus leprosis virus nuclear type reveals a close association with orchid fleck virus’. Contacts have been made with collaborators in Mexico, Colombia and Panama for further shipment of infected leprosis samples to continue the transmission experiments with all 3 types of CiLV. Using newly developed PCR primers we will determine the viruliferous status of the B. yothersii after acquisition from infected leprosis tissue. Based on these results we will determine if Florida endemic healthy Brevivalpus Florida mites are able to acquire the various citrus leprosis viruses. In addition the utilized mites will be sent to USDA cooperators to continue to compare their taxonomic status with those that do transmit in Colombia elsewhere. Mite transmission work in Colombia continued with work by our collaborator Guillermo Leon. In acquisition experiments it was found that B. yothersii (phoenicis) mites effectively acquired CiLV-C2 after 30 minutes feeding on leprosis symptomatic leaves of Valencia orange. In transmission experiments with mites that were allowed an acquisition period of 24 h, positive virus transmissions were accomplished after only 10 minutes of feeding. Transmission rates increased incrementally up to a maximum after 24 hours feeding. However in using PCR to detect CiLV-C2 in the mites variable results were found. It appears that a minimal level of virus must be present in the mite for positive detection however PCR negative mites may still acquire and transmit the virus.
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