As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF and are growing well in the greenhouse. These will be planted in the spring of 2011, and 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. An experimental attract/kill product, to disrupt citrus leaf miner (CLM) without disrupting ACP, was not effective last year. Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Several applications of Admire are being used to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance.
Work continued to determine the suitability of Rutaceae species for the development of the Asian citrus psyllid and as hosts for the various species of Candidatus Liberibacter. The psyllid successfully colonize and reproduce on Choisya ternata, C. arizonica, and Helietta baretta in no-choice tests, but reverted back to its preferred hosts, orange jasmine (Murraya paniculata) and curry leaf (Bergera koenigii) in choice tests. On Amyris madrensis, A. texana and Zanthoxylum fagara eggs were laid and hatched but no nymphal development beyond first instart was found. Psyllid reproduction did not occurred on Esenbeckia berlandieri, Ptelea trifoliata nor Casimiroa edulis, although adult survived on these species. Severinia buxifolia is a frequently grown ornamental on which psyllids feed and reproduce. As previously reported we were able to transmit Ca.Liberibacter asiaticus to it by grafting. Symptoms in S. buxifolia were very nondescript with only thickened leaves and some vein corking. Live bacterial populations were measured monthly in the plants using a newly devised qPCR method. Transmissions of Candidatus Liberibacter asiaticus were successfully done from S. buxifolia to both healthy sweet orange and to healthy S. buxifolia in two separate tests. To date we have a 50% transmission rate from S. buxifolia to sweet orange and a 72% rate from S. buxifolia to S. buxifolia. Data showed that the bacterial populations in S. buxifolia declined over time if not exposed to infective PCR positive psyllids. Generic PCR primers developed were successful in detecting all know species of Candidatus Liberibacter. In S. buxifolia the titer of the Ca. Liberibacter asiaticus began getting lower like that seen with M. paniculata. In testing these plants with the generic primers a good titer of Ca. Liberibacter was found. The identification of this bacterium is ongoing. Esenbeckia sp.was identified as qPCR positive after psyllid transmission tests making it a new host for Ca. Las. Ca. Liberibacter asiaticus infected periwinkle and dodder were found to have higher live bacterial levels than source citrus plants. These studies will determine if certain plants are good hosts of live bacteria. Attempts to transmit Ca. Liberibacter africanus with D. citri failed. The number of psyllids used, length of acquisition and inoculation periods had no effect. The psyllid colony were pre-tested for presence of the bacterium and were tested again after acquisition and after inoculation and all tested negative for the bacterium. Ca. Laf source plants had Cq values in the low 20’s. Dodder transmission of Ca. Laf from sweet orange to Choisya X Aztec Pearl, periwinkle, and sweet orange was obtained but not to Severinia buxifolia. Dodder was highly infected as was the sweet orange and periwinkle, but Choisya had lower bacterial levels. Two sets of general ITS primers and new specific Ca. Laf ITS primers (Postnikova 2011) were used to generate PCR products for cloning and sequencing. Five clones from Laf infected Choisya were confirmed to be Laf. Murraya paniculata was confirmed to be a host of Laf, by PCR, cloning and sequencing. Tests were initiated to dodder transmit Laf to Bergera koenigii and M. paniculata. C. ternata, Choisya X. Aztec Pearl, and Choisya ‘Sundance’ were inoculated with the B239 Taiwanese isolate of Las with psyllids and Choisya X Aztec Pearl with dodder. To date all PCR assays have been negative with psyllid transmission to C. ternata. Fortunella polyandra, Clausena harmandiana, and C. excavata were obtained for testing. Work to further test the susceptibility or resistance of the cultivar IAPAR73 continued using psyllid transmissions and low numbers of qPCR positive plants were obtained.
Rutaceae species are indigenous in North America. Work continued to determine the suitability of some of these plants for the development of the Asian citrus psyllid as hosts for the various species of Candidatus Liberibacter. Using no-choice and choice experiments the psyllid was successfully colonize and reproduce on Choisya ternata, C. arizonica, and Helietta baretta in no-choice tests, but reverted back to its preferred hosts, orange jasmine (Murraya paniculata) and curry leaf (Bergera koenigii) in choice tests. On other species (Amyris madrensis, A. texana, Zanthoxylum fagara) eggs were laid and hatched but no nymphal development beyond the first instart was found. No psyllid reproduction occurred on Esenbeckia berlandieri, Ptelea trifoliata nor Casimiroa edulis, although adult psyllids survived on these species for a few days. Severinia buxifolia is a frequently grown ornamental on which the psyllids feed and reproduce. As previously reported we were able to transmit Ca.Liberibacter asiaticus to it by grafting since it is graft compatible with citrus. Symptoms in S. buxifolia were very nondescript with only thickened leaves and some vein corking. Live bacterial populations were measured monthly in the plants using a newly devised qPCR method. Data showed that the bacterial populations in S. buxifolia declined over time once they were no longer inoculated with Ca. Liberibacter asiaticus positive psyllids. Generic PCR primers developed were successful in detecting all know species of Candidatus Liberibacter in PCR. In S. buxifolia the titer of the Ca. Liberibacter asiaticus began getting lower like that seen with M. paniculata. In testing these plants with the generic primers a good titer of Ca. Liberibacter was found. The identification of this bacterium is ongoing. Ca. Liberibacter asiaticus infected periwinkle and dodder were found to have higher live bacterial levels (as determined by newly devised qPCR) than source citrus plants. These studies will determine if certain plants are good hosts of live bacteria. Attempts to transmit Ca. Liberibacter africanus with D. citri failed. The number of psyllids used, length of acquisition and inoculation periods had no effect on transmission. The psyllids were pre-tested for presence of the bacterium and were tested again after acquisition and after inoculation and all tested negative for the bacterium. Source plants had Cq values in the low 20’s indicating high levels of Ca. Liberibacter africanus. Dodder transmission of Ca. Laf from sweet orange to Choisya X Aztec Pearl, periwinkle, and sweet orange was obtained but not to Severinia buxifolia. Dodder was highly infected as was the sweet orange and periwinkle, but Choisya had lower bacterial levels. Two sets of general ITS primers and new specific Ca. Laf ITS primers (Postnikova 2011) were used to generate PCR products for cloning and sequencing. Five clones from Laf infected Choisya were confirmed to be Laf. Murraya paniculata was confirmed to be a host of Laf, by PCR, cloning and sequencing. Tests were initiated to dodder transmit Laf to Bergera koenigii and M. paniculata. C. ternata, Choisya X. Aztec Pearl, and Choisya ‘Sundance’ were inoculated with the B239 Taiwanese isolate of Las with psyllids and Choisya X Aztec Pearl with dodder. To date all PCR assays have been negative with psyllid transmission to C. ternata. Fortunella polyandra, Clausena harmandiana, and C. excavata were obtained for testing. Work to further test the susceptibility or resistance of the cultivar IAPAR73 continued and PCR testing is underway.
Previous work in our lab showed that LAS concentrations are the highest in the seeds of LAS-infected fruit. Since then, our focus has been on: 1) developing a method to aseptically obtain an inoculum from the seeds and 2) implementing a method to estimate the viability of LAS cells extracted from the seeds. Initially, an inoculum was obtained from LAS seeds by bead-beating whole seeds with metal beads and combing the seed pulp with a basic liquid media to obtain a suspension. An equal amount of this suspension was added to different media treatments and the relative concentrations of LAS cells in each treatment were determined by quantitative PCR. However, qPCR of replicate treatments had high variability. Since this was most likely due to the non-homogenous nature of the suspension (still contained some visible seed particles), efforts were made to create a more homogenous inoculum. Future suspensions were filtered through a 100 ‘m filter to remove large particles. Subsequent qPCR analyses of replicate treatments were much less variable. Next, a method was implemented to estimate LAS cell viability in the inoculated treatments over time. Ethidium monoazide (EMA) was used in conjunction with qPCR to determine the percentage of viable LAS cells. EMA binds to non-viable DNA so that it is not amplified by qPCR. Initial cell viability is variable, but is on average about 5% from the seed inoculum. After one week of incubation in flask containing various media treatments, however, no viable cells could be detected. Previously, LAS bacterial inoculum was also placed in sterile microfluidic chambers for observation under the microscope. Bacteria were observed in the samples in the microfluidic chambers. After about 2 weeks, the contents of the microfluidic chambers were eluted. LAS concentrations were determined by qPCR. Some LAS DNA was detected in the channels, but it is unknown whether this DNA was viable or not because the EMA viability assay was not implemented at that time. Future work will try to assess the viability of cells in the microfluidic chambers.
In this second quarter, 400 plants of Valencia orange on Kuharske Carrizo rootstocks were grown from rooted cutting in 1-gal plastic nursery containers and kept in the greenhouse under natural light conditions at 17-25’C. The plants will be used to conduct three different experiments that will include infected and non infected plants as well as treated non treated plants. For the fist experiment we will apply therapeutic compounds to HLB infected and healthy plants that will enhance Systematic Acquired Resistance (SAR) response and counteract ethylene in early infected tissues. The therapeutic compounds that we will be using in this experiment are Benzothiadiazole (BTH), Actigard, Salicylic acid, .-Aminobutyric acid (BABA). We will use untreated plants as controls. This will equal 5 treatments x 2 disease status (healthy and infected) x 10 biological replicates = 100 trees. For the second experiment we will be looking the regulation of glucose transport on HLB infected and non infected plants using gibberellin (GA3), the cytokinin 6-benzyladenine, KNO3 and a combination of the GA3, 6 benzaladenine and KNO3. We will use untreated plants as controls. This experiment will include 5 treatments x 2 disease status (healthy and infected) x 10 biological replicates = 100 trees For the third experiment we will examine sucrose + atrazine at two different rates along with an untreated control. The treatments will be applied at two different times to HLB infected and healthy plants, after infected plants become PCR+ for HLB infection. This will equal 3 treatments x 2 (healthy and infected plants) x 2 application times x 10 biological replicates = 120 trees . Plants infection will be done on the 3rd week of April by grafting infected bark pieces which will be obtained from citrus infected trees from commercial orchards. HLB infection for the bark pieces will be confirmed by qPCR 3 months after infection.
In this second quarter, 400 plants of Valencia orange on Kuharske Carrizo rootstocks were grown from rooted cutting in 1-gal plastic nursery containers and kept in the greenhouse under natural light conditions at 17-25’C. The plants will be used to conduct three different experiments that will include infected and non infected plants as well as treated non treated plants. For the fist experiment we will apply therapeutic compounds to HLB infected and healthy plants that will enhance Systematic Acquired Resistance (SAR) response and counteract ethylene in early infected tissues. The therapeutic compounds that we will be using in this experiment are Benzothiadiazole (BTH), Actigard, Salicylic acid, .-Aminobutyric acid (BABA). We will use untreated plants as controls. This will equal 5 treatments x 2 disease status (healthy and infected) x 10 biological replicates = 100 trees. For the second experiment we will be looking the regulation of glucose transport on HLB infected and non infected plants using gibberellin (GA3), the cytokinin 6-benzyladenine, KNO3 and a combination of the GA3, 6 benzaladenine and KNO3. We will use untreated plants as controls. This experiment will include 5 treatments x 2 disease status (healthy and infected) x 10 biological replicates = 100 trees For the third experiment we will examine sucrose + atrazine at two different rates along with an untreated control. The treatments will be applied at two different times to HLB infected and healthy plants, after infected plants become PCR+ for HLB infection. This will equal 3 treatments x 2 (healthy and infected plants) x 2 application times x 10 biological replicates = 120 trees . Plants infection will be done on the 3rd week of April by grafting infected bark pieces which will be obtained from citrus infected trees from commercial orchards. HLB infection for the bark pieces will be confirmed by qPCR 3 months after infection.
This project is focused on understanding the phloem disruption response from infection with the HLB bacteria, Candidatus Liberibactor asiaticus (LAS). Phloem distruption leads to starvation of the roots and tree decline. Callose and phloem protein 2 plugging are present in about a 3 to 1 ratio in field plants. Some virulence factors of the bacteria may cause necrosis responses. In the third year we want to fully understand if both phloem plugging and direct virulence of the bacteria play a role in decline development or by only one of these factors. Grapefruit trees with PAPETALA3-IPTgp (isopentenyl transferase) showing elevated (1, 3)-.-glucanase expression (callose polymer forming enzyme) were subjected to LAS by graft or psyllid inoculation and grown in the greenhouse for over one year. By January 2011 all plants had some HLB symptoms, but intensity did vary. Phloem samples will be prepared and examined for plugging quantity and types. This will tell whether multiple copies of the gene did block callose formation and the plants declined due to other bacterial induced virulence or the multiple copies or the elevated (1, 3)-.-glucanase expression caused more callose formation. Transformation experiments were continued in efforts to produce plants that over-express the citrus ‘-1,3-glucanase gene, both with the constitutive 35S promoter (p35S ‘ BG-35T) and the Suc 2 phloem specific promoter (pSuc2-BG-35T). Approximately 40 transgenic plants (Valencia and precocious sweet orange Vernia) have been successfully micrografted to Carrizo and are growing well in the greenhouse. Continued protoplast transformation experiments attempting to transfer the ‘-1,3-glucanase gene resulted in the recovery of GFP-positive somatic embryos as follows: 331 of sweet orange OLL#20, 39 of sweet orange ‘Jin Cheng’, and 10 of ‘Valencia’. Approximately one third of these embryos are normal and healthy, and should allow for transgenic plant recovery. LAS causes severe symptoms in plant compared to the psyllid. We hypothesized that a number of pathogenicity/virulence related genes of the bacterium would be overexpressed in planta, compared to the psyllid. To test this hypothesis, quantitative real-time PCR assays using total RNA isolated from infected plants and psyllids were conducted. Gene specific primers were used to check the expression of 560 genes in LAS. Genes showing a differential expression of two fold or more in either the plant (167) or psyllid (108) were categorized into Clusters of Orthologous Groups of proteins functional categories. Those genes are being further confirmed using different batches of samples to guarantee its accuracy. Two potential virulence genes that were overexpressed in planta along with 10 more constructs of putative virulence genes are being generated. Differential expression of these selected genes were also evaluated in more or less susceptible citrus types infected with LAS. Two potential virulence related genes were then screened on Nicotiana benthamiana plants for symptom expression, using transient assays and showed significant effects on tobacco.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. Results showed that the use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime, and a manuscript reporting this was accepted with revision for publication in PCTOC. Recovered transgenic Mexican lime trees 1.5 years after removal from tissue culture were girdled, which induced flowering about 4 months later; with complete flowering and fruit set in less than two years. Experiments to test this with commercial sweet oranges are underway. We continued with experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency, including: kanamycin, hygromycin, mannose and phosphinothricin. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and Faryna Harvesting, yield and fruit quality data was collected from the St. Helena project. Approximately 10 acres of trees planted 3.0 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The best rootstocks identified to show positive affects on rapid tree growth with precocious bearing and good early fruit quality (higher lb. solids) were somatic hybrids Changsha mandarin+trifoliate orange 50-7, white grapefruit+trifoliate orange 50-7, and sour orange+Carrizo, and tetrazygs White#4 (Nova+HBPummelo x Succari sweet orange+Argentine trifoliate orange), Orange#13, Orange#14,Orange#18 and Orange#19 (Orange series all Nova+HBPummelo x Cleo+Argentine trifoliate orange). Trees on these rootstocks averaged more than a half-box of fruit per tree with juice brix values great than 11. These rootstocks will now be tested to determine if they can shorten juvenility in transgenic plants produced from juvenile explant. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL and Vernia sweet oranges were successfully micrografted to Carrizo citrange or experimental Tetrazyg rootstocks and are growing well in the greenhouse. These will now be clonally propagated onto the rootstocks mentioned above for further study of early flowering and transgene expression. Horticultural manipulations on these plants will include the RES (Rapid Evaluation System) growth method plus girdling. Transformation with early-flowering genes: ‘Citrus has at least 3 FT genes. Cloning and characterization of all 3 (genomic clones) has been completed. We have put them into transformation vectors with a constitutive promoter and performed transformation experiments with Carrizo and Duncan. Although only a few transgenic plants were recovered, we know the constructs work because we had previously tested them in tobacco, where we recovered early flowering phenotypes, as well as some other phenotypic alterations. Our current theory is that expression with the 35S promoter is too strong in citrus (in some cases we got flowering directly on the initial explant!). We are currently testing a poplar FT with a weaker inducible promoter (a heat shock promoter shown to be inducible in Oregon); which will hopefully solve the promoter problem.
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 now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. ‘ We have greatly improved our efficiency of screening . ‘ We are modifying the vector to express more than one anti-HLB gene. ‘ We are modifying the vector to allow addition of a second vector. ‘ We are preparing to put trees into the field for testing as soon as potential freezes are over. ‘ We continue to supply infected and healthy psyllids to the research community.
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 now are focusing on when and how psyllids acquire Las from newly infected plants. This information is necessary of the epidemiology models for managing HLB. 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. We have greatly optimized conditions to allow exposed plants to become rapidly inoculated by infected psyllids. We continue be a resource of healthy and infected psyllids and plants for other laboratories and we are building CTV expressed genes to control psyllids or Las for other labs.
The results of the analysis that estimates the annual HLB infection rate that switching from the traditional HLB management program to an enhanced foliar nutrient program maximizes profits were presented at the 2nd International Research Conference on HLB in Orlando, Florida on January 12, 2011. Results of the grove replacement model were published in Citrus Industry Magazine in April of 2011. This model is in the process of being developed into a more user-friendly version that will enable growers and other users to determine when groves with declining fruit yields should be replanted. The Asian Citrus Psyllid (ACP) and HLB have spread rapidly throughout all citrus growing counties in Florida. At this time, control of ACP through intense use of pesticides remains the only viable means of managing HLB. Evolution of pesticide resistance in ACP has already been documented in Florida. Stakeholders are alarmed that the best defense against ACP and HLB may be eroding. At the recommendation of the National Academy of Sciences ‘Citrus Health Management Areas (CHEMA)’ were created in Florida. These contiguously-managed areas of groves improve the effectiveness of psyllid spray programs Florida. The costs of ACP management programs will be monitored in several CHEMAs each year of this five year project. The first two years will provide a benchmark of these costs for the standard management protocol, while the remaining three years will provide a comparison of the increased costs and hopefully reduced ACP populations and resulting reduction in tree mortality from the implementation of these programs. Costs will be based on recommended application rates, frequency of application, and material and application costs. Estimation of the returns associated with key rotation spray modules and binary mixtures will involve estimation of production not lost assuming ACP control reduces the likelihood of resistance development and spread of HLB in a particular block. We will utilize a block-level HLB Gompetz function spread model developed in Brazil (Gottwald et al. 2008) to depict the spread of HLB as a function of tree age at time of first infection, years elapsed since first infection, ACP control, and the level of external pressure of surrounding infections. Yield will be predicted as HLB progresses through a block. The value associated with varying levels of premature fruit drop and tree mortality will also be estimated. Costs and returns computed for key rotation spray modules and binary mixtures will be compared to those associated with conventional HLB cultural management and with the benchmark costs obtained in the first two years. Costs in CHEMAs are expected to be reduced from increased aerial pesticide applications and greater economies of scale in ground applications. Costs are expected to be increased from the use of more expensive pesticides in the pesticide resistance rotation program. Economic benefits are expected from lower tree loss and greater fruit production as a result of reduced ACP populations and infection rates.
When dsRNA targeting either a psyllid cathepsin or a psyllid vacuolar ATPase gene are fed in artificial diets to the Asian citrus psyllid, an increase in psyllid mortality is realized. The oral uptake of ~300 bp dsRNA fragments matching the coding region to either psyllid Vacuolar ATPase or cathepsin can induce mortality in the Asian citrus psyllid. Comparisons were made to determine the optimal dsRNA size. Psyllids were fed either the ~300 bp dsRNAs directly or after processing to siRNAs with the Dicer enzyme. Results showed that the 300 bp dsRNAs induced greater mortality and than that observed with processed siRNAs. Furthermore, non-linear dose dependent toxicity of the ~300 bp dsRNAs suggesting complex interactions that have not yet been characterized with respect to dsRNA induced toxicity in insects.
1-The first objective of the second year was to build and start the operation of a plant growth room at the Citrus Research and Education Center in Florida (CREC). The growth room construction started on October 22nd 2010 and the projected finish date was February 11th 2011. There was a delay of a few weeks and the main contractor will finalize on March 25th, but the computer system contractor is still finishing the programming that will control the environmental conditions. We checked the growth room and it is working as expected however disposal of the waste stream will be a concern when the growth room is in full operation since the water that we will dispose needs to be collected in an external tank and test by the county to guarantee that we are not disposing contaminants that can affect the environment. We already started furnishing and placing equipment inside the building. Because of the delay in the construction, the growth room is not in fully operation yet. We believe this will take at least one additional month. The cost of the construction was higher than the original budget plan; the extra funding was provided by CREC and IFAS facilities as agreed initially. 2- A full time technician with nursery experience was hired after several months of searching. The process of hiring was slow. A first candidate was hired and the offer of employment was rejected due to a low salary offer. A second candidate was found but he quit two months after hiring. We hired a third technician with limited experience and he will start the first week of April. It seems like salary will be an important issue in the future to recruit and retain personal. 3- Training of the manager Dr. Zapata was completed at the IVIA under the supervision of Dr. Pena. It was emphasized during the training the improvement of transformation methods for more recalcitrant types, molecular analysis of the regenerants and plant material preparation at the greenhouse/growth room, including micrografting, phytosanitary treatments, fertilization and pruning. An annual schedule for completion of planting, transplanting, grafting and obtaining budsticks to transform was developed. 4- The Mature Transformation Laboratory was established, using an existing laboratory located at CREC. Two technicians with limited experience were hired and are currently being trained in the first tissue culture techniques, including culture media preparation, grafting, micrografting, explant preparation, culture and regeneration, etc. 5-Our selected varieties Hamlin 1-4-1, Pineapple S-F-60-3 and Valencia S-SPB-1-14-19 were subjected to cleaning through shoot tip-grafting at the Florida Department of Agriculture and Consumer Services (DOACS) with the help of Dr. Peggy Sieburth. They are kept at out lab and ready to be grafted on rootstocks when the growth room is fully operative. 6-Dr. Leandro Pena and his greenhouse and growth room manager Josep Peris made a visit of one week in March 2011 to supervise the last steps of the growth room construction before been finalized and suggested minor details to make the facility more reliable and helpful for operators. They also short-trained the tissue culture technicians in horticultural practices. They checked substrate, seed stock and nutrition issues with the manager Dr. Zapata.
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. Here are the major achievements: 1. We are currently assessing citrus genes modulated by Las infection in 1) Comparison of citrus leaves, stems and roots to Las infection (completed, paper in writing), 2)Comparison of healthy vs. infected leaf samples in citrus grove (microarray data collected and QRT-PCR is underway),3) Comparison of different citrus varieties that are different in tolerance and susceptibility (in progress). 1) Comparison of citrus leaves, stems and roots to Las infection (completed, paper in writing The alteration of gene expressions by Las in leaf, stem and root tissues of Valencia sweet orange was investigated using Affymetrix microarray analysis. Out of 30,279 probe sets, a total of 8667, 2795 and 1142 showed significantly altered (p< 0.05) expression in leaves, stems and roots, respectively. Using 2 fold change as cut-off value, 1008, 580 and 58 transcripts were significantly up-regulated in leaf, stem and root tissues, respectively, whereas, 1109, 350 and 58 were correspondingly down-regulated in Las infected plants. Differences were observed for genes involved in cell wall synthesis and remodeling, lipid metabolism, photosynthesis, secondary metabolism, and starch and sucrose metabolism. Biotic stress induced signaling and transcription factors, PR-proteins, heat shock proteins, hormones and genes involved in protein modification, redox reactions and secondary metabolism were affected more in leaves and stems than in roots. PR genes were mainly repressed in roots but showed both patterns in leaves and stems; JA genes were up-regulated in stems, down-regulated in roots but up- and down-regulated in leaves; Calvin cycle genes were mainly altered in roots; SA and heat shock proteins were not significantly altered in roots. Transcriptional factors with WRKY, AP2/EREBP, MYB, bZIP, bHLH and Zinc finger domains were differentially regulated in all tissues, but least in roots. Differences were shown by C2C2(Zn) DOF zinc finger family proteins, affected in leaves and stems only; homologs of MEE47, nuclear factor PBF-2 and ATRR1 were regulated in roots only while MADS box transcription factor family and several unclassified transcriptional factors were down-regulated only in leaf tissues. We are further analyzing the data to understand how Las affects leaves, stems, and roots since they have distinct roles and function and how they contribute to the HLB disease development. 2)Comparison of healthy vs. infected leaf samples in citrus grove (microarray data collected and QRT-PCR is underway using multiple tools including MapMan. 3) Comparison of different citrus varieties that are different in tolerance and susceptibility. This work is in progress.
The goal of this proposal has been to investigate whether Ca. Las is transmitted between infected and uninfected ACP adults in a sex-related manner to better understand the mechanisms by which disease is spread in field. We carried out a series of experiments to evaluate if Ca. Las is transmitted from male to female psyllids during routine mating. Our preliminary investigations indicated that Ca. Las may be transmitted from male to female psyllids but not from females to males or among psyllids of the same sex. Pairs of Ca. Las infected male and healthy female, Ca. Las infected female and healthy male, Ca. Las infected female and healthy female, Ca. Las infected male and healthy male, Ca. Las infected female and Ca. Las infected male (positive control) healthy female and healthy male (negative control) adult psyllids were introduced separately in Petri dishes filled with agar medium. The insects were allowed to mate for 72 hrs. After 72 hrs, the insects were transferred to Ca. Las resistant Murraya koenigii plants for 12-14 days for multiplication of bacteria in recipient psyllids. DNA was prepared from each of the female and male psyllids separately and analyzed for Ca. Las presence utilizing a real time PCR assays. Our subsequent investigations confirmed that Ca. Las is sexually transmitted from Ca. Las-infected male psyllids to healthy females but not from infected females to healthy males or among psyllids of the same sex. Ca. Las was transmitted from Ca. Las-infected male psyllids to roughly 3% of healthy females. Ca. Las was not detected in the recipient sex immediately after mating but required a minimum incubation period of 2 weeks in psyllid bodies for PCR detection. These results also suggested multiplication of bacteria within psyllid bodies. No Ca. Las was detected in recipient psyllids when the recipient psyllids were maintained on HLB-resistant (Murraya koenigii) plants for longer than 4 weeks suggesting that psyllids may lose infectivity if they continuously live on HLB-resistant plants. We were able to detect Ca. Las bacteria in ACP ovaries of recipient females with PCR. However, we were unable to detect the presence of bacteria in genital parts of male and female psyllids with scanning and transmission electron microscopy perhaps due to washing of bacteria during sample preparation procedures. Ca. Las was also not detected in psyllid salivary glands using electron microscopy. More precise and accurate procedures such as in situ hybridization may be required to detect the presence of bacteria in psyllids. Also, we were able to detect Ca. Las in eggs of recipient females with PCR but not with electron microscopy. PCR detection of Ca. Las in psyllid ovaries suggested transovarial transmission of bacteria. Transovarial transmission was also confirmed in F2 generations of Ca. Las-recipient females which were produced on M. koenigii plants. We continue to evaluate if the Ca. Las-recipient females are capable of infecting new citrus plants. The experimental procedures for this have been completed; however, in some cases we are still awaiting to collect the results because a minimum 10 week period is required for detection of HLB in newly infected plants. In some cases this period may be longer; therefore, we have asked for a short extension of this 1-year project. These results show a new mechanism for C. Las transmission in addition to the known mechanisms (through plant feeding, and transovarial transmission). Although the level of this type of transmission is low; it is present. These results further underscore the importance of effective psyllid control for HLB management.
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