A series of transgenics scions and rootstocks, produced in the last several years, continue to move forward in the testing pipeline. It appears prudent to replicate plants of each transgenic event and conduct challenges that last 10-14 months. Most of these plants in our program have been transformed with AMPs driven by several constitutive and vascular specific promoters. Plants from the initial round of scion transformations are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (D. Hall collaboration) is being conducted on a replicated set of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. Several events grew better than all controls at 14 months after initiating the challenge, with 35% greater trunk-cross-sectional area increase than the overall experimental average and 64% greater growth than the mean of the controls, but do not show immunity to CLas development. These will soon be placed in the field for further evaluation. Forty four AMPs were screened in-vitro, a number of which were synthetics specifically designed to enhance efficacy against alpha-proteobacters. There appeared to be a ceiling of activity which could not be exceeded. The most active AMPs included Tachyplesin 1 from horseshoe crab, SMAP-29 from sheep, D4E1 and D2A21. A series of promoters were tested with the GUS gene. The three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than the other phloem-specific promoters citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. D35S produces the highest level of expression but with great variability between events. Anthocyanin regulatory genes, give bright red shoots (UF Gray collaboration) and were tested as a visual marker for transformation, as a component of a citrus-only transgenic system. When antibiotics were left out of regeneration media, almost no red shoots were recovered. However, high anthocyanin apples are reported to have field resistance to bacterial fire-blight. Red citrus transgenics will be tested for HLB, ACP, and canker resistance. CLas sequence data target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. In E. coli expressing the CLas translocase, two exterior epitope-specific peptides suppressed ATP uptake by 60+% and significantly suppressed CLas growth in culture. After verification these will be used to create transgenes. In our program, new constructs and resulting transgenics are in process, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), and a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA).
A transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, to support 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 more than two years. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 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 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 will be 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. Additional plantings are welcome from the research community.
The goal of this proposal is to examine how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. We are on schedule for the second year of the project. There was a delay for getting the funds for this year, however they are being released now. We have set trials using sweet orange and grapefruit plants that have young growing flushes and plants that have only matured flushes. These plants have been exposed to HLB-infected psyllids. Leaves on which psyllids fed were analyzed by PCR to see if the HLB bacterium could be detected soon after the exposure of leaves to infected psyllids. As a result in this experiment, we were able to detect presence of the bacterium fairly early after the initial exposure. Plants exposed to infected psyllids have been transferred to greenhouse and further monitored for the development of infection. We are in a process of analyzing and comparing infection rates of plants with young flushes versus plants with only matured flushes. Additional trials are in progress. To characterize potential inoculum sources of the bacterium available for psyllids within an infected tree we are examining presence of viable bacterium in different types of flushes that are produced during the development of the disease after psyllid-mediated inoculation of a tree and evaluating the proportion of psyllids that acquired the bacterium after their exposure to different types of flushes during infection development and their ability to transmit infection to new trees. We conducted several trials in which healthy psyllids 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 and after 21 days 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. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. These plants are being monitored for the development of infection. Currently we are analyzing numbers of plants that became infected upon inoculation with psyllids fed on different types of flushes. Another goal is to understand whether the HLB bacterium can have different forms or can be present at different stages in different types of tissues. One of the approaches that are being undertaken is to compare levels of the HLB bacterium genes expression in old symptomatic and young pre-symptomatic flushes. This work is ongoing. The next objective is to examine psyllid transmission rates from and to citrus varieties that are highly tolerant to HLB. We have propagated 6 different varieties of citrus: Valencia sweet orange, Duncan grapefruit, Persian lime, Eureka lemon, Carrizo citrange, and Poncirus trifoliata. Those varieties represent plants with different degrees of susceptibility to HLB. Currently these plants are being exposed to HLB-infected psyllids. After 1-month exposure, plants will be moved to greenhouse and monitored for the development of HLB infection. We are analyzing infection rates for these varieties. Later infected plants will be used as inoculum donors to examine psyllid transmission to new plants.
This is a proposal to find ways to optimally deploy the superinfecting vector to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. This is a new project and the funds for this project are currently being released. The research is in progress. We already have designated personal who would conduct the proposed research. Plant material that will be used in this project is being prepared. Using plant material and inoculum sources that are already available we are setting up initial experiments with the purpose to examine the levels of multiplication of the superinfecting CTV ector in trees infected with different field isolates of CTV to investigate how previous infection of trees with the virus affects the ability of the vector to infect and multiply in those trees and to examine the effect of various rootstock/scion combinations on the superinfecting ability of the vector in order to evaluate what combinations would support high levels of vector expression. These two objectives are our main focus for the first year of funding.
This project has been an administration challenge from the beginning. It was impossible to get funds to our Brazilian colleague and difficult to get funds to our USDA partners. Dr. Machado had only requested funding for the first two years of the project. Since he could not get funding in Brazil, he asked me to support a “sandwich” student in my lab, which Dr. Turpin approved. This student is returning to Brazil this month after one year in my lab. She worked on controlling tissue contamination, which is our main problem with mature transformation, but with only partial success. Drs. Grosser and Gmitter have had the same difficulties at the CREC and do not feel that they should continue on this project without improved facilities. The part of the project that has been successful is the work on small penetrating peptides (CPPs) in the Moore lab. This technology allows an alternative to Agrobacterium-based transformation of citrus. The Agrobacterium method produces a low rate of stable transformants (~9%), takes several months, and commercialization is potentially difficult because of the negative views of bacterial genetic modifications. We propose an alternative method of transformation using cell penetrating peptides (CPPs). CPPs are positively charged short amino acid sequences able to simultaneously bind proteins and nucleic acids and deliver them across cellular membranes and cell walls. Many applications are available using CPPs including transient expression assays and gene silencing. We have developed a standard method for the transient expression of reporter genes (GUS and GFP) in citrus. Our data indicate that up to 50% of treated explants express GUS when CPPs are used alone. Several optimization steps have been tested. For instance, the efficiency is increased to 100% when CPPs are used in conjunction with a lipid reagent. Our main goal is to use this method to improve stable citrus transformation efficiency compared to the Agrobacterium method. We have produced 84/163 segments which survived kanamycin selection and produced shoots. We will use PCR and reporter gene analysis to confirm their stable integration. Further experiments, comprising RNAi and protein expression, will also be performed.
New outbreaks of invasive fruit flies (Diptera: Tephritidae) continue to threaten agriculture world-wide. Establishment of these pests often results in serious economic and environmental consequences associated with quarantine, control, and eradication programs. Early fruit fly detection and eradication in the United States requires deployment of large numbers of traps baited with the highly attractive male specific parapheromone lures trimedlure (TML), cue-lure (C-L), and methyl eugenol (ME) to detect such pests as Mediterranean fruit fly, Ceratitis capitata (Wiedemann), melon fly, Bactrocera cucurbitae (Coquillett), and oriental fruit fly, B. dorsalis (Hendel), respectively. The current study compared the performance of solid single lure cones and plugs in conjunction with DDVP insecticidal strips; liquid lure with naled formulations; and single, double, and triple solid lure wafers impregnated with insecticide. Treatments were placed in AWPM and Jackson traps under Hawaiian climatic conditions in habitats where B. dorsalis, C. capitata, and B. cucurbitae occur together. The overall goal of this study was to develop a more convenient, effective, and safer means to use male lures and insecticides for improved detection and male annihilation of invasive fruit flies. In survey trials near Kona, HI captures of C. capitata, B. cucurbitae, and B. dorsalis with Mallet TMR wafers were equal to those for the standard TML, ME, and C-L traps used in Florida and California. A solid Mallet TMR wafer is more convenient to handle, safer, and may be used in place of several individual lure and trap systems, potentially reducing costs of large survey and detection programs in Florida and California, and male annihilation programs in Hawaii. With confirmatory trials completed in Hawaii, further testing will be conducted in citrus orchards under California weather conditions. Through Dr. Joseph Morse of the University of California, Riverside, we will conduct weathering trials of the novel TMR dispensers in California (Riverside, Lindcove, Bakersfield, Ventura, and Costa Mesa, CA) beginning in July 2012. Climate data will be obtained from Hobo weather recorders maintained at each location. Weathered dispensers will be sent to Hawaii and Washington for bioassays and chemical analyses, respectively. Roger Vargas of US PBARC will oversee bioassays in Hawaii. Peter Cook of Farmatech and John Stark of Washington State University will collaborate on chemical analysis of wafers in North Bend, WA. Currently, approximately 30,000 sets of TML, ME, and C-L traps are maintained throughout the state. From a worker safety, convenience, and economic standpoint, Farma Tech TMR Mallet solid wafers with DDVP may be more cost effective, convenient, and safer to handle than current liquid lure and insecticide formulations (e.g. naled) used for detection programs for TML, ME and C-L responding flies in California. Cost/benefit analyses of Mallet TMR vs. standard trapping systems will be done.
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. Money was just released in last week so no results to report.
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. The field survey of tangerine hybrid blocks is nearly finished with approximately 1000 isolates collected. Pathogenicity testing and collecting monoconidial isolates is underway. Analysis of data from 2008-2011 is under way and a manuscript is in preparation and is waiting for the 2012 results. Plants have been prepared for a series of experiments to look at the fitness characteristics among sensitive and resistant isolates.
The first objective of this project was to hire a Florida-based faculty scientist that could be trained under Dr. Leandro Pena in Spain, for the purpose of learning the mature tissue transformation technique and transferring the technology to Florida. The scientist (Dr. Cecilia Zapata) was hired, at the end of the first year of the three year project, and traveled to Dr. Pena’s lab at the IVIA, Spain, where she was trained in all tissue culture techniques associated with citrus mature transformation, starting with preparation of the source of material at the greenhouse and ending with the acclimatization of transformants in the greenhouse. It was emphasized that the preparation of plant material needed for mature transformation is the key to successfully and consistently obtaining mature transformants, and this can only be achieved by producing budsticks in a highly controlled and clean environment. The second objective of the project was to build a greenhouse at the Citrus Research and Education Center in Florida for the purpose of creating and growing citrus for mature transformation and to establish a Mature Transformation Laboratory. A growth room was constructed instead of a greenhouse due to budget constraints. It took approximately 7 months to construct the growth room. It is currently operational after more than a year of troubleshooting. The water filtration system still needs some adjustments to be able to obtain a better water quality. The water quality is affecting the plant growth and the humidifiers. A generator needs to be purchased; without it, any prolonged electricity failure could jeopardize the whole project. The laboratory is fully operational. The third objective of the project was to obtain mature transgenic plants from the most important Florida citrus cultivars. We started using the growth room and planted the rootstocks at the beginning of April 2011. Three (3) sweet orange varieties were indexed in vitro and micrografted. The cultivars introduced were Hamlin 1-4-1, Valencia SPB 1-14-19 and Pineapple F-60-3. A calendar was established in October 2011 and firsts mature transformation experiments were performed in November 2011, all the protocols developed at the IVIA were adjusted to our specific environmental conditions and clone specificities. Mature Valencia, in our conditions, was very responsive to organogenic regeneration. We obtained positive plants, checked by PCR, and they are currently growing in the growth room. Hamlin was also transformed but was less responsive to organogenic regeneration, but we were able to obtain a plant currently growing in the growth room. Pineapple did not respond and we discarded the cultivar after the last batch in the production calendar was used. We are still waiting from results on this last experiment. We are currently introducing another clone of the same Hamlin cultivar 1-4-1 to improve its quality. A few of the initial mother plants were not as cleaned as previous determined and we introduced more plants using antimicrobial to guarantee cleanliness. It seems like yearly introductions may be necessary to maintain the quality of the material desired in the experiments. We are also cleaning the rootstocks Swingle Citrumelo and Carrizo to be transformed in future experiments.
The overall objective of this project is to develop and use a high-throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps) which causes psyllid yellows of tomato. Previous work focused on development of a system for the model plant Arabidopsis thaliana which has the best developed genetics of any plant and has been used in previous chemical genomics experiments. However, repeated attempts to infect Arabidopsis plants grown in solid culture media, liquid culture media, or hydroponics were not successful. Only plants grown in soil were infected by psyllid nymphs. The most recent experiments gave a low percentage of infected plants (about 15%), and differences between Arabidopsis lines observed previously were not repeatable. We believe that it will still be of interest to analyze the tolerance (lack of symptoms) seen in CLps infected Arabidopsis plants and are accumulating samples from CLps-positive plants for gene expression analysis. Because of the problems with Arabidopsis system, in late 2011 we began to develop a system for tomato which, as a natural host of CLps, is more easily infected. Compound leaves along with petioles from tomato plants were placed in 50 ml culture tubes with the cut end immersed in water in a microfuge tube. This design was adapted from one shown on a poster by Ammar et al. at the Citrus Health Research Forum in Denver in October. Adult psyllids are placed in the culture tubes and within 7 days most tomato leaf petioles are qPCR positive for CLps. After two weeks Ct values are typically less than 25 and about 80% of plants are infected. This system appears promising since chemicals can be introduced into the water for plant uptake. We will investigate methods to induce rapid rooting of these leaf petioles since this may increase chemical uptake. Experiments to test chemical uptake were initiated. We also began testing a transgenic tomato carrying a marker gene (GUS) driven by a pathogen responsive promoter (CaBP22). This system has facilitated detection of chemicals that activate defense pathways in Arabidopsis. Sweet orange seedlings were grown to test chemical application methods during the next quarter. This work is supported by a separate funding source, but little progress on the chemical genomics project is expected while this work is carried out.
Previously, we have shown that specific psyllid dsRNAs can be toxic to Asian citrus psyllids when the psyllids feed on citrus that have been engineered to produce these dsRNAs using a Citrus tristeza virus (CTV) expression vector. In this work, variability of dsRNA present within the tissues on which the psyllid is feeding effects toxicity to the psyllid and we have identified a threshold concentration of dsRNA needed to be toxic to adult psyllids. We have also identified a number of dsRNAs matching specific psyllid genes to which the psyllid are hypersensitive to (active at relatively low concentrations) when these dsRNAs are taken up orally through artificial diet feeding. Experiments have been initiated to express these dsRNAs in citrus and test their effects on all life stages of the psyllid when fed on these engineered plants.
The focus of this research has been to clone, express, and test the effect of suggested RNAi molecules on psyllid vectors using an artificial feeding system. We have developed a gut gene library, have isolated several sequences for critical proteins, and constructed RNAi molecules based on these sequences that will kill Asian citrus psyllids in controlled feeding experiments. We have also constructed and tested contest entrants. Several molecules have potential to control ASP. We have expressed two of the molecules in citrus using Dr. Dawson’s CTV vector, and have shown leaves from these trees are highly toxic to psyllids. There is a good correlation between RNAi expression and psyllid mortality. We will be testing other RNAi molecules using the CTV vector shortly. The sequences of the RNAi molecules and the genes they target are intellectual property. This project has given hope for a specific, environmentally friendly field control of Asian citrus psyllid without transforming trees. We are in the process of securing intellectual property rights for UF and USDA that will allow licensing, production, and marketing of this technology.
In FL nurseries, rootstock seed trees are located outdoors and only protected from psyllid transmission of Candidatus Liberibacter asiaticus (Las) by insecticide applications. In 2008, a survey detected two Carrizo citrange trees as HLB+. Given the potential risk for seed transmission and introduction of Las into nurseries by seed from source trees, assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008 seed were collected from mature Pineapple sweet orange trees in Collier Co. and in 2009 from Murcott tangor trees in Hendry Co., FL. For Pineapple orange, 415, 723 and 439 seedlings and for Murcott, 332 seedlings were tested at least twice by qPCR using 16S primers. In 2007, a single Pineapple seedling was suspect HLB+ but upon repeated testing was negative. From nurseries in 2008, 290 seedlings were recovered from fruit located on symptomatic branches of 2 Carrizo trees, and in 2009, 125 seedlings were recovered from 2 trees of Swingle citrumelo, 649 from 4 trees of ‘Kuharske’ Carrizo, 100 from 1 tree of Cleopatra mandarin and 100 from 1 tree of Sekwasha mandarin. In 2008, one suspect HLB+ Carrizo seedling was detected but HLB+ status was not confirmed after repeated testing. In 2009, a single questionable PCR detection for Cleopatra mandarin was obtained. Despite the occasional HLB+ test results, no plants have ever developed HLB symptoms and repeated testing has never confirmed anything other than the transient presence of Las in seedlings grown from seed obtained from Las-infected trees.
Huanglongbing (HLB) was first discovered in Florida in 2005. In response, Florida citrus nurseries began treating rootstock seed trees located outdoors with insecticide applications to reduce risk of psyllid transmission of ‘Candidatus Liberibacter asiaticus’ (Las), the putative causal agent. In 2008, a survey identified two ‘Carrizo’ citrange trees with symptoms of HLB. To assess the potential for seed transmission from HLB-affected seed source trees, assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008, 1557 seedlings germinated from ‘Pineapple’ sweet orange seeds from trees in Collier County were assayed by quantitative polymerase chain reaction (qPCR) using 16S rRNA gene primers. Of these seedlings, a single plant was positive for (Las+), although additional tests were negative. In 2009, no Las+ plants were detected among 332 ‘Murcott’ tangor seedlings from trees in Hendry County. From nurseries in 2008, one Las+ seedling was detected in 290 seedlings from fruit located on symptomatic branches of two ‘Carrizo’ citrange trees, but its Las+ status was not confirmed after repeated testing. In 2009, a single Las+ result was obtained for one of 100 Cleopatra mandarin seedlings, whereas no Las+ seedlings were detected for 125 seedlings from seeds from two trees of ‘Swingle’ citrumelo, 649 seedlings from four trees of ‘Kuharske’ citrange, or 100 seedlings from one tree of ‘Shekwasha’ mandarin. Despite the occasional Las+ qPCR tests, no plants developed HLB symptoms. The most probable explanation for these results is transient transmission of Las from seed obtained from HLB-affected trees with no subsequent disease establishment.
Over the past year, our research has focused on the following areas: (i) Isolation and sequencing of TAL effectors from additional citrus canker strains Sequencing of TALE genes is especially difficult due to the presence of between14 and 20 repeats of the highly sequence-related DNA binding domain. However with considerable effort, we have now determined the sequences of eight proteins from five novel strains: A44 (Argentina), Etrog (Florida), 2090 (Florida), Miami (Florida), and 93 (Brazil), with four more protein sequences nearing completion. Although these strains show variation in phenotype or host range, our engineered promoter constructs containing 14 TALE recognition sites conferred recognition to TALEs in four of the strains, with the fifth pending analysis. These results further support our aim of engineering a resistance construct that will be triggered by a broad range of canker strains. Differences do occur in some of the sequences, and we plan to investigate how these differences may influence the behavior of strains in various assays. (ii) Production and testing of stable transgenic citrus lines: As of the end of this year, we have transformed a total of 21,504, 747, and 173 explants of ‘Duncan’ grapefruit, ‘Ruby Red’ grapefruit, and sweet orange cultivars, respectively, and have 446 plants in 4 inch pots. We have tested epicotyl and cotyledon explant material and find that epicotyls are the most efficient material for use with grapefruit, whereas cotyledons appear to work best for sweet orange. The Ruby Red cultivar is the most difficult to work with, because of the difficulty in sourcing seed. Each transformant is grown through shooting, rooting and transfer to soil, and then it is analyzed by PCR for each of the construct components – promoter, gene and selectable marker. Plants are further tested by pathogen inoculation. All stable and transient transformations were made with eight distinct gene constructs and a negative control. Overall, we find the broadest and best induction using the 14 box promoter, relative to other promoter versions. We have tested three HR-inducing genes – the Bs3 gene from pepper, and the AvrGf1 and 2 genes from Xanthomonas We have not observed activity of Bs3 in citrus to date. AvrGf1 has worked well in transient assays, but we have not yet analyzed enough stable lines to identify reproducible disease resistance. We continue to test lines as they mature, and AvrGf2 lines will also be tested when they become available. (iii) A third gene option for conferring resistance The type 3 effector AvrGf2, identified from X. fuscans subsp aurantifolii strain C, is being tested as another resistance gene option because it has been observed to cause a more robust HR on grapefruit than AvrGf1. The coding sequence was fused with the Bs3- PIP14 box promoter and used in transient and stable transformation assays. In transient assays, the avrGf2 construct did confer a robust HR within 3 days as compared to 4 days using AvrGf1. Stable transformation experiments involving epicotyls of ‘Duncan’ grapefruit, ‘Ruby Red’ grapefruit, and sweet orange segments, had 42, 16 and 32 plants transferred to rooting media, respectively. More transformants are in the pipeline, and all will be subject to molecular characterization and testing.
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