This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, as proof of concept to determine the root specific nature of the promoters (RB7, C1867 or SLREO), we have incorporated the promoter-gus sequences into N. benthamiana and several plantlets have been regenerated. These constructs were also incorporated into Carrizo citrange in the previous quarter, but we have had a severe Mould mite infestation in our tissue culture room which destroyed most the promoter-gus citrus plantlets. A repeat experiment to produce fresh carrizo citrange plants expressing the root specific promoters were carried out in this quarter and plantlets are being regenerated. The Carrizo citrange plants expressing a root specific promoter – insecticidal gene construct (GNA, APA or ASAL genes individually or stacked with the CpTI gene) were not affected by the mite infestation and several plants have been hardened and transferred to the greenhouse for growth.
The goal of this project was to create canker-resistant citrus through a strategy of an engineered cell death response to Xanthomonas citri pv. citri TAL effectors. During the project, we defined sequences of 14 distinct effector binding elements (EBE) recognized by Xcc TAL effectors. Constructs were created that incorporated these EBEs into the Bs3 promoter driving a pathogen effector gene (either avrGf1 or avrGf2) that triggers a hypersensitive cell death reaction in several citrus cultivars. Two key constructs were one with all 14 binding sites (14EBE) and one with four binding sites (4EBE) that was expected to be bound by at least two TAL effectors from each X. citri strain characterized. These constructs were shown to function as expected in a transient assay in citrus leaves: they produced a hypersensitive response and reduced bacterial growth when co-inoculated with a Xanthomonas strain carrying a TAL effector recognizing one of the EBEs. In the absence of pathogen inoculation, the promoters are tightly “off” in the transient assay. However, despite the tight regulation of these constructs in a transient assay and multiple attempts to produce stable transformants in Duncan grapefruit or sweet orange, we have been unable to recover transgenic lines. We also tested Carrizo citrange, a citrus variety that does not exhibit a hypersensitive response to AvrGf1 or AvrGf2, and we were able to recover transformants. These results suggest cryptic induction of the construct at some point during the transformation process, causing transformants to be selected against. A second promoter derived from the citrus Lateral Organ Boundaries (LOB) gene also failed to produce transformants when driving AvrGf2 expression. Therefore, despite the theoretical promise of the approach, more work would be needed to define a promoter and executor gene combination that could be efficacious in citrus.
Seasonal root sampling continues in two field sites for root density and root growth. We are collecting a second year of root growth data from Hamlin/Swingle and have 1 year of root growth data on Valencia/Swingle. Results so far emphasize the need to use treatments that improve root longevity as the main method of managing HLB root loss. Root growth stimulation is unlikely to improve root density. Preliminary tests of root tubes are almost complete. This will allow for more rapid quantification of root growth and death with less damage to observed trees. Field site selection for the first set of tubes is underway. Sampling at a rootstock trial site continues. Only one rootstock tested to date has shown a significant difference in response to HLB we will be installing root tubes to compare root growth and root death data to rhizotron experiments. We continue to monitor the most promising rootstocks identified in the field trial to HLB using rhizotrons in the greenhouse. The first experiment is nearing completion and data analysis of root growth and root longevity in response to Las is currently underway. The analysis is taking longer than expected because of some variations introduced by the original setup that need to be corrected for. Fine tuning is being done to the setup to reduce the data analysis steps in future experiments. Additional rootstocks are being considered for the second round of rhizotron experiments and should be planted and inoculated shortly. Method development to characterize the mechanism by which Liberibacter causes root death is underway and the experimental samples will be collected shortly.
To confirm that treatments with acidified irrigation water reduce the impact of bicarbonate stress on root health, we surveyed 8 ridge groves in Highlands county and 4 flatwoods groves in Hardee county with high bicarbonate stress as detected in our 2013 survey. All the blocks are less than 10 year old Valencia trees on bicarbonate sensitive rootstocks, Swingle and Carrizo. The survey is bimonthly to follow the recovery of these blocks and at harvest to compare 2014 season block yields 1.0 to 1.5 years after acid treatments began. From May to November root density fluctuated but was maintained at about the same level throughout the season. Root density was 5-10X higher in the ridge than in the flatwoods groves. Phytophthora populations were low or non-detectable in ridge groves but were always present and about 2-3x higher in the flatwoods groves. Soil pH ranged from 5.0-6.0 in the ridge after 1-1.5 years of acidification. pH in the flatwoods groves was initially in this range but rose above 6.5 and remained at that level. Flatwoods groves with higher bicarbonates in irrigation wells and soil require higher rates of acidification treatment to reduce below 6.5 as recommended for Swingle and Carrizo rootstock groves. Block yields will be evaluated for response in production compared to previous seasons. Surveys are continuing in 2015 with the addition of 4 groves without acidification in Highlands and Hardee counties to serve for comparison with an un-managed check. Recommendations based on these results were published in the May 2015 issue of Citrus Industry magazine.
Recent evaluation of root production on HLB-affected trees compared to presumed healthy trees confirms that root loss is due to reduced root longevity. This root loss is exacerbated by biotic and abiotic stresses in the rhizosphere. Increased susceptibility of Las’infected roots to Phytophthora spp. is evidenced by statewide populations that have fluctuated from unprecedented highs in the 2011 season to an unprecedented low in 2013 compared to 25 years of pre-HLB soil populations. Phytophthora propagules per soil volume and per root resurged in 2014 in response to a more than doubling of root density based on intensive (i.e., local repeated measures) and extensive (i.e., Syngenta statewide survey) sampling compared to 2013. Substantially higher root density in Florida groves in spring and summer 2014 has so far this harvest resulted in less fruit drop than in the previous two seasons. Both field surveys and greenhouse experiments show that HLB increases the susceptibility of the citrus root system to the damaging root pathogen P. nicotianae. Increased P. nicotianae infection suggests that root system damage occurs more quickly in the presence of both pathogens. Chemical control of P. nicotianae slows infection of the root system and may slow yield decline in HLB-affected trees in Phytophthora-infested groves, however, HLB reduces the effectiveness of fungicides for control of Phytophthora root rot as a consequence of increased root susceptibility to P. nicotianae infection.
For 2015 season, FireWall 50WP (50% streptomycin; Agrosource, Inc.) has been granted an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey is to apply our published protocol (Behlau et al., 2012) for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc. The survey for 2015 season will be conducted in November 2015 and the report of results submitted to FDACS. Greenhouse trials to measure the residual systemic activity of streptomycin against Xcc in leaves after foliar spray will be repeated as in 2014 to confirm trans-cuticular and upward movement of streptomycin into new foliage via the xylem.
Objective 1. As reported previously, methyl accepting chemotaxis proteins (MCPs) were identified for wide (Xcc62, Xcc306) and narrow host range strains of Xanthomonas citri subsp. citri (Xcc) compared to X. fuscans subsp. aurantifolii B and C strains, X. alfalfae sbsp. citrumelonis (Xac) or X. campestris pv. campestris (Xc). All Xcc strains showed similar MCP content except Xcc306 which lacked some of the MCPs. MCP analysis was expanded to include Xcc strains from different geographical areas. 37 strains from Argentina (27%), Uruguay (32.5%), Brazil (27%) and 5 isolates from citrus of uncertain origin (13.5%) were analyzed for 15 MCPs. Only one of the MCPs was conserved among all Xanthomonas strains (XCV1951), and two of them were specific of Xc (XCC0324 and XCC1954). Objective 2. The role of extracellular DNA (eDNA) was evaluated in the formation and stabilization of the biofilm matrix. The presence of eDNA in biofilm was confirmed for several Xcc strains and Xac. DNAse treatments of Xcc strains and Xac reduced biofilm formation at the initial stage of development as well as disrupted preformed biofilm. The DNAse effect on formation or disruption varied among Xcc strains and Xanthomonas species and indicates differing roles for eDNA. Differences in fiber structures containing eDNA in biofilms, bacterial cultures, and in twitching motility assays were detected by SYTO-9 staining and fluorescence microscopy. The proposed roles for eDNA in the early stages of biofilm formation are as an adhesin and in mature biofilms as a structural component. .
The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped from the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL, to Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, in early October, 2014. An additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped to Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. At Penn State, all the transgenic lines are being propagated as vegetative cuttings. We have achieved over 50% success rate in rooting cuttings from the plants, and rooted cuttings are now growing for all of the independent lines. The rooting process for cuttings takes 3-4 months in our facility at Penn State. We have performed two protein immunoblots with an antibody that will detect the FLT-antiNodT fusion protein (anti-cmyc antibody). We detected protein of the correct size (~50 kD) as a single band without degradation products in two different lines, and not in control plant samples. These results are very promising and are consistent with successful expression of full-length FLT-antiNodT fusion protein in ‘Duncan’ grapefruit. During the next quarter, we will be repeating these experiments with all of the transgenic lines and using improved protein extraction techniques to develop better western blot images and documentation.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we continued optimizing the NAD+ treatment conditions. Both soil drench and foliar spraying of NAD+ were tested again. Foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. We previously observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes and are repeating the experiment to confirm the systemic effects. We are also testing different recipes for stabilizing NAD+ on the surface of leaves or in soil, since this chemical is not stable under aqueous conditions. For objective 2, transgenic citrus plants expressing the Arabidopsis nonhost resistance genes are growing in greenhouse and will be tested for canker resistance. We are also propagating the transgenic plants to produce progenies for HLB resistance test. Citrus homologs of the Arabidopsis nonhost resistance genes have been cloned and sequenced. To test their functionality, we have cloned the citrus homologs into plant expression vector and have transformed into the corresponding Arabidopsis mutants. T1 plants are growing.
Analysis and writing continue in preparation for the end of this project in June. One student is completing her MS degree, and with her data completed and analyzed, manuscripts are being developed. These may not be completed by the termination date of the project, but we hope to have at least first drafts done. The FT3 gene is also being put into the CTV vector and is being put into CRISPR/Cas systems in collaborations with others.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This greenhouse is completed and operational. – The first cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation are currently being propagated. – The second cohort of advanced, tetrazygous citrus rootstock materials for en masse propagation have been identified and are being prepared to have cuttings taken from them. – In addition to the 1st & 2nd cohorts of tetrazygous rootstocks, promosing diploid rootstocks have also been identified and are being prepared to have cuttings taken from them. As of March 2015, plants are still growing in the green house and no significant changes to report.
A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. Exposure to canker inoculum showed remarkable resistance in chimera compared to control. RNA was isolated from 16 transgenic Hamlin containing Chimera. RT-qPCR showed 50% of them have relative high gene expression. One of them showed over hundred times higher expression compare to plant expressing the lowest level of chimera. These promising transgenic lines were replicated by grafting for HLB challenge. About 30 Hamlin transformed with thionin also were obtained. Twenty transgenic lines were confirmed containing thionin gene by PCR. RNA was isolated from 16 transgenic Hamlin containing thionin. Six of them have relative high gene expression by RT-qPCR. These transgenic lines will be replicated for HLB challenge. Two new chimeral peptide have been developed and is used to transform citrus. Many transformed Carrizo shoots with new chimera construct were obtained. Some were transferred to the rooting medium. Replicated trangenic lines expressing chimera, thionin and D4E1were grafted with HLB infected rough lemon. Las tilter will be checked by qPCR periodically. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was 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 for HLB and other diseases. Many putative transformants were generated on the selective media. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slight canker resistance by infiltration test. Spray inoculation was tried and some of them show obvious canker resistance. To confirm that high ROS production was not due to variability in Hamlin, we examined 40 Hamlin seedlings and no or very low level ROS production was detected. In contrast, relatively higher ROS production was detected from wild-type Carrizo seedings compared to Hamlin seedlings. Two potential FLS2 orthologues were identified in Hamlin and their expression was shown much lower compare to nbFLS2. Replicated trangenic Carrizo lines expressing nbFLS2 were challenge with ACP. Las titer will be checked by qPCR periodically. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. 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. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter is being used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only.
The overall aim of this project is to develop and evaluate oil-in-water (o/w) microemulsion (ME) formulations to deliver antimicrobial essential oils (EOs) to the phloem of HLB-infected trees. In the previous quarter we improved the oil loading and temperature stability of our formulations, performed method development for estimating essential oil uptake and transport in citrus plants and also developed dye doped formulations to investigate penetration of essential oils into citrus leaves by fluorescence microscopy. Previously we had reported that the formulations had minimum inhibitory concentrations (MIC) in the range of 50-100 ppm of oil concentration against L.crescens. Since the minimum bactericidal concentrations (MBC) are about 4 times greater than MIC, we tested the formulations at 500 ppm oil concentration in order to estimate the MBC. All the oil formulations as well as controls (formulation with the oil containing just surfactant) were effective indicating that MBC of the formulations is less than 500 ppm of oil concentration. Tests to assess efficacy of the formulations in HLB infected citruses via a bud graft technique were undergoing at Indian River Citrus and Education Center, Ft. Pierce. The results showed the formulations as well controls (formulation with the oil containing just surfactant) killed the bacteria in the bud stick. EO-A and thyme oil were also phytotoxic to the buds at the concentration evaluated (5000 and 500 ppm), since none of them grew. However, the scions treated with just control did grow indicating that the surfactants used are not phytotoxic to the plants. In field tests using trunk application EO-A and EO-B were ineffective. We tested the potential of combining the currently used thermal therapy (Dr. Ehsani, UF) with our EO formulations which could lead to enhanced essential oil penetration into leaves and kill the bacteria in plant parts like roots where thermal therapy is ineffective. Thyme oil formulations were diluted to 2000 ppm of thyme oil in field and sprayed using a hand sprayer till an even coverage was achieved which was followed by thermal treatment. QPCR tests are undergoing to assess the efficacy of such a combined technique. A leaf assay test is also being conducted with EO-A formulation at Dr. Gonzalez’s lab as a quick study to evaluate the performance of the formulation at different EO-A concentrations.
Image acquisition and PCR analysis: Individual leaves (213 total) from seedlings from the first run (Optical Sensing Project-1; OSP-1) were photographed using three emission filters (500nm, 690nm and 730nm) using three excitation wavelengths (UV 364nm, Blue 470nm, Green 527nm) in addition to polarization images at 594nm Approximately 1,500 images were examined for correlations between HLB infection and fluorescence emissions and optical rotation of 594 nm light (polarization images). The leaves were processed by isolation of the midveins and the tissue pulverized in liquid nitrogen and stored at -80 C for PCR analysis at a later date. A second trial (OSP-2) was started Dec 15th by placing the seedlings in William Dawson’s psyllid room at the CREC at Lake Alfred, FL.
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