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 have repeated NAD+ treatment experiment. Again, both soil drench and foliar spraying of NAD+ have been conducted. The plant defense activator Actogard, which is highly effective against citrus canker was included in the experiment as a control. We found that while foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. Interestingly, we observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes. We are planning to repeat the experiment to confirm the systemic effects. Meanwhile, we are still trying to find the best approach for NAD+ application. NAD+ analogs are under test for identifying potential chemicals to control citrus canker. For objective 2, 30 transgenic lines expressing ELP3 and 22 lines expressing ELP4 have been generated. The transgenic lines have been molecularly characterized to confirm the presence and expression of the transgenes. The transgenic plants are growing in greenhouse and will be tested for canker resistance. Citrus homologs of ELP3 and ELP4 have been cloned and sequenced. We are cloning the two genes into T-DNA vector and will be transformed into the Arabidopsis elp3 and elp4 mutants, respectively, to confirm their functionality.
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 have repeated NAD+ treatment experiment. Again, both soil drench and foliar spraying of NAD+ have been conducted. The plant defense activator Actogard, which is highly effective against citrus canker was included in the experiment as a control. We found that while foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. Interestingly, we observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes. We are planning to repeat the experiment to confirm the systemic effects. Meanwhile, we are still trying to find the best approach for NAD+ application. NAD+ analogs are under test for identifying potential chemicals to control citrus canker. For objective 2, 30 transgenic lines expressing ELP3 and 22 lines expressing ELP4 have been generated. The transgenic lines have been molecularly characterized to confirm the presence and expression of the transgenes. The transgenic plants are growing in greenhouse and will be tested for canker resistance. Citrus homologs of ELP3 and ELP4 have been cloned and sequenced. We are cloning the two genes into T-DNA vector and will be transformed into the Arabidopsis elp3 and elp4 mutants, respectively, to confirm their functionality.
Seasonal root sampling continues in two field sites for root density and root growth. We have completed a second year of root growth data from Hamlin/Swingle and are continuing to collect a second year of root growth data on Valencia/Swingle. Results continue to emphasize the need to use treatments that improve root longevity as the main method of managing HLB root loss. Additional root growth appears to occur at the expense of older roots and is unlikely to provide sustained improvement in root density. Root tube images taken with the root scanner to assess root growth, lifespan, and death continue to be collected monthly. Visual observation shows that the method is working well, but data analysis has been delayed due to an unexpected software glitch, a new version has been released and we are working through a backlog of images. Only one rootstock tested to date has shown a significant difference in response to HLB. It remains the only rootstock with significantly better root density (increased) when infected by HLB. Root loss has not been observed yet in this rootstock. Fruit drop data has been collected to determine if the increased root density is associated with reduced fruit drop. We continue to monitor the most promising rootstocks identified in the field trial to HLB using rhizotrons in the greenhouse. The second set of rootstocks has been grafted with Hamlin scion or left ungrafted to monitor the effects of the scion rootstock interaction on the rootstock response. Phytohormone analysis has been completed on roots, but no clear consistent change has been observed in old roots or new root tips. Microscopy of the root samples to identify the mechansim of root dieback is awaiting the arrival of a new confocal microscope.
We monitored stem water potential using thermocouple psychrometers on trees in a commercial Hamlin grove in Lake Alfred (Gapway Grove) that has an established experiment comparing Advanced Citrus Production System (ACPS) and conventional grower practices (C). We found no statistical difference in the mean minimum daily water potential between the two treatments during the three sampling periods between April 4-10, April 30-May 9th, May 21-May 23, however we did find greater drought stress in ACPS trees on the hot days in May. There was a borderline statistical difference (p=0.065) in the slope of the water potential curves, where trees in the conventional grower irrigation treatment did not experience as rapid of an increase in drought stress compared to the ACPS trees. These data support xylem vulnerability curves generated in the lab that estimate the loss of hydraulic conductivity resulting from drought stress, where the ACPS trees suffered greater losses in conductivity during moderate drought conditions. We found no significant differences in xylem vessel diameters, but we did note that the pit membranes that prevent losses in hydraulic conductivity were significantly thicker in the C trees compared to ACPS trees. Thus, we found that ACPS trees were more vulnerable to the effects of drought stress on a daily basis during the early part of the growing season, presumably due to the fact that they receive more regular irrigation and have adapted to a wetter, more consistent soil moisture environment, and have not invested in additional resistance to the effects of drought compared to the C trees that are irrigated on a less frequent basis. This study highlights the importance of maintaining consistent irrigation for field grown trees, and that clogged or damaged irrigation systems in ACPS trees are a significant risk. A peer-reviewed publication with these data is currently in progress for submission to Tree Physiology. A second component of this study was to determine whether plant growth regulators (PGRs) could be used in a heavily infected Hamlin grove in Lake Alfred to mitigate preharvest fruit drop. Our trial focused on replicating data from colleagues in Brazil who reported benefits of using frequent, low doses of 2, 4-D throughout the growing season. We monitored fruit drop every two weeks starting in September through harvest in December, and found no significant differences between the different treatments. HLB symptoms were somewhat variable in this block, and we saw several mature trees die during this single season, but our study included enough trees to control for tree health. In some cases, applying the commercially-available adjuvant product EcoAgra showed a slight decrease in preharvest fruit drop, but there was enough variability in the data to suggest that none of the treatments we applied to these Hamlin trees was sufficient to reduce drop. At this point we do not recommend 2,4-D at the concentration and frequency applied in this study to be used to mitigate preharvest fruit drop, but these data should be considered within the scope of other PGR trials occurring simultaneously around the state. Symptom severity, local climate, soil differences, and existing pest or disease issues could account for success in some areas but not others.
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 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 have found that three different lines appear to be giving strong tolerance against HLB. We are propagating the plants for more extensive analysis.
Objective 1. To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface. The importance of motility and chemotaxis in the infection process for citrus Xanthomonas strain varying in host range is under investigation. Membrane protein receptors (MCP) profiles were predicted from complete genome sequence analysis and PCR amplification and nucleotide sequencing for wide (Xcc62, Xcc306) and narrow (XccIran2, XccIran10, Xcc12879) Xanthomonas citri subsp. citri (Xcc) strains compared to B and C canker strains (Xfa69, Xfa341), X. alfalfae subsp. citrumelonis (Xac) and X. campestris pv. campestris (Xc). All Xcc strains showed similar MCP content except Xcc306 which lacked some of the MCP sequences. To confirm differences in MCP profiles among wide and narrow host range Xcc, the evaluation will include 40 addtional strains. These results should confirm whether there is a relationship between MCP profile and chemotaxis differences among Xcc strains. Objective 2. To investigate biofilm formation, composition and structure in relation to motility for different Xcc strains and non-canker xanthomonads. Previously, we detected extracellular DNA (eDNA) in the initial stages of the biofilm formation. Differences between wide and narrow host range strains of Xcc were found when the biofilms were treated with DNAse which reduced biofilm formation and disrupted preformed biofilms. To evaluate the potential direct effect of eDNA on bacterial aggregation, cultures of Xcc, Xac and Xc were amended with genomic DNA (gDNA) from each of the strains. In most cases no differences were found in biofilm formation between cultures treated and non-treated with gDNA. Therefore, the role of the DNA in biofilm formation is more complex. The next objective was to determine the origin and nature of the DNA detected in biofilm structure. To characterize such DNA, gDNA and eDNA were analyzed by BOX and ERIC PCR and DNA profiles compared but no differences were found. Futures assays will use RAPDs and several primer sets to detect any differences in DNA. In addition to characterizing biofilm, exopolysaccharide (EPS) production was evaluated in the different strains after growth in nutrient broth medium amended with 2% sucrose. No quantitative differences in EPS were detected among strains but narrow host range strains showed qualitative differences which may be due their high DNA content. Further assays are planned to clarify this difference.
FireWall 50WP (50% streptomycin; Agrosource, Inc.) was granted by 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 was 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 applications was conducted in January 2016. After sprays with FireWall, no Xcc colonies resistant to streptomycin were detected from two grove locations that received two sprays of FireWall in spring- summer 2015. Xcc in one of the locations has been exposed to streptomycin for three seasons without development of resistance. A report of our findings will be submitted to FDACS.
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 about twice as high in the ridge as in the flatwoods locations. 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 th 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.
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., statewide survey) sampling compared to 2013. Substantially higher root density in Florida groves in spring and summer 2014 is predicted to result in less fruit drop than in the previous two seasons. Both field surveys and greenhouse experiments show that Huanglongbing (HLB) increases the susceptibility of the citrus root system to the damaging root pathogen Phytophthora nicotianae. This increased P. nicotianae infection suggests that root system damage may occur more quickly in the presence of both pathogens. Chemical control of P. nicotianae may slow infection of the root system and subsequent yield decline in HLB-affected trees in Phytophthora-infested groves. To test the value of P. nicotianae control in slowing tree decline in the presence of both pathogens, 100 P. nicotianae-infested nursery trees were inoculated or mock-inoculated with HLB-affected budwood. After HLB inoculation, trees were treated bimonthly with Ridomil Gold SL (mefenoxam) or Aliette WDG (Aluminum tris O-ethyl phosphonate) compared to untreated controls. HLB status, P. nicotianae infection and propagule counts, and visible symptoms were assessed bimonthly. After 12 months, the trees were harvested and biomass of roots and shoots was measured. As expected Ridomil and Aliette significantly reduced P. nicotianae root infection and increased fibrous root mass compared to untreated controls for HLB(-) trees. Neither Ridomil or Aliette significantly reduced P. nicotianae root infection or increased fibrous root density for HLB(+) trees, although both measures quantitatively improved with Ridomil performing slightly better than Aliette. These data suggest that HLB reduces the effectiveness of fungicide control of Phytophthora root rot as a consequence of increased susceptibility to P. nicotianae.
FireWall 50WP (50% streptomycin; Agrosource, Inc.) was granted by 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 was 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 2014 season was conducted in November and the report recently submitted to FDACS. After sprays with FireWall, no Xcc colonies resistant to streptomycin were detected from two grove locations that received one or two sprays of FireWall in summer 2014. Thus far, Xcc in the Felda location has been exposed to streptomycin for two seasons and the Vero Beach location for one season without development of resistance. Two greenhouse trials measured the residual systemic activity of streptomycin against Xcc in leaves to be 10 weeks after foliar spray application. Trans-cuticular movement of streptomycin explains the consistent performance in field trials against canker in that the penetration of the leaf insures that streptomycin is protected from wash-off by rainfall, weathering and degradation by UV light.
The Core Citrus Transformation Facility (CCTF) continued to provide service for production of transgenic citrus plants. The work load that included: co-incubation experiments, explants incubation, shoot harvesting and inspection, PCR testing, micro-grafting, and care of plants in the greenhouse was kept at maximum level allowed by the number of holidays within the last quarter of the year. The work done by the CCTF from October through December was concentrated on older orders as five new orders were placed to CCTF from two different clients in late December. Production of transgenic rootstock plants ordered by the CRDF continued at slower pace. One out of 29 plants that were previously growing in the greenhouse died. Nineteen more ‘pot-adapted’ plants were moved from the laboratory to the greenhouse bringing the total number up to 47. The growth of first plants moved to the greenhouse was not as vigorous as expected and they were not cut into explants for propagation. That may be done in the middle of February. In the period covered by this report, CCTF produced plants for the following orders: pNPR1-17 plants, pNPR1-G-five plants, pELP3-G-12 plants, pELP4-G-five plants, pMG36-eight plants, pX7-2- five plants, pHGJ27- one plant. Three orders that are being serviced at this time require detection of transgenes in transgenic plants by microscopy. The methodology for this detection is still being worked out with the EM lab personnel. If this was solved at earlier date, the output of the facility would have been higher. Plants produced in this quarter were mostly Duncan grapefruit and Carrizo citrange with the exception of one Valencia sweet orange plant. As it was anticipated in the previous report, the quality of Duncan fruits and seeds from the CREC’s grove has deteriorated further. Before the end of January, CCTF will contact local grower to get supply of better quality Duncan fruit.
All of the research described in the previous report is being analyzed or active research is being transferred to a new grant from CRB. The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype is concluded and analyzed. There were some surprises. RNA levels of FT3, the FT homologue from citrus that other research by us and others indicates is most closely associated with flowering, were low in leaves at bloom time, whereas we expected high levels of expression. This may indicate that high levels of FT protein are produced at that time and sent to apices and mRNA is depleted. A study of CiFT3 transgenic tobacco plants treated with various growth regulators has been performed and the data is now being analyzed. The growth hormones produced striking and individually different phenotypes in each treatment. The data includes plant height and leaf number, size, and area. The endogenous ciFT3 promoter from sweet orange was successfully cloned to be used in the transcription activator-like (TAL) effector system inducible by methoxyfenozide that will hopefully activate the naturally present FT3 gene in citrus. Large numbers of citrus seeds are being germinated for transformation studies with this construct.
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 almost 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. We have installed tubes for the preliminary tests of a new root scanner to nondestructively measure root growth and root death. Preliminary tests are being used to determine the optimum depth of tube needed for future experiments. This will allow for more rapid quantification of root growth and death with less damage to observed trees. These will be used in future experiments of treatments used to increase root longevity and for observing the effects of HLB on young plantings that are too easily damaged by extensive soil core sampling. Sampling at a rootstock trial site continues. As some of the rootstocks initially chosen for observation have not performed better than Swingle, these rootstocks will no longer be sampled and others that show positive canopy responses are under consideration for root sampling. The response the root system of the most promising rootstocks identified in the field trial to HLB is currently being observed in 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. Additional rootstocks are being considered for the second round of rhizotron experiments. Method development to characterize the mechanism by which Liberibacter causes root death is underway.
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 last quarter leaf uptake and phloem transport studies of the EO formulations were initiated using two approaches: (i) fluorescence microscopy with dye doped formulations and (ii) GC/MS spectrometry. Previously we have reported the development of o/w formulations with maximum oil loadings of 7% for thyme oil and EO-A and 5% (w/w) for EO-B showing high inhibition of L. crescens. Further modifications in the interfacial properties of the formulations, by varying the surfactants, co-surfactant and the ionic strength have led to increased oil loadings from 7 to 16% for thyme oil and from 5 to 10% for other oils. This will make the formulations more economically viable and reduce the amount of surfactant applied on the plants. Experiments to determine the change in the droplet size and stability of the formulations due to increased oil loading are underway. Method development experiments were performed to estimate EO uptake and transport in citrus plants by using GC/MS and LC/MS. Initial GC/MS analysis of thyme oil revealed its major components to be thymol, p-cymene, .-terpinene with carvacrol in small amounts. Macerated leaves from Valencia orange and Duncan grapefruit plants were also analyzed by GC/MS for headspace analysis to establish the background and interference from molecules with similar emission as EO components. Additionally the detection limit of the EO (or component) was determined by GC/ MS headspace analysis by mixing known amount of EO to macerated citrus leaves. Preliminary experiments with thyme oil formulation show that thymol component can be detected in the headspace of leaf samples upto 1mg/ L thyme oil. Future studies include estimation of EO transport in the citrus plants. To investigate the penetration of formulations into citrus leaves, dye doped formulations were prepared with the lipid soluble dyes e.g. Bodipy 505/515, Vybrant DiO etc. having fluorescence emission in 475-525 and 550-650 nm range. Initial studies done by foliar application of the dye doped formulations showed non-uniform uptake of the microemulsions and photo-bleaching during imaging. Further studies are underway to study the uptake of EOs.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over four years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/8th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Data and tissue samples were collected from almost every tree in the test site during the last quarter. Additional plantings are welcome from the research community.
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