Objective 1. To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface. A new assay was used to evaluate the chemotaxis response to apoplastic fluids from sweet orange, Mexican lime, kumquat, grapefruit, lemon, cabbage and Prunus spp. for strains Xcc A, A*, Aw, X. alfalfae subsp. citrumelonis, X. campestris pv. campestris, and X. arboricola pv. pruni. No significant differences were found in chemotaxis response to host versus non-host apoplastic fluids when the strains were exposed in the same culture plate. The apoplastic fluids from Mexican lime produced a significant and positive effect on the swarming motility for strains Xcc A 306 and 62, Xcc A* Iran2, and Xcc Aw 12879. Objective 2. To investigate biofilm formation and composition and its relationship with bacteria structures related with motility in different strains of Xcc and comparison to non-canker causing xanthomonads. Extracelullar DNA (e-DNA) was confirmed to be important in the initial stages of biofilm formation for citrus xanthomonads. Currently, the effect of DNAse treatments on surface movements on semisolid culture medium plates is being evaluated for the citrus strains. Aggregation is disrupted by DNase treatments at a very early stage of the biofilm formation, i.e., within 24 hours. e-DNA may be involved in the initial structural development of the biofilm. DNase treatments of preformed biofilm reduced aggregation by 60% for citrus xanthomonads and 25% for X. campestris. e-DNA was visualized using SYTO-9 staining and fluorescence microscopy. Differences in aggregation of DNase treated and non-treated bacteria were also visualized by TEM. In addition, e-DNA fibers were detected by SYTO9 staining of cells undergoing twitching motility. The role of e-DNA in twitching motility is under further investigation. Another biofilm component, EPS, is also being compared for wide and narrow host range strains of Xcc. No differences in EPS production have been detected thus far.
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, both soil drench and foliar spraying of NAD+ have been performed. In the side-by-side experiment with the plant defense activator Actogard, soil drench provided good protection against citrus canker, whereas foliar spraying had limited effects. We are repeating the experiment and trying to find the best approach for NAD+ application. We have also been testing NAD+ analogs to identify potential chemicals for citrus disease control. For objective 2, newly generated transgenic plants are growing in greenhouse. Presence and expression of the transgenes have been tested. All transgenic plants are growing in the greenhouse and will be tested for canker resistance. Citrus homologs of the defense genes have been cloned and sequenced. The will be used for functionality test through complementation experiment.
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, both soil drench and foliar spraying of NAD+ have been performed. In the side-by-side experiment with the plant defense activator Actogard, soil drench provided good protection against citrus canker, whereas foliar spraying had limited effects. We are repeating the experiment and trying to find the best approach for NAD+ application. We have also been testing NAD+ analogs to identify potential chemicals for citrus disease control. For objective 2, newly generated transgenic plants are growing in greenhouse. Presence and expression of the transgenes have been tested. All transgenic plants are growing in the greenhouse and will be tested for canker resistance. Citrus homologs of the defense genes have been cloned and sequenced. The will be used for functionality test through complementation experiment.
The objective of this research will 1) characterize Pr-D (FP3) and its role and disease suppression; 2) investigate the dynamics of the prophages/phages in Las bacteria by revealing the variations in gene expression and recombination; and 3) identify critical elements, such as heat and chemical stress that facilitates lytic activities of the prophages. In addition, we will demonstrate whether or not if the ‘cross protection’ using mild strains of Las bacteria will work for the HLB pathosystem along with quantitative detection protocols for prophage-based strain differentiation. We have propagated more Las-infected periwinkle and citrus plants that contain high titers of prophage/phage FP3, which will be used for isolation and characterization of prophage/phage FP3. Different varieties of citrus plants inoculated with a mild strain have been evaluated in greenhouse. Intriguingly, different varieties showed different response to the “mild stains/isolates”. However, in a given variety, the mild stain status was maintained. We are evaluating the factors that affect the symptoms and titers and determining if a mild strain can be maintained in major commercial citrus varieties. We have developed a digital PCR (dPCR) system for early detection of HLB and tracking of lysogenic and lytic activities of the Las prophage/phage. We show that as few as 1 to 2 copies of the targeted DNA molecules per microliter can be detected, with the prophage probe providing the best sensitivity. The copy number measurement of the targeted DNA molecules can be statistically differentiated from the healthy sample and negative water controls. We are optimizing dPCR-based assay for differentiation of Las populations that carry different prophages/phages.
The objectives of this research are 1) to develop cost effective thermotherapy protocols for field application by optimizing temperature and relative humidity conditions in the tent; 2) to develop a mathematical model derived from our data and grower’s data which will be used to determine the best treatment duration in future applications; and 3) to study gene expression of HLB-affected citrus plants that received heat treatment, and identify critical citrus genes that may be induced by heat stress for the benefit of suppressing HLB. We are continuing the field samples and data analysis on heat-treated HLB-affected trees. Field trials to monitor Las titers and fruit drop/production in Valencia and grapefruit are ongoing with the latest leaf samples and fruit counts taken in August and September. We are still in the process of determining an algorithm that relates environmental conditions with decreases in Las titer. In term of transcriptome analyses, we have conducted a comparison study between heat-treated and non-heat-treated citrus plants from a field. There were 31 consistent up-regulated genes and 47 down-regulated genes in the the citrus trees treated with heating. We also conducted heat-treatment with constant temperature at 40’C, 85 % relative humidity, and a 12 hr photoperiod for 4 days. The RNA-seq data revealed 22 up-regulated and 20 down-regulated differentially expressed genes in LB-affected plants that received controlled heat treatments. We are currently verifying the putative important genes with differential expression (DE) by RT-PCR. Additionally, chemical treatments were applied via gravity-based injection or trunk wrapping with bio-degradable sponges to several trees that had received prior heat treatment at Picos farm. Trees are being monitored for growth and Las titer.
All of the research described in the previous report is still ongoing or is still being analyzed. 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 is being analyzed. A study of CiFT3 transgenic tobacco plants treated with various growth regulators has been performed and all of the data have been collected except for the flowering dates of the nontransgenic control plants that have not yet flowered. 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. The complete construct is completed and is being tested in tobacco for a rapid test before citrus experiments are started. This research was presented at the ASHS national meeting.
An extensive survey of HLB-affected groves indicated that greater decline in fibrous root health and greater expression of HLB symptoms is observed where irrigation water is high in bicarbonates (> 100 ppm) and/or soil pH > 6.5. Affected groves employ micro-sprinkler irrigation that concentrates fibrous roots in the wetted zone and soils often have a history of excessive dolomite liming to manage high residual copper. Affected orchards have off-color foliage, thin canopies due to excessive leaf drop, twig dieback and more severe HLB symptoms in leaves and fruit. HLB symptom expression of trees on different rootstocks is ranked Swingle citrumelo > Carrizo citrange > sour orange > Cleopatra mandarin which follows rootstock intolerance of bicarbonate. To identify the relationship between HLB decline and bicarbonate stress, fibrous roots of 8-15 yr old Valencia orange trees on Swingle or Carrizo rootstock were sampled in 37 orchards with varying soil pH and irrigation water quality. Lower root density was correlated to irrigation water pH and soil pH > 6.2. Fruit production over three seasons (2009-2012) during which HLB incidence was accelerating revealed that groves under high bicarbonate stress declined 20% in yield compared to groves under low bicarbonate stress with a 6% increase in production. Yield loss under bicarbonate stress was correlated with reduced fibrous root density compared to the non-stress condition. To confirm that treatments with acidified irrigation water reduce the impact of bicarbonate stress on root health, we are surveying 8 grove ridge locations in Highlands county and 4 flatwoods locations in Desoto 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. This survey will continue 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.
Stress intolerance of HLB infected trees is a direct consequence of greater than 30 percent loss of fibrous root density compared to non-diseased trees. HLB-induced 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. To understand the possible interaction between these two pathogens and host tolerance, two rootstocks, Cleopatra mandarin (Citrus reticulata) and sour orange (Citrus aurantium) were inoculated with Las, Phytophthora nicotianae (P.n.), both or neither. P.n. infection, root loss, and carbohydrate content of fibrous roots were assayed. P.n. infection increased on both rootstocks indicating that Las reduced their tolerance to P.n. Differences in P.n. infection on Las positive seedlings at 5 and 11 weeks after inoculation suggest the interaction changed over time. Both pathogens caused significant root loss alone, but Las in combination with P.n. did not cause additional root loss compared to Las alone. Based on these results, we hypothesize that 1) early in disease development, Las increases susceptibility to P.n. infection by increasing zoospore attraction and/or facilitating penetration; 2) Las induces root loss resulting in a temporary drop in P.n. population by reducing available food supply; 3) As new root flushes occur, P.n. starts the infection cycle again and this cycle repeats until there is a complete loss of fibrous root system. To test the value of P. n. control in slowing tree decline in the presence of both pathogens, P.n.-infested nursery trees were inoculated or mock-inoculated with HLB-infected 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.n. 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.n. root infection and increased fibrous root mass compared to untreated controls for HLB(-) trees. Neither Ridomil or Aliette significantly reduced P.n. 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.n.
Seasonal root sampling continues in two field sites for root density and root growth. Sampling has been completed for a full year for Hamlin/Swingle and 6 months for Valencia/Swingle. Differences in root growth patterns for different scions are already becoming apparent. The role of HLB and Las in modifying quantity and timing of root growth between these two scions is being analyzed. Results so far emphasize the need to use treatments that promote root longevity as the main method of managing HLB root loss. Root growth stimulation is unlikely to improve root density. As more of the healthy trees in the field trial become PCR positive for Las it is increasingly difficult to find sufficient presumed healthy trees for comparison. This is especially difficult for trees of sufficient age for seasonal root sampling. While field sampling continues, the results will become more descriptive of the decline of HLB affected trees than a comparison to healthy trees. We will soon be purchasing equipment to allow non-destructive measurements of root density and root growth, so that young plantings can be sampled allowing for direct comparison of healthy and HLB affected trees in the field. Sampling at the UF-CREC St. Helena rootstock trial site has been underway for 1.5 years to evaluate the effects of HLB on new experimental rootstocks. These data demonstrate how the new rootstock lines respond to Las infection. The findings been presetned for presentation at recent grower meetings (Citrus Expo and OJ Breaks). The most promising rootstocks have been included in greenhouse trials to understand why they are responding differently with the objective of finding and validating parameters for rapid screening for new rootstocks. The two most promising rootstock breeding lines and Swingle as a standard rootstock control have been graft inoculated in the greenhouse and transplanted to rhizotrons to monitor root growth and death. Data analysis of root growth and root longevity in response to Las is currently underway. Dying roots are becoming apparent, so sampling for comparative physiological responses will begin soon.
FireWall 50WP (65.8% streptomycin sulfate; Agrosource, Inc.) has been granted a second year of 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 of FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance. This survey for 2014 season will be conducted in November and the report submitted to EPA by the end of the year. A recently completed greenhouse trial 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 aginst canker in that the penetration of the leaf insures that streptomycin is protected from wash-off by rainfall, weathering and degradation by UV light.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time (~50%) to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of September 2, 2014, a total of 6,208 transgenic plants have passed through inoculation process. A total of 122,855 bacteriliferous psyllids have been used in no-choice inoculations.
The overall aim of this project is to develop and evaluate soft nanoparticles (SNP) to deliver natural biocides to the phloem of HLB infected trees by foliar and/or bark application. In the last quarter (July ‘ Oct ’14) the efficacies of formulations of all three EOs employed (EO-A, EO-B and Thyme oil) were evaluated through multiple methods. Subsequent to a careful selection of formulations based on efficacy against L. crescens, phytotoxicity and EPA registration of surfactants used, preparation of selected formulations have been scaled up. Currently, tests to assess efficacy of the formulations in HLB infected citruses via a bud graft technique at undergoing at Indian River Cirrus and Education Center, Ft. Pierce. Normally about six months are required for the assay to give results. To investigate the penetration of formulations into citrus leaves, dye doped microemulsion formulations have been prepared and applied by foliar application. From a number of oil soluble dyes, formulations were initially developed with fluorescein and Nile Red dyes. However, upon application to plants and successive dilution in the leaf, significant background interference was observed in the form of auto-fluorescence of chlorophyll and other leaf components (red region). Presently, new dyes have been selected between blue and green fluorescence. Formulations have now been developed with the new dyes like Bodipy 505/515, Vybrant DiO etc. which show fluorescence in 475-525 and 550-650 nm range. Leaf penetration experiments will begin in the upcoming weeks. In another approach we are aiming to developing methods for detection of EO transport to the phloem. Duncan grapefruit and Valencia orange plants have been acquired in order to do so. As an additional impact, zebra chip disease on tomato and potato caused by Ca. Liberibacter solanacearum (CLso) a close relative of CLas was identified as a rapid disease surrogate. The assays were all done at Texas A&M University. Some SNP formulations were successful in relieving zebra chip symptoms on tomato plants in a period of 30 days. QPCR tests are currently being conducted to get a quantitative evaluation.
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. Additional plantings are welcome from the research community.
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 remarkabe resistance in chimera compared to control. Canker infiltration showed greatly increased resistance in two chimera AMP and several thionin transgenics, at 107CFU/ml. RNA was isolated from transgenic plants containing chimera and thionin. RT-PCR showed gene expression in the transgenic plants. Further gene expression level was evaluated with RT-qPCR. Our results showed gene expression variation between different transgenic lines, from several fold to 35 fold. Transgenic lines containing D4E1 were evaluated with Xcc infiltration. All the transgenic lines with canker development at 105 CFU/ml while some transgenic lines show less canker development at 104 CFU/ml. Bacterial growth rate in transgenic lines containing D4E1, chimera and thionin was investigated by qPCR. Our results showed some transgenic lines containing chimera and thionin had low Xcc growth rate. More transformed Hamlin carrying chimera were generated and over 30 were confirmed positive by PCR. About 20 Hamlin transformed with thionin also were obtained. They will be tested by RT-PCR and replicated for HLB challenge. Putative transgenic plants of PP-2 hairpins (for suppression of PP-2 through RNAi to test possible reduction in vascular blockage even when CLas is present) and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. 40 putative transgenic plants transformed with citGRP1 were tested by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 had increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. 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 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.
This quarter we have continued to make progress on our transformation approaches: 1. Stable transformation in citrus using new vectors. We previously found that the original vector system used to create the Bs3 promoter constructs was contributing to low transformation efficiency in citrus, and switched to a pCAMBIA-based vector system. Two ProBs314EBE:avrGf2 transgenic plants created using the new vectors in citrus cultivar Carrizo have now been confirmed by PCR to contain the transgene, and these will be examined for appropriate gene expression by RT-PCR. An ongoing pipeline of transformants are being generated with the new vectors. To date a total of 2,056 putative transgenic shoots of grapefruit, sweet orange and Carrizo were screened for this period. Results show that no GUS positive has been observed for the sweet orange cultivar transformed with any of the constructs analyzed, however, 3 shoots were chimeric for the pCAMBIA2201:NosT:Bs3super::avrGF2 construct. In general, Grapefruit had a combined total of 12 and 41 shoots being GUS positive and chimeric for GUS, respectively for all constructs anlayzed while Carrizo citrange had 185 and 180 shoots being GUS positive and chimeric for GUS, respectively. These GUS and chimeric shoots will later be screened via PCR once rooted and transferred to soil for acclimatization. 2. Stable transformation in tomato test system: A tomato test system was previously designed and tested in which the 14 EBE promoter was fused to the avrBs4 gene capable of inducing a hypersensitive reaction in tomato. T1 generation of Bonny Best and Large Red Cherry transformed with ProBs3_14EBE:avrBs4 were screened for pathogenicity reaction with X. euvesicatoria strain (Race 9). Promising resistant transgenics have been selected for T2 generation analysis to confirm that this test system, in which resistance is induced by the effectors AvrBs3 and AvrHah1, is functional for conferring stably-transformed transgenic disease resistance.
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