The current main priority is hiring the project postdoctoral fellow in the Zipfel laboratory. Whereas an initial candidate was identified, the hiring was not completed and additional candidates are currently being interviewed. The intention is to make an offer to a qualified postdoc by mid-November. Others in the lab are progressing the initial objectives: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. We have prepared constructs for EFR, XA21, EFR-XA21 and XA21-EFR chimera. We have carried out transient expression assays in N. benthamiana and found that the proteins express well. We have shown that the XA21-EFR chimera is functional, as exemplified by a gain of responsiveness to elf18 in a ROS burst assay. We are currently testing the functionality of XA21 and XA21-EFR by treatment with the axS17 peptide. This protein is unstable and difficult to synthesize, so we have obtained wild-type and ax21-minus strains of Xanthomonas euvesicatoria so that we can make extracts to test XA21-induced responses in N. benthamiana.
In previous reports we have described the preparation of a scFv library prepared in phagemid vector pKM19. The basic scFv library contains 2 x 10_7th unique phage that bind to different antigens present in ‘Ca. Liberibacter asiaticus’ and the psyllid vector. We have also reported that we have isolated scFv from this library that bind to epitopes contained in proteins of ‘Ca. Liberibacter asiaticus’ that are likely to be related to host pathogen interactions and virulence. These epitopes are found on two flagellar proteins, the major outer membrane protein, a pilus protein, a protein believed to polymerize the capsular polysaccharide surface layer of the bacterium, the TolC protein required for survival in a plant host, and InvA, the invasiveness protein that prevents an infected cell from undergoing programmed cell death by apoptosis. During work reported previously we have found that these scFv bind to their targets in extracts of infected plants and can be easily detected in a dot blot format. However, there is often a weaker reaction with uninfected plant material. One of the powerful aspects of scFv technology is that the binding affinity of any scFv for its target can be improved by mutagenesis. During this time period we have prepared mutated scFv libraries for several of our antigen targets. We have followed the procedures described by Pavoni et al., (Gene 391 (2007) 120’129). In summary, the antigenic diversity of the scFv is encoded in defined hypervariable regions designated ‘CDR3’ in both the Vh and Vl segments. The hypervariable regions were targeted by PCR mutagenesis using degenerate primers KM144’KM143 and KM148’KM145 to introduce random mutations in CDR3 regions of both the heavy or light chains. These mutated fragments were then assembled with the remaining portions of the scFv genes by combination with the products of amplification with primers KM148’KM157 and KM158’KM143 primers for HC and LC, respectively. The result is secondary mutated libraries that contain large numbers of phage with related sequences likely to recognize antigens potentially relevant to virulence. We have created five such mutated libraries, based on scFv1289 (TolC; 1.3 x 10_6th TU); scFv734 (Omp6f; 4.2 x 10_6th TU); scFv968 (FlhA; 4.0 10_6th TU); scFv932 (FlgL; 1.4 x 10_6th TU); scFv1202 (kpsA; 1.5 10_6h TU). Future work will be performed to isolate scFv with the greatest binding specificity (low cross reaction to uninfected plants) and affinity. These scFv will then be available for labeling proteins for in vivo visualization by various microscopic techniques, for testing as diagnostic reagents in various applications, and for introduction into plants to determine if they can confer resistance to HLB.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, transgenic citrus plants expressing the Arabidopsis MKK7 (AtMKK7) gene are under canker resistance test. These plants have been propagated and will be used for citrus greening test. As an extension of the project, we tested whether exogenous NAD+ could induce resistance to citrus canker. Exogenous NAD+ has recently been found in our lab to be a strong inducer of systemic acquired resistance (SAR). Since SAR has been shown to be effective against citrus canker, we expected exogenous NAD+ would induce resistance to canker. Indeed, our preliminary result showed that exogenous NAD+ activated strong resistance to citrus canker. We are confirming this promising result. For objective 2, we are continuing the direct genetic screen for citrus varieties with increased resistance to citrus greening. Seedlings from gamma ray-irradiated Ray Ruby grapefruit seeds have been inoculated with psyllids carrying greening bacteria. Seedlings developing greening symptoms have been removed. The remaining seedlings will be re-inoculated with psyllids carrying greening bacteria.
Our objective is to determine how Asian Citrus Psyllid (ACP) behavior is affected by Las-infection in healthy and diseased citrus. In previous experiments we have determined that ACP adults initially settle on Las-infected plants. We hypothesized that while the Las-infected plants are initially attractive to ACP, after prolonged feeding the psyllid experiences imbalanced nutrition and choose to seek a better host. To examine this hypothesis we planned to determine how ACP settles on plants with known nutrient deficiencies. In previous experiments we used asymptomatic Las-infected Valencia seedlings to show ACP movement and host acceptance. However, in this quarter we chose 4-year old Valencia trees for our settling experiments with (1) late-stage symptomatic Las-infections, (2) nutrient deficiencies, or (3) healthy trees as control. To ensure that we still had the same settling behavior that we found in previous work, we began our experiment with control vs Las-infected tree choice tests. Surprisingly, the older Las-infected trees were no longer initially attractive, and nearly all of the ACP settled and remained on healthy trees. We attributed this to the poor condition of the Las-infected trees that were no longer suitable hosts. We conducted the same experiment using Las-infected pineapple sweet orange trees that were highly symptomatic yet still actively growing and in relatively good condition. In this experiment, ACP settled evenly over both Las-infected and healthy trees. Over the course of the experiment, ACP moved; however, there was no clear pattern of movement to or from healthy or Las-infected trees. We hypothesized that as the disease progresses, the attractiveness of the Las-infected tree changes. To continue this line of questioning, we have obtained sweet orange trees that are (1) healthy, (2) asymptomatic, newly Las-infected, and highly symptomatic, Las-infected trees. We will conduct three way choice tests between these trees to determine ACP settling preference over time. We are also establishing nutrient deficiencies in seedling Valencia trees for use in future settling experiments.
Data analysis of the 2011 LAS experiments is currently being completed. A publication on this data is being written concurrently and should be ready for submission by the end of the year. For this publication, we are attempting to obtain more information about the apparent biofilm that was formed at the air-liquid interface of cultures supplemented with citrus juice in 2011 experiments. From one of the experiments, biofilm was harvested from all of the flasks and DNA extracted from these samples. PCR was used to amplify the V4 region of the 16S rDNA gene from these samples as well as from the initial seed coat-based inoculum originally used to inoculate the culture flasks. Amplification was verified by gel electrophoresis, and there are now confirmed PCR products (with concentrations determined by flurospectrophotometer) from the inoculum and biofilm samples. These PCR products will soon be sequenced via next-generation sequencing to determine the bacterial composition of the biofilm. We anticipate that the biofilm could be composed of LAS, a “helper” bacterium that provides necessary nutrients to the culture, or a combination of both. Exhaustive sequencing of these samples will answer this question. New experiments for the fall 2012 season are underway. Since the concentrations of LAS in citrus fruit seed coats are the highest from ~September-November, the experiments are being conducted during this time. The experiments are of a similar design to the 2011 experiments, but with only two media types: 100% King’s B media and 100% commercial grapefruit juice. Viability will be measured over time with EMA-qPCR as before but with lower frequency, as the viability pattern has already been established in previous experiments. The focus this time will be on examining biofilm formation, nutrient utilization as measured by ICP-OES, and attachment via microfluidic chamber observations. We are also trying to determine the best method for long-term storage of LAS cells by storing the same inoculum under different conditions and subsequently reviving the cells and comparing cell viability between the different storage methods. This would hopefully allow us to determine the best way to store the cells so that experiments with LAS can be conducted during times of the year when cell concentrations are not high enough in the fruit seed coats.
Data was prepared and a poster developed for the American Society for Horticultural Sciences Annual Meeting in Miami, FL in August (Albrigo). Data included field and greenhouse comparisons of plugging types and new photomicrographs of phloem plugging and necrosis in different size scaffold limbs. All tissues in diseased plants showed similar phloem disruption symptoms and more phloem cells were laid down in HLB affected plants, young and old. Material for an oral presentation was prepared for delivery at the American Society for Horticultural Sciences Annual Meeting in Miami, FL in August concerning the transformation of citrus with the beta 1, 3-glucanase gene, their propagation and eventual testing by challenging with HLB (Ahmad Omar).
Sampling of field trees for further evaluation of phloem plugging continued. Trees of citrus relatives were evaluated for HLB and psyllid activity in field rootstock trees. No HLB was detected in Poncirus, but HLB did occur in Carrizo. Further, no Poncirus was ever found to have any psyllid stages on the leaves in two locations examined 3 times. Plants were transformed with virulent genes from the Liberibacter bacteria. Preparations were continued for transformation of citrus with then glucanase gene. Sept-Oct 2010 report
Locations and HLB affected trees were identified, confirmed by PCR and sampled for phloem plugging evaluations. Some of the needed samples were taken and fixed for EM work. Continued transforming plants for virulence factor evaluations and also plants for glucanase additions for evaluations. Different types of citrus and transformation techniques are now being used. Arrangements for testing transformed plants being made. Sept-Oct 2011 report
3rd Quarter (final funding period): This quarter was largely devoted to the development of experimental approaches to be employed in the assessment of resistance to Las infection in the transformed lines. These experiments were aimed at developing protocols that can detect and quantify the survival of Liberibacter in the early stages of infection in our transformed lines expressing various constructs of R proteins. 1-Test for possible feeding preference between transformed versus non-transformed citrus: Preliminary analyses were conducted to determine whether the introduction of inducible and constitutively expressed resistance R genes affected the feeding preferences of uninfected psyllids. The cuttings of all transgenic citrus plants were subjected to uninfected psyllids feeding. There was no observable difference in psyllid feeding behavior preference between transformants and control citrus plants, which is a condition that will facilitate the assessment of resistance in the transformants. 2-Development of a one-step DNA extraction protocol for PCR analysis of Las infection: Our strategy was to develop a facile and sensitive assay using heavily infected citrus leaves from nontransformed citrus initially, before subsequent application to transformed lines. A variety of genomic DNA extraction procedures were tested with an emphasis on limiting the quantities of plant material to the smallest possible, as well as assessing protocols that involved addition of plant material to extraction solutions followed by a brief heat treatment and then direct addition to PCR reactions. Detection of Las rDNA was reproducibly obtained using 1 mm and 0.5 mm midvein cores. Overall, extraction procedures that did not require prior genomic DNA purification (one-step approach) gave better results at lower extraction solution volumes; however, quantitative real time PCR was adversely affected to some extent by some of the extraction solutions utilized in the one-step approach. In order to precisely quantify the copy number of Las in infected citrus plants we established standard curves for Las using the plant mitochondrial cytochrome oxidase (Cox) gene as a control to measure the amount of plant material in the sample. Likewise, the wingless Wg gene served as a control in psyllid extractions. Standard curves were based on calibration curves constructed using purified PCR amplicons obtained from plant and psyllid genomic DNAs. Based on the assumption that cloned Las, Cox, and Wg amplicons may represent more accurate copy number reference, all three standards were cloned in pUC19 (Las and Wg) and pUC119 (Cox). In construction of a heat map of infection using a heavily infected leaf, Las copy number (16S rDNA) varied across midvein sections, with the secondary vein and a non-vein section of the blade showing the lowest amount of Las. The ability to detect Las 16S rDNA in 0.5 mm midvein cores suggests that fine-scale mapping of the early infection is feasible. 3-Netted single-leaf clip cages used to detect initial infection stages: Ten psyllids from an infected population (furnished by the Dawson laboratory) were placed in single-leaf clip cages and allowed to feed for a period of 7 days and then removed for PCR determination of Las infection. A total of 10 leaves were exposed to infected psyllids and were harvested at one week intervals for PCR analysis of Las copy number. Las/Wg copy number ratios varied from 2,238×10-5 to 23,575×10-5 in the psyllids. Early detection of Las from midveins was feasible; however, the sensitivity of the assay in its present form was still needs improvement. We continue to make adjustments to our testing conditions including the modification of psyllid-containment cages.
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 2,750 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. 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. 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 one week, 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. This report marks the end of the first quarter of the project, during which we have established the infrastructure for the screening program. A technician dedicated to the project is being hired, two small greenhouses for rearing psyllids are almost completed, and general supplies including insect cages have been procured. USHRL dedicated an existing conventional greenhouse for the project, erected two new hoop houses for the project, and assigned a support scientist to the screening project. Additional ARS funds were used to increase the bio-security of the existing greenhouse to guard against invasion of parasitoids of the psyllid. This screening program supports two USHRL projects funded by CRDF for transforming citrus.
Our project was extended for four months, as we applied for a no cost extension. The end date of the project will be end of August, 2012. As we are nearing the completion of our project, we are continuing with our analysis of the collected data under different objectives and preparation of manuscripts for reporting the important findings.
Initial funding for this project was finally obtained on June 20, 2012. Construction of the rapid flowering system (pvc pipe scaffolding system) in the greenhouse has been completed. Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks and growing in airpots have been entered into this RES system. The plants have been single stemmed, and some are already approaching 6 feet in height. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. The first experiment combines our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene). The two-transgene Gateway based cloning system was employed to build the 2-gene construct. The NPR1 gene is under control of the rolD promoter while the CEME gene is under control of the d35S promoter. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance. Experiments to combine the NPR-1 gene with other lytic peptide transgenes including CEMA and AttacinE are underway, also using the new Gateway technology.
Greenhouse Study: A potted hydroponics nutrient study was established with ‘Valencia’ orange in early 2010 for the purpose of developing specific plant nutrient deficiencies through exclusion of certain chemicals from the nutrient solution. The major objective of the study was to examine the response of these nutrient deficient trees to selective bud-inoculation with HLB, and compare them with uninoculated trees and fully fertilized trees (controls). The experiment was conducted in a completely randomized design (CRD) with seven nutritional treatments and ten replications. The treatments included full strength Hoagland’s nutrient solution (T1), one tenth concentration Hoagland’s nutrient solution (T2), full strength Hoagland’s solution minus Mg (T3), minus Ca (T4), minus B (T5), minus Mn (T6), and minus Zn, Cu, Mo, Fe (T7). The results of the first PCR analysis in the spring of 2011 showed a significantly higher Ct value (38) from the calcium-starved trees than from the full Hoaglands nutrition trees (33), suggesting minimal to no HLB transmission in the -Ca treatment despite their exceptional bud take during graft inoculation. In the fall PCR testing, the Ct value for the Ca-deficient trees could not be detected, again suggesting a lack of HLB transmission to those trees. In the same fall test, the other nutrient-deficient treatments tested with Ct values ranging from 20 (-Mg) to 31 (1/10 strength Hoaglands solution), all indicating highly successful transmission of HLB to the host trees by bud grafting. The somewhat strange lack of Ct detection with PCR tests in the trees with Ca deficiency, coupled with the significant 21% reduction in leaf Ca concentration on average due to the HLB inoculations, suggests that these observations could be due to a strong competition for Ca resources between the host plant and the cLas pathogen apparently causing the HLB disease. Critically low leaf Ca concentrations resulted from Ca starvation at the roots (1.648%), but coupled with HLB inoculation, they dropped even lower to 1.218%, which is a 26% reduction. A healthy citrus leaf typically has high Ca concentrations relative to all the other mineral nutrients, often in the 4-5% range. Field study: The HLB/nutrition field study was established with an existing mature block of ‘Hamlin’ oranges near Lake Alfred in early 2010 for the purpose of comparing high and low levels of foliar nutrient spray amendments and high and low pesticide application intensities. Treatments imposed in March 2010: 1 ‘ Std Pesticide, Std foliar nutrition, 2 ‘ High Pesticide, Std foliar nutrition, 3 ‘ Std Pesticide, High foliar nutrition, 4 ‘ High Pesticide, High foliar nutrition [‘Std’: Growers Fertilizer FeMnZn foliar spray 2x/yr, ‘Reduced’ pesticide program (+scouting); ‘High’ nutrient sprays: G.P. Solutions products; DKP, phosphite, salicylate, micronutrients, slow release nitrogen, Bacillus subtilis biofungicide -during growing season, aiming for flushes]. Observations: – HLB symptomatic trees were all variably impacted by the disease – most infected trees remain productive after 3 years; average yields of 336 to 484 boxes/acre not related to treatments – infected trees on low foliar nutrient program also doing well. – Few Zn deficiency symptoms visible on infected trees. Low leaf Zn, Mn measured suggests an increased dose of those nutrients needed. With a no cost extension we hope to add 2012 yields to our data set and be able to draw complete conclusions.
Objective 1 (To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface) Assays to determine the ability of canker strains to move in response to different stimuli have been started. Different strains that include types A, Aw and A* canker bacteria, citrus bacterial spot and black rot xanthomonas were exposed to organic acids, amino acids and polysaccharides. Swimming motility was evaluated in microtiter plate/tip assays by counting bacterial abundance in the tip with the stimulus as compared to the control. Evaluation of the results is in progress for by analysis of the global behaviour of the strains and by analysis of each strain for specific behavior for each of the compounds. Differences among the strains will compared with measurements of responses to different carbon compounds using Biolog to find if there are relationships between chemotaxis and metabolism characteristics of the bacterial strains. 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). As a continuation of the former project (NAS-85) studies of biofilm formation are in progress. The first investigation is focused on the study of bacterial appendages putatively involved in biofilm matrix composition. Purification of these appendages has been performed by extraction, centrifugation and isolation in acrylamide gels.The major protein has been identified as pilus type IV in Xanthomonas citri subsp. citri A strain. Initially, no variation among the other strains has been detected. The next experiments will focus on the EPS production by the strains and biofilm structures as observed by scanning electron microscopy.
The objective of this research has been to identify repellents for Asian citrus psyllid (ACP) and also to develop practical tools from these identifications that may eventually be used for ACP management. Throughout the project we have engaged in partnerships with private industry; therefore, our focus has remained on practical end-products that have been under development. The pest management company, ISCA Technologies, was one private company that made significant progress with their SPLAT dispenser, in terms of developing a practical repellent tool for ACP using our discoveries. However, other companies, such as Alpha Scents also made progress in the development of tools for ACP repellency during this project. We determined that volatiles from guava leaves signi’cantly inhibited attraction of ACP to normally attractive host-plant (citrus) volatiles. A similar level of inhibition was recorded when synthetic DMDS was co-released with volatiles from citrus leaves. In addition, the volatile mixture emanating from a combination of intact citrus and intact guava leaves induced a knock-down effect on adult ACP. Compounds similar to DMDS including dipropyl disulphide, ethyl-1-propyl disulphide, and diethyl disulphide did not affect the behavioral response of ACP to attractive citrus host plant volatiles. Head-space volatile analyses were conducted to compare sulphur volatile pro’les of citrus and guava, used in our behavioral assays, with a gas chromatography-pulsed ‘ame photometric detector. DMDS, produced by wounded guava in our olfactometer assays, was not produced by similarly wounded citrus. The airborne concentration of DMDS that induced the behavioral effect in the 4-choice olfactometer was 107 pg/ml. In a small plot ‘eld experiment, populations of ACP were signi’cantly reduced by deployment of synthetic DMDS from polyethylene vials compared with untreated control plots. Our results verified that guava leaf volatiles inhibit the response of ACP to citrus host plant volatiles and suggested that the induced compound, DMDS, may be partially responsible for this effect. Also, we showed that ‘eld deployment of DMDS reduces densities of ACP and thus may have potential as a novel control strategy. Also, we found that volatiles from crushed garlic chive leaves, garlic chive essential oil, garlic chive plants, wild onion plants and crushed wild onion leaves all repelled ACP adults when compared with clean air, with the ‘rst two being signi’cantly more repellent than the others. However, when tested with citrus volatiles, only crushed garlic chive leaves and garlic chive essential oil were repellent, and crushed wild onions leaves were not. Analysis of the headspace components of crushed garlic chive leaves and garlic chive essential oil by gas chromatography-mass spectrometry revealed that monosul’des, disul’des and trisul’des were the primary sulfur volatiles present. In general, trisul’des (dimethyl trisul’de) inhibited the response of ACP to citrus volatiles more than disul’des (dimethyl disul’de, allyl methyl disul’de, allyl disul’de). Monosul’des did not affect the behavior of ACP adults. A blend of dimethyl trisul’de and dimethyl disul’de in 1 : 1 ratio showed an additive effect on inhibition of ACP response to citrus volatiles. The plant volatiles from Allium spp. did not affect the behaviour of the D. citri ecto-parasitoid Tamarixia radiata. Thus, Allium spp. or the tri- and di-sulphides could be integrated into management programmes for ACP without affecting natural enemies. In addition, we investigated volatiles from essential oils of coriander, lavender, rose, thyme, tea tree oil and 2-undecanone, a major constituent of rue oil repelled ACP adults compared with clean air. Also, coriander, lavender, rose and thyme oil inhibited the response of ACP when co-presented with citrus leaves. Volatiles from eugenol, eucalyptol, carvacrol, b-caryophyllene, a-pinene, a-gurjunene and linalool did not repel ACP adults compared with clean air. Practical tools from this under development.
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