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.
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). In the early spring after the second winter of 2011/12, we assessed and analyzed all trees for leaf greenness (SPAD chlorophyll index meter), leaf tissue nutrient concentration, and growth performance by measuring the stem diameter (caliper). The caliper results were significantly different across nutrient deficiency treatments but not for HLB inoculations or for HLB x deficiency interactions. Nevertheless it is noteworthy that the -Mg treatment caused significantly lower stem growth (11%) in the presence of HLB infection. Not surprisingly, the biggest reduction in growth occurred in the 1/10 Hoaglands treatment with HLB inoculation. The leaf SPAD measurement was significantly lower than the full Hoaglands control for all nutrient deficiencies except the low B treatment. As expected, all nutrient deficiency treatments produced significantly lower nutrient concentrations in the leaves for the respective elements. In some cases there were also overlapping induced deficiencies in the leaf tissues caused by nutrient imbalances from another element. HLB inoculation significantly reduced leaf concentrations of Ca, Mg, Zn, and B (severe), and K, S, and Fe (moderate). Unfortunately there were no significant deficiency x HLB interactions evident in this analysis. The question remains whether elevated nutrient levels created by selective nutrient additions would have created a significant nutrient x HLB interaction. Such an experiment would be a worthwhile follow-up study to undertake. 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. Details of the field experiment are the following: 5 ac ‘Hamlin’ on Carrizo and Swingle, 10×25 feet (174 trees/acre), Planted 1997, 15% cumulative HLB symptomatic trees in 2009-2011, 10.7 gph microsprinklers automated daily irrigation, 180-200 lb N/ac/yr, granular N-P-K + Ca(NO3)2 + B + Mg + Fe, 4x equal fertilizer splits per year 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. During the first season the treatments were applied as scheduled and allowed to equilibrate, as is recommended for any experiments with large mature trees. In the second season (2011), leaf samples were collected during the late summer season and analyzed for nutrient content. Only Zn and Mn concentrations in the leaf tissue were significantly increased by the intensive foliar nutrient sprays (21 and 20 mg/kg versus 18 and 17 mg/kg, respectively for the controls), but nevertheless these concentrations were all in the low to deficient range. Iron concentrations in the leaf tissue were lower in the intensively sprayed treatment (76 mg/kg) compared with the controls (102 mg/kg). In 2011 unproductive ‘Hamlin’ trees were also clipped and removed from the block as part of the routine grove maintenance. The primary decline symptoms visible in the canopies of removed trees appeared to be due to blight. There were no significant effects of the experiment treatments 1-4 on the incidence of removed blighted trees. However we established that 31 removed trees (4.6%) were on Carrizo rootstock, and only 9 trees (1.4%) were on Swingle rootstock. Total = 6%. 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. Questions: – Is our comprehensive ground fertilizer program masking the effects of foliar nutrition? Soil factors?
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 main 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). It took about 9-12 months for visible nutrient deficiency symptoms to develop on the trees, and results were confirmed with leaf tissue analyses and published norms. Magnesium deficiency developed the fastest, showing classic chlorotic symptoms on the leaves. Other nutrients like calcium developed deficient levels much more slowly but nevertheless their effects on citrus health were still significant. By the first winter (2010/11) after inoculation with HLB on half of the trees and after most of the nutrient deficiencies had started to develop, the greenhouse became infested with greasy spot, a common fungal disease of citrus. Apparently the high humidity which was maintained in the greenhouse during the winter months was particularly conducive to greasy spot spore germination and natural leaf infection. We immediately noticed a differential incidence and severity of greasy spot among the different nutrient treatments and decided to explore the relationships. After treating the entire experiment with a curative spray of strobilurin fungicide, we scored all the trees for incidence and severity of greasy spot and analyzed the results statistically with respect to nutrient treatments and controls.. Greasy spot incidence was up to 90% lower in the full strength balanced nutrient treatment (T1) as compared to the nutrient deficient treatments. Electrolyte leakage, which is an easily measured lab parameter and an indicator of compromised cell membrane integrity, was higher in T2 and T4 as compared to the full strength nutrient treatment. Leaf sap pH was lower in different nutrient deficient treatments (pH 6.0-6.2) compared to the balanced nutrition (pH 6.3). Leaf nutrient concentrations, principal component analysis (PCA) and canonical discriminant function analysis (DFA) revealed that omitting any one of the nutrients can increase the susceptibility to greasy spot. The results confirm Liebig’s law of minimum, and suggest that a complete balanced nutrition supply for citrus may significantly reduce the occurrence of greasy spot, reduce pesticide spray requirements, promote overall tree health. Although these results were not repeated in the field yet, and the greasy spot arrived by accident, we learned new and valuable information about the modification of plant host resistance with mineral nutrition, especially the importance of calcium. We hope to see similar results with the HLB-inoculated trees over time.
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). It took about 9-12 months for visible nutrient deficiency symptoms to develop on the trees, and results were confirmed with leaf tissue analyses. Two buds from HLB+ trees were grafted onto the scions of half the trees in the experiment, so that the interaction of HLB with or without each nutrient deficiency could be measured. In Fall of 2011 the bud take of each graft-inoculated tree was scored on the following scale, and a sample of the main canopy was tested by PCR: 1 Brown or dead bud 2 Green bud 3 Bud sprouted 4 Less than 5 cm 5 More than 5 cm branch The analyzed results showed that, surprisingly, the full strength Hoaglands nutrition (no missing nutrients), and the 1/10 strength Hoaglands nutrition treatments had significantly lower bud scores (1.7 and 1.4, respectively) than the treatments with single missing nutrients. Also surprising was the discovery that the calcium deficient treatment had the highest bud performance score (4.0), and was significantly higher than all except the -Mn treatment. Since calcium plays an essential role in cell wall development and function, as well as callose formation, it is possible that the lack of calcium actually benefitted the fusion of vascular systems between the grafted bud and the host scion. 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 Core Citrus Transformation Facility (CCTF) continued to produce transgenic plants at expected rate of about a 100 per quarter. Plants for the following orders were produced: eight Duncan plants (ELP3 gene); ten Duncan plants (ELP4 gene); 15 Duncan plants (p7 gene); three Duncan plants (p10 gene); three Duncan plants (pWG19-5 vector); three Duncan plants (pWG20-7 vector); five Duncan plants (pWG21-1 vector); 15 Duncan plants (pWG22-1 vector); five Duncan plants (pWG24-13 vector); 12 Duncan plants (pWG25-13 vector); five Duncan plants (pWG27-3 vector); four Hamlin plants (pLC220 vector); eight Duncan plants (p35 gene), 11 Duncan plants (35S-TRX vector), and two Duncan plants (SUC-TRX vector). CCTF received three new orders in this period. The decision was made to replace the soil to which plants get transferred to following successful grafting. Use of soil that would get contaminated occasionally resulted in a loss of plants in previous periods. A special, commercially available, foam product is being used instead of commercial potting soil. The problem of loss of plants due to soil contamination has now been remedied.
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