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.
The antibody developer, Creative Biolabs, Inc., has nearly completed screening for antibodies against the Candidatus Liberibacter asiaticus NodT protein. They have identified six high-affinity binding antibodies to the 30 amino acid peptide antigen used. The materials will be shipped to Dr. McNellis’ lab at Penn State University within the next few weeks. We anticipate beginning to screen the antibodies for their ability to detect the native NodT protein produced by Candidatus Liberibacter asiaticus starting in mid-August, working through the fall.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in Col-0 background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers. We have been in the process of testing disease resistance of these plants to P. syringae. If a citrus SA gene confers broad disease resistance, we expect to see enhanced resistance to P. syringae when the citrus gene is overexpressed in Col-0 and/or to see the complementation of the phenotypes exhibited by its corresponding mutant. For ctNDR1, we have so far obtained 29 independently transformed US942 transgenic citrus plants carrying ctNDR1 overexpressing construct (US942::d35S::CtNDR1). The presence of the transgene in the plants was confirmed by transgene-specific primers. We performed disease resistance assays with Xanthomonas citri subsp (Xac), using excised leaf discs from 24 US942::d35S::CtNDR1 plants. Twelve leaf discs (that was all we could sacrifice for now) from each independently transformed line were used in the infection. Leaf disks, each from a single leaf, were tested in three infection groups and each infection was conducted independently on a separate day. Our preliminary results indicate that US-942::d35S::ctNDR1 transgenic clones had significantly reduced growth of Xac, as compared to untransformed controls. Therefore, these results suggest that overexpression of ctNDR confers enhanced resistance to citrus canker disease.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in Col-0 background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers. We have been in the process of testing disease resistance of these plants to P. syringae. If a citrus SA gene confers broad disease resistance, we expect to see enhanced resistance to P. syringae when the citrus gene is overexpressed in Col-0 and/or to see the complementation of the phenotypes exhibited by its corresponding mutant. For ctNDR1, we have so far obtained 29 independently transformed US942 transgenic citrus plants carrying ctNDR1 overexpressing construct (US942::d35S::CtNDR1). The presence of the transgene in the plants was confirmed by transgene-specific primers. We performed disease resistance assays with Xanthomonas citri subsp (Xac), using excised leaf discs from 24 US942::d35S::CtNDR1 plants. Twelve leaf discs (that was all we could sacrifice for now) from each independently transformed line were used in the infection. Leaf disks, each from a single leaf, were tested in three infection groups and each infection was conducted independently on a separate day. Our preliminary results indicate that US-942::d35S::ctNDR1 transgenic clones had significantly reduced growth of Xac, as compared to untransformed controls. Therefore, these results suggest that overexpression of ctNDR confers enhanced resistance to citrus canker disease.
Objective 1. Develop a simple in-field system to raise citrus canopy temperature. This objective is driven by the need of such system to achieve objective 2 and test our central hypothesis. A moving greenhouse was developed to cover single trees during the summer of 2012. Four trees (~ 2.5.2.5.2.5 m) were treated, one tree per day, during the months of September (trees T1 through T3) and October (tree T4). From each tree, three symptomatic branches were sampled to determine microbial kill before (0 h) and 2, 3, 4, and 5 h during the treatment. Temperature distribution throughout the canopy and on the sampled branches was also recorded. Maximal temperatures in the ranges 50 to 53 ‘C were reached at the top (2.4 m) of the canopy whereas at the bottom of the canopy (i.e., 0.6 m) maximal temperatures ranged from 36 to 43 ‘C. Due to varied micro-meteorological conditions during the treatment, temperatures of the T1 through T4 sampled branches reached above 40.C for 217, 166, 35, 228 min, respectively. For T1, T2 and T4 trees, average temperatures of the sampled branches reached above 45 ‘C for 87, 35, and 49 min or more. Attempt to quantitatively determine microbial kill by determining percent live bacteria at selected time intervals during thermal treatment was unreliable due to the very uneven distribution of initial proportion of live-to-dead bacteria and analysis variability. However, overall, after thermal treatments, live microbial populations decreased. These findings indicate that practical application of heat will require supplementing solar heat with electrical heating elements and fans to produce adequate, fast, uniform thermal treatment of trees.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to (repel) ACP, 4. Provide economic analysis of costs and projected benefits and 5. Extend results to clientele. Paperwork was not completed and funds made available by UF-IFAS until 13 March 2012. This quarter the experiment was established on a 10-acre block on the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The land is part of the old outside nursery that is now being converted to production grove. Already provided with 4 inch drain tile, it was releveled flat, bedded with 18 inches elevation between swale bottom and bed top with the swales having a 0.1% grade south to north. Irrigation lines were installed in April with a 20HP electric pump and two 48′ sand media filters which will provide water originating from the nearby Townsend canal. Each tree is provided with 2 Toro Turbo-Plus II Drip Emitter 2 Lph with pressure compensation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each plot is split into two subplots, mulch and no mulch. Mulch is metalized (aluminized/reflective) polyethylene film shown in preliminary evaluations to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. Planting density is 23 ft between rows and 9 feet within the row giving 210 trees per acre. There are 16 main plots each 5 rows wide and 26 trees long, each divided into two subplots of 5 rows x 13 trees. All plots were treated with Allion herbicide at a rate of 5 fl oz/ac. The metalized mulch specially prepared by the ImaFlex company was 6 ft wide, 2.5 or 3 mil thick protected on top. with a clear coat layer. Mulch was laid 11-12 June using a Kenco plastic mulch layer with 4.5 exposed above the soil and holes punched with sharpened post hole diggers. Trees, ‘Hamlin’ orange on ‘Carrizo’ citrange rootstock, were planted 2-3 July and Scott’s Suncote 15-8-11 12-14 month complete control release fertilizer (1 lb) added to each tree hole. Imidacloprid (Nuprid 2F) at a rate of 22.4 oz/ac was applied in designated plots on 9 July 2012 by spraying 8 oz of solution onto bare soil within 6 inches of all sides of the trunk of the tree using an EZ-Dose’ sprayer with a pressure of 45 PSI and a flow rate of 3.7 gpm.
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