Currently, objective 1, investigating the specificity and efficacy of immune priming response in ACP, is underway. The objective of this experiment is to identify if ACP will survive a lethal dose of pathogenic bacteria, Serratia marcescens, if they have had prior immune challenge by a sublethal dose of the same or other species of bacteria. We are currently investigating the effect of: a) pathogen and b) non-pathogen to determine whether immune priming regulates the response of ACP to subsequent infections. Fifty experimental 3 day old ACP adults were collected and starved for 3 h. Half of the insects were orally primed by feeing on an artificial diet solution containing a sublethal dose priming treatment, corresponding to a lethal dose response of 1% (LD1) of the insect pathogen S. marcescens, while they other half were fed a control artificial diet solution containing bacterial media only (Tidbury et al. 2011) (Table I, Objective 1a; Fig 2A). The diet solution consist edof 15% (w:v) sucrose and 1/10 (v:v) green food coloring (McCormick & Co.) sandwiched between two parafilm layers, as described in Hall et al. (2010). After 24 hours, each treatment group was enclosed on separate branches of a potted citrus plant (var. ‘Swingle’). Seven days after priming, insects were removed from plants, starved for 3 h, and either injected (experiment 1) or orally inoculated (experiment 2) on a diet solution containing a lethal dose pathogen challenge or control treatment. After feeding or injection, the ACP were returned to the ‘Swingle’ plants for 14 days or 100% mortality. Mortality throughout the experiment was recorded every 24 hours. After 14 d, ACP were examined for the presence of S. marcescens by plating crushed insects on Petri dishes containing LB nutrient agar. Samples of colonies formed after 3 d were collected for DNA extraction and confirmation of bacterial identity using conventional PCR. Each experiment was replicated six times. We are currently analyzing the results of these experiments. Subsequent experiments to address the effects of non-pathogen immune priming to pathogenic bacteria (objective 1b) are still underway. Additionally, we have initiated the second project objective, to determine the specificity of RNAi immune priming in D. citri. We have begun construct dsRNA using a GFP sequence. GFP is not naturally present in ACP; therefore, introduction of the dsRNA for this target should activate the RNAi response without inducting psyllid mortality. Additional targets will be constructed during the current project quarter.
The objective of this project is first to identify a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian citrus psyllid (ACP). The toxicity of the selected toxin will then be enhanced by addition of a peptide that binds to the gut of ACP. This peptide addition to the toxin is expected to enhance both binding and toxicity against ACP. Seven Bt strains showed toxicity against ACP with significant ACP mortality at 500ug/ml relative to control treatments. One Bt strain was selected for further identification of individual toxins by LC-MS/MS analysis. Based on the results, three specific Cry toxins were identified. The identity of these toxins was confirmed by comparison with the Bt Cry toxin holotype list, using ClustalW2-nucleotide. Further ACP bioassays with two of the toxins from the selected strain indicated that both toxins show toxicity against ACP at 500ug/ml. One of these toxins was selected for modification with the gut binding peptide. A phage disulfide-constrained heptapeptide library was screened and four ACP gut binding peptides were isolated. These peptides were expressed as peptide-mCherry fusion proteins as described in the previous report. Pull down assays were used to assess the binding of peptide-mCherry fusion proteins to ACP brush border membrane vesicles (BBMV) to confirm binding and to assess the relative strength of binding of the different peptides. mCherry alone and a random sequence peptide-mCherry fusion protein were used as negative controls for these assays. Specific binding of peptides to BBMV proteins was supported by the results of binding competition assays with mCherry or with a competitive synthetic peptide. Two dimensional gel electrophoresis coupled with ligand blot analysis showed that four peptide-mCherry fusion proteins bind to 50kDa, 37kDa and 25kDa BBMV proteins all with the same pI (~9). The negative control, mCherry showed non specific binding to an abundant 50kDa protein with a pI ~5. Analysis of peptide binding to the psyllid gut in vivo using fluorescence microscopy indicated that peptide 15-mCherry and peptide 18-mCherry bind the ACP gut, with minimal background fluorescence from the mCherry negative control.
Two citrus groves, one – 20 year-old Hamlin sweet orange trees predominately on Swingle rootstock and a second consisting of three year old Hamlin sweet orange trees on Swingle rootstock have received acid injection to selected blocks with and without sulfur applications for fifteen months. Irrigation water was acidified at one of four target water pH (7.5, 6.0, 5.0, and 4.0). A controlled release form of elemental sulfur was applied to half of the trees in each pH treatment (main effect) including the non-acidified control (pH~7.5). A controlled released form of elemental sulfur (Tiger 90) was allied at a rate of 500 pounds per treated acre to plots receiving either acidified irrigation water or control plots receiving irrigation water that was not acidified in June. Soil samples collected in August indicate that soil pH in the plots recieving sulfur applications remained near target pH level at both sites. However, plots not receiving sulfur applications increased in soil pH levels by an average of 0.75 pH units with plots to have pH between 6.0 and 7.0 returning near the pH of the irrigation water . This result is similar to data collected in 2014. The cause of soil pH is the low amount of irrigation required during the summer months. At both the mature and young tree site, no significant difference in root density was found in samples collected in August 2015. Significant increases in nutrient concentrations of leaves collected in June 2015 were been found in plots at both sites with reduced water and/or soil pH. The greatest increase in leaf nutrient concentrations were found for Mg, Mn, Zn, and B. These results may indicate increased nutrient uptake from soils with soil solutions below 6.5. Nutrient deficient symptoms consistent with HLB positive trees have lower nutrient concentration in their woody tissues and thus can not provide nutrients to leaves until these reserves are replenished by higher nutrient availability presumed at lower soil pH levels (5.5 to 6.5). Average Hamlin and Valencia tree water uptake under greenhouse conditions were not significantly different from one another. However, water uptake by trees affected with HLB were 20%-25% lower than healthy trees. These data have been consistent for the past year. There is increasing evidence of reduced water uptake for trees receiving water supplemented with calcium bicarbonate. The cause of reduced water uptake appears to be lower but non-significant reductions in root density and soil pH increases in soil irrigated with higher concentrations of calcium carbonate. Reduced water uptake by trees receiving calcium carbonate in irrigation water would account for reduced leaf area and trunk diameter.
Two citrus groves, one – 20 year-old Hamlin sweet orange trees predominately on Swingle rootstock and a second consisting of three year old Hamlin sweet orange trees on Swingle rootstock have received acid injection to selected blocks with and without sulfur applications for fifteen months. Irrigation water was acidified at one of four target water pH (7.5, 6.0, 5.0, and 4.0). A controlled released form of elemental sulfur (Tiger 90) was allied at a rate of 500 pounds per treated acre to plots receiving either acidified irrigation water or control plots receiving irrigation water that was not acidified in December. Significant increases in nutrient concentrations of leaves collected in November 2015 were been found in plots at both sites with reduced water and/or soil pH despite increases in soil pH during the summer months (reported in September 2015 report). The greatest increase in leaf nutrient concentrations were found for Mn, and Zn. These results may indicate increased nutrient uptake from soils with soil solutions below 6.5 and is presumed to be because of lower soil pH levels (5.5 to 6.5). Average water uptake by trees affected with HLB continue to be 20%-25% lower than healthy trees. Water uptake for trees receiving water supplemented with calcium bicarbonate was significantly reduced compared with health trees. Despite higher soil pH, tree root densities were not significantly different for HLB affected trees irrigated with water supplemented with calcium carbonate when compared with healthy trees irrigated without supplemental calcium carbonate. Tree size remain similar for all treatments.
The objective of this research project is to investigate and develop a potential non-phytotoxic, environmentally-friendly film-forming ACP repellent solution for preventing HLB infection. In the last reporting period, OS-SG 11, 12 and 13 were studied for plant safety and rainfastness and these formulations were delivered to our collaborators for ACP infection trials. In this reporting period, a new series of formulations (OS-SG 14-A,B,C,D) were developed using a new EPA approved polymer and environmentally friendly silica source. Optimization process involved different ratios of polymer to silica. The objective was to have the best combination for formulation stability and rainfastness. A clay based commercial control (Surround WP) was used for comparison purposes and this product is available to growers. Safety analysis of OS-SG 14 series was conducted using sour orange citrus as a model plant. Phytotoxicity studies were conducted using a Panasonic Environmental Test Chamber (Model MLR- 352H) to control light intensity, humidity and temperature cycling to simulate summer conditions (85% RH, 34oC). OS-SG 14 series did not cause any plant tissue damage at the applied rates, neither the Surround WP control. Temperature testing was conducted to determine if the presence of polymer posed a risk of increasing the heat build up on the plant surface. The OS-SG 14 series did not exhibit significant increases in temperature over commercial control. The composition and interactions between the components were confirmed using Fourier transform infrared spectroscopy (FT-IR), which confirmed the presence of silica from Si-O stretching, SiO-H stretching, Si-H bending and the polymer was detected from C=O stretching. The morphology of the new silica core used in OS-SG 14 series was observed using Scanning Electron Microscopy (SEM) and revealed large micron and sub-micron size irregular shaped particulates. The crystallinity of the silica core was studied using X-ray diffraction and it was found to be mostly amorphous with some crystalline structures. The commercial control material was amorphous in nature. The most optimized version from the OS-SG 14 series will undergo further studies including additional SEM, XRD and NMR characterizations. Film adherence (rainfastness) will be studied using AAS and atomic force microscopy (AFM). One or two best performing formulations will be selected from the from the OS-SG 14 series for future trials.
This project was initiated in 2014 and is focused on understanding the effect of nutrients applied through foliar fertilization programs (FFP) on HLB-affected trees in the Indian River marketing district. Two research trials have been established in commercial mature grapefruit groves in St. Lucie County and a young tree trial is being conducted at the UF-IRREC grove. Grove 1 has ~25 years old of Flame grapefruit on Swingle rootstock. Grove 2 utilizes ~7-year-old Ruby Red on Sour orange trees. Trial 3 is looking at the effect(s) that foliar fertilizers have on young tree growth and their ability to protract HLB disease symptoms in 2-year-old Ray Ruby grapefruit on Kuharske rootstock. Combinations of macro and micronutrient treatments initiated on all three trials in February 2014 and applications have been made quarterly since. The populations of the psyllids have been stable in both groves where the experiments are being conducted. The traps located in the different treatments are no showing significant differences among them, with an average between 0.03 and 0.06 individuals per trap per month. These values are low in comparison with the initial evaluations where the averages were between 1-3 individuals per trap. Fruit drop counts taken at about 3-week intervals beginning in July in all treatments for both groves showed no significant differences with the control treatment in terms of total amount of fruit drop or average fruit drop per tree. These results follow the same pattern observed during the evaluation in the 2014/15 season. The average of fruit drop was 53.2 and 26.2 fruit per tree Grove 1 and Grove 2, respectively. The grapefruit in Grove 2 was commercially harvested in mid-December. Four trees within each plot were individually harvested and all the fruit from each tree was run through the mobile Autoline sorting machine to determine fruit count and size distribution. Treatments 2B (application of micros, phosphite and DKP) and 4A (application of phosphite and KNO3) had significantly more larger fruit (27, 23 and greater than 23) in comparison with 1A (Control) and treatment 1-Ca ( control with soil application of CaNO3). In terms of Gross Packet Value (GPV), the treatment 2B showed a higher value per tree ($127.15) relative to the control treatment ($76.68). In general the GPV was superior for all treatments in 2015 than in 2014. The amount of boxes per tree was not statistically different among treatments, however the highest value was found for treatment 2B (average of 4.62 boxes per tree) and the lowest for the control treatment (average of 2.98 boxes per tree).
This project was initiated in 2014 and is focused on understanding the effect of nutrients applied through foliar fertilization programs (FFP) on HLB-affected trees in the Indian River marketing district. Two research trials have been established in commercial mature grapefruit groves in St. Lucie County and a young tree trial is being conducted at the UF-IRREC grove. Grove 1 has ~25 years old of Flame grapefruit on Swingle rootstock. Grove 2 utilizes ~7-year-old Ruby Red on Sour orange trees. Trial 3 is looking at the effect(s) that foliar fertilizers have on young tree growth and their ability to protract HLB disease symptoms in 2-year-old Ray Ruby grapefruit on Kuharske rootstock. Combinations of macro and micronutrient treatments initiated on all three trials in February 2014 and applications have been made quarterly since. Foliar nutrient applications were made according to schedule during the quarter. Materials are sprayed with a hand gun to all trees within plots, averaging about 2.3 gallons of solution per tree. Sticky traps are changed out every month and the number of psyllids counted. No differences between treatments have been observed. Fruit drop counts began in each grove in mid-July. During each count, the number of fruit on the ground under each measurement tree in each plot were counted. After counting, fruit were raked into the water furrow or bed middles to ensure an accurate count on the next measurement date. During the four measurement dates in the quarter, fruit drop in Grove 2 was minimal, ranging from 0 to 18 fruit per tree, averaging 4.1 fruit per tree. There were no differences among treatments. In Grove 1, drop was also minimal with an average of 3.0 fruit per tree. Drop ranged from 0 to 20 fruit per tree. No differences were found with respect to treatment. Leaf samples were taken in each grove for nutrient analysis in mid-August. In Grove 1, trees in the control plots and those not receiving phosphite had lower K than other treatments. Treatments not receiving the minor element sprays had lower leaf Mg, FE, Cu, Mn, Zn, and B than trees in treatments receiving the applications. In Grove 2, most leaf N and K levels overall were in the optimum range compared to mostly below optimum in Grove 1. With the exception of Zn and Mn, the minor element concentrations were similar among treatments. Leaf Mn and Zn concentrations were lower in control trees and the treatments not receiving the minor element sprays. Soil samples were taken in all plots in July. In grove 2, pH ranged from 7.2 to 7.6, averaging 7.4. Soil P ranged from 166 to 244 lb/ac, averaging 208 lb/ac. In Grove 1, soil pH ranged from 7.4 to 7.9, averaging 7.6. Soil P ranged from 148 lb/ac to 209 lb/ac, averaging 172 lb/ac. No differences in any soil parameter were evident with respect to treatment in either of the groves.
The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). Our previous study indicate that Las contains a functional SA hydroxylase that degrades SA and its derivatives. SA and its derivatives play important roles in plant defenses. Las employs SA hydroxylase to suppress plant defenses. Our central hypothesis is that we can improve HLB management by counteracting SA hydroxylase. We will focus on counteracting SA hydroxylase using inhibitors based on structure based design. This project contains two objectives: 1) Control HLB by optimization of application of SA and its analogs. We are testing the control effect of SA and its analogs, e.g., ASM, Imidacloprid, DL-2-aminobutyric, 2,6-dichloro-isonicotinic acid, and 2,1,3 Benzothiadiazole via trunk injection in field trial. Oxytetracycline is used as a positive control, whereas water was used as a negative control. SA, Acibenzolar-S-methyl (ASM), benzo (1,2,3) thiadiazole-7-cabothionic acid S-methyl ester (BTH), and 2,6-dichloroisonicotinic acid (INA) have also been applied once onto selected trees by foliar spray in November, 2015 during fall flush. 2) Control HLB using a combination of SA, SA analogs or SA hydroxylase inhibitors. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design are being tested for their inhibitory effect against SA hydroxyalse.
The goal of the proposed study is to characterize the effect of using endophytic microbes in controlling HLB. Our hypothesis is the outcome of the interaction among Las, psyllid and citrus is affected by the citrus phytobiome. In order to achieve the goal of this study, the following objectives will be conducted: Objective 1. To characterize the phytobiomes and endophytic microbes from HLB survivor trees and HLB diseased trees. Three healthy and three HLB infected trees were selected for phytobiome analysis from Gapway grove based on the Las QPCR detection results. The microorganisms collected from this experiment were classified as three types: rhizosphere, rhizoplane and endosphere communities. Rhizosphere, rhizoplane and endosphere DNA and RNA preparation was done using powersoil RNA extraction kit. The DNA and RNA samples were sequenced. Around 10 Gb clean reads data was generated per metagenome sample. Analysis of the metagenomic data is ongoing. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. We have begun to test different grafting methods of roots. We are also in the process of isolating endophytic microbes associated with survivor trees.
Our hypothesis is that application of antibacterial-producing bacteria directly to citrus root could suppress Las population in the roots and control Las. Application of antibacterials in this manner will avoid the strict restrictions of application of antibiotics on crops and ease public concerns since those bacteria are naturally present in the soil and are associated with plant roots. In order to achieve the goal, the following objectives will be conducted: Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria which are closely related to Las. We will mainly test the antagonistic effect of Bacillus, Paenibacillus, Streptomyces and Pseudomonas strains Agrobacterium tumefaciens, Sinorhizobium meliloti, and L. cresens; Control HLB using antibacterial-producing bacteria. For the field test, we will investigate how antibacterial-producing bacteria affect HLB disease severity, Las titres, and citrus yield, survival of the antibacterial-producing bacteria in the rhizosphere and expression of the antibacterial biosynthesis genes in vivo. We have isolated Streptomyces spp. Bacillus spp. Paenibacillus spp., and Pseudomonas spp. from Florida groves. Multiple isolates showed antimicrobial production activity. We tested 27 antibacterial compound producing bacteria. These strains had been recovered, purified and confirmed by 16S rDNA sequencing. The antagonistic activity against Agrobacterium, Sionrhizobium meliloti and Xanthomonas citri pv. citri was determined. 5 strains, belonging to Paenibacillus, Burkholderia, Paenibacillus, Streptomyces and Streptomyces showed good antagonistic activity. Three bacteria showing high antimicrobial activities have been sequenced to help us understand the mechanism. Currently, the genome sequencing was finished and we are analyzing the results. Four bacterial strains: two Burkholderia, one Pseudomonas geniculata, one Rhodococcus strains have been tested for their activity in and all showed induced plant defenses and against infection by Xanthomonas citri. To further study the antimicrobial producing bacteria, tow Burkholderia strains have been labeled with GFP tag. Seven other strains are being labeled with GFP or RFP tag. We also investigated the antibiotic genes in nine antimicrobial producing bacteria that we isolated previously. These strains were inoculated to citrus roots and the colonization was determined by inoculation and recover method in lab condition using small citrus seedlings. Around 10E8 cfu were inoculated to each seedling. Approximately 10E4 cfu were recovered from roots 20 days after inoculation (dpi). In a separate experiment, two Burkholderia strains were tested and up to 10E5 cfu/g soil was recovered at five days post inoculation. For the field trial, we have selected the grove and conduct survey on HLB disease severity. We compared different delivery methods to improve the efficacy of beneficial bacteria. A root drench delivery method has been established. Pseudomonas geniculata strain 95 was applied to trees in Block 9, CREC on Oct 22, 2015. And the bacteria are applied every two months. Three concentrations: 10E6, 10E7, and 10E8 CFU/ml were used and 1 gallon bacterial culture was applied per tree using soil drench method. We are investigating HLB disease index, bacterial survival rate and Las titers.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgeniclines showed delayed canker symptom development. Further detailed analysis of transgenic plants is being carried out.
This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (Oct 2015 to Dec 2015), we continue to evaluate 1) the effect of Pen and SD on control of HLB disease by gravity bag infusion in the field; 2) the efficiency of effective chemical compounds (Pen, SDX, Pcy and Carv) against HLB disease by gravity bag infusion; 3) the effectiveness of a combination of chemotherapy, thermotherapy and nutrient therapy against HLB in the field trials. The chemical compounds (Pen and EBI-602) and additional nutrients were applied to the heat-treated citrus for two times by foliage spray, using our optimized nano-delivery system. The variance analysis results indicated that there were significant differences in the chemical treatments, the heat treatments and their interactions. Pen was the more effective to control Las bacterium than EBI-602. In this quarter, ten chemicals were prepared in two different concentrations of the nano formulations (0.1% and 1.0%) in the greenhouse test. The preliminary results showed that the concentrations of nano formulations had no different effects on the chemicals for control of Las bacterium. PEN and ZS were the most effective to eliminate the Las bacterium. The effects of the integrated approach on the citrus fruits will be reported in next quarter.
The use of antimicrobials is one of the few effective treatments against HLB in citrus trees. However, penetration of substances into trees is hindered by the presence of protective layers such as the thick cuticle on leaves, and cork on stems. To overcome the obstacles imposed by the cuticle to increase penetration of externally supplied substances, we have successfully tested laser light. Laser light technology involves the use of low level light energy to disperse the cuticle creating microscopic and superficial indentations of approximately 250 m. In doing so, infiltration of substances into the leaf is greatly enhanced. Once inside the leaf tissue, substances can follow the natural transport pathway through the apoplast, absorbed by phloem cells, and transported throughout the tree. Specific goals are 1. To build and test a more flexible and elaborate laser machine that will allow for more decisive experiments in the greenhouse; 2. Test for the effectiveness of several antimicrobials; 3. Carry out initial field experiments with young trees. The laser machine was delivered and tested under laboratory conditions. A series of initial experiments were conducted in efforts to fine tune the machine in terms of energy, distance, speed of laser and striking angle. Additional experiments have been conducted comparing the effectiveness of laser in enhancing penetration of oxytetracycline compared to untreated leaves. We have also tested the effect of waxing the lasered area to prevent dessication. In some key experiments we have tested the changes in titer numbers of samples treated with antimicrobial in the presence and absence of laser. At the moment, the data collected indicate the superior penetration and effect of antimicrobials when leaves are subjected to a laser treatment. Additional experiments to quantify the penetration improvement and movement of oxytetracycline down the phloem are being conducted.
This is a 3 year study to provide a side-by-side comparison of 5 soil-applied commercially available products as well as an organic mulch as recommended by growers. The impact of treatments on tree health, foliar nutrition, disease rating, HLB status, root density, yield and fruit quality will be evaluated. Trials will be located in 3 Florida sites: Central Ridge, East Coast and Southwest and will incorporate well-managed trees with HLB but that are as healthy as possible. Each trial will be large enough to be statistically significant with treatments within label guidelines to avoid need for crop destruct or permitting. Objectives of the Project: Test 5 soil-applied products, with mulch subplots, plus an untreated control (6 treatment plots) on health and HLB status of orange trees over 3 years. The 6 treatment plots of 20 trees each will be replicated 4 times plus mulch subplots at the 3 sites will include: 1. BioFlourish (Triangle Chemical).3 applications per year; 2. Ecofriendly Citrus Soil Amendment. 8 x per year; 3. Serenade Soil (Bayer CropScience) 3 x per year; 4. Aliette (Bayer CropScience) 3 x per year 5. Quantum product line 12 x per year; 6. Untreated control (3 mulched trees only 1/year) Our responsibility is to analyze microbe products, analyze root mass, Las in the root, survival or colonization of microbes in the roots, and indicator strains of microbial diversity in the soil. Totally 5 products, including Bioflourish, Ecofridendly citrus soil amendment, serenade soil, aliette and quantum product were involved. The bacteria ingredients in the product Bioflourish and Ecofridendly citrus soil amendment were determined. There were several Lactobacillus species in Bioflourish, and Bacillus subtitis was detected in Ecofridendly citrus soil amendment. The bacteria in serenade soil and quantum product were mentioned by the producer and thus not tested in lab. The field trails were carried out at three sites around Florida (south west, central ridge and east coast) as described by the proposal. The root density was determined in December 2014. Roots were extracted from 500 cc aliquant of each soil sample over a 2 mm mesh sieve by hand. The dry weight of roots was measured. There were totally 504 samples (3 sites*6 treatments*4replicates*7 trees). The average root mass was 1.14 g/500 cc soil. The root mass did not show any significant difference between treatments, neither for each site nor pulled together (ANOVA, P>0.05). When mulch treatments were taken into account, the results suggested mulched treated trees tended to have higher root mass, but not significant (ANOVA, P>0.05).
This is a 3 year study to test 5 soil applied treatments to prevent HLB infection in newly planted citrus trees and also the test the ability of treatments to maintain tree health should they become infected. The materials are to be Product A – Numbered Compound. Product B – Aliette Product C – Serenade Soil as well as 1 combination of A+B. The impact of treatments on new tree health, foliar nutrition, disease rating, HLB status, root density, subsequent yield and fruit quality will be evaluated. The trial will be located at a single Florida site and will incorporate well-managed trees from a commercial nursery in a new planting. This trial will be large enough to be statistically significant with treatments within label guidelines for products B and C which are registered for citrus. Our main responsibility is to examine annual root density and Las population in the root in year 2 and year 3. Product C contained Bacillus subtilis as active ingredient, so the root colonization and survival rate in soil were tested under greenhouse conditions. 15 ml of product C was applied to Valencia seedlings. The roots and rhizosphere soil were harvested at 1dpi (days post inoculation), 7 dpi and 15 dpi for root colonization and survival analysis. The analysis for other products is ongoing. Product A was not available, product B did not contain bacteria. The samples from project 934.1C were just received. The root mass and bacterial community of each treatment will be determined
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