A trial was initiated in July for evaluation of Magna-Bon (copper sulfate pentahydrate) by trunk injection/infiltration on expression of HLB. The trial is located in Southern Grove in Clewiston, FL in a block of 8 year-old Valencia orange trees. There are 7 treatments with 20 trees per treatment and each treatment has 6 blocks of four trees in a completely randomized block design. Treatments: 1) Untreated Check; 2) One infiltration @ 500 ppm plus Magna-Bon 47; 3) One injection/year of Magna-Bon @ 1,000 ppm; 4) Two injections/year of Magna-Bon @ 1,000 ppm; 5) One infiltration @ 250 ppm plus Magna-Bon 47; 6) Two infiltrations @ 250 ppm plus Magna-Bon 47; 7) One infiltration Magna-Bon 47. Measurements are assay of flush leaves by qPCR at the Southern Gardens Diagnostic Lab(SHDL), sampling of flush leaves for Copper. Tree health assessment twice per season (April and October). First treament was made July 2011.
Objective 1 is to conduct a field evaluation of nutritional sprays for control of HLB and HLB symptom expression and yield. The field study was set up May 2010 in Southern Grove, Hendry Co., FL. Six treatments were located in 4 plots of 150 trees per treatment (interior 10 trees in each block were identified for PCR, leaf nutrition sampling, tree health and yield evaluation). Treatments were 1) non-treated check; 2) Nutri-Phite sprayed 4 times bimonthly; 3) N-Sure sprayed bimonthly; 4) Agra Sol Mn/Zn/Fe plus Nutri-Phite plus triazone urea sprayed bimonthly; 5) Keyplex 1400 DP plus Nutriphite plus triazone urea sprayed bimonthly; 6) Wettable powder nutrients (Diamond R #2) plus Nutri-Phite P+K sprayed bimonthly. The materials were applied to both sides of the tree in 125 gallons per acre with an airblast sprayer driven at 2 mph to obtain thorough coverage. Three disease ratings have been taken so far and a slight decline in tree health has been observed, but no significant treatment effects have been observed. There were no significant treatment differences in yield at the first harvest, after the initiation of treatments the previous April. Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application, Hamlin sweet orange trees have been inoculated and are being treated bimonthly with the nutritional sprays treatments 1, 2, 3, and 5 from objective 1. Infection rate and progress are being monitored by qPCR monthly. By 6 months post inoculation (MPI), most inoculated trees tested Las positive by qPCR within the expected graft inoculation success rate. No treatment differences were apparent. At 6 months post inoculation Las detection was lost in multiple trees in treatments 2 and 5. Root samples were taken for DNA extraction and 7/7 and 6/7 trees were positive for Las in root tissue for treatment 2 and 5 respectively, while only 4/7 were positive in treatments 1 and 3. After pruning trees at 6 MPI for canopy management, sampling at 7 MPI showed a slight reduction in titer in the new flush of all treatments except treatment 1, where no Las was detected until 8 MPI. This suggests that treatments 2,3, and 5 may potentiate movement of Las to new flush where psyllids are most likely to feed and acquire Las. Sampling and fixing of plant material for microscopy of phloem and leaf blade tissue began at two months and continues monthly. Fixed samples were observed at 6 and 8 MPI and sectioning of 9 MPI samples is underway. At 6 MPI reduced phloem plugging and necrosis was observed in treatments 3 and 5, however these treatments had some symptomatic leaves without detectable Las. These leaves had abnormal starch buildup preferentially in phloem tissue instead of mesophyll cells. At 8 MPI no difference in phloem plugging or starch accumulation was observed among treatments. At 6 MPI disease symptom and tree health ratings were routinely taken. Initially no treatment differences were observed. By 8 MPI at least one tree in each treatment had begun to decline and treatment 2 showed the most severe symptoms. At 9 MPI all HLB inoculated trees in treatment 2 had significant decline, while there was no treatment effect in uninoculated trees. In the other three treatments symptom development was variable, but a strong decline of tree health was observed in HLB inoculated trees of all treatments. At 10 MPI most HLB inoculated trees were dead, so this replication was terminated and treatment of a second replication of the experiment was initiated. An additional full nutrient treatment without phosphite was added to test the nutrient treatment without phosphite.
Objective 1 is to conduct a field evaluation of nutritional sprays for control of HLB and HLB symptom expression and yield. The field study was set up May 2010 in Southern Grove, Hendry Co., FL. Six treatments were located in 4 plots of 150 trees per treatment (interior 10 trees in each block were identified for PCR, leaf nutrition sampling, tree health and yield evaluation). Treatments were 1) non-treated check; 2) Nutri-Phite sprayed 4 times bimonthly; 3) N-Sure sprayed bimonthly; 4) Agra Sol Mn/Zn/Fe plus Nutri-Phite plus triazone urea sprayed bimonthly; 5) Keyplex 1400 DP plus Nutriphite plus triazone urea sprayed bimonthly; 6) Wettable powder nutrients (Diamond R #2) plus Nutri-Phite P+K sprayed bimonthly. The materials were applied to both sides of the tree in 125 gallons per acre with an airblast sprayer driven at 2 mph to obtain thorough coverage. Three disease ratings have been taken so far and a slight decline in tree health has been observed, but no significant treatment effects have been observed. There were no significant treatment differences in yield at the first harvest, after the initiation of treatments the previous April. Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application, Hamlin sweet orange trees have been inoculated and are being treated bimonthly with the nutritional sprays treatments 1, 2, 3, and 5 from objective 1. Infection rate and progress are being monitored by qPCR monthly. By 6 months post inoculation (MPI), most inoculated trees tested Las positive by qPCR within the expected graft inoculation success rate. No treatment differences were apparent. At 6 months post inoculation Las detection was lost in multiple trees in treatments 2 and 5. Root samples were taken for DNA extraction and 7/7 and 6/7 trees were positive for Las in root tissue for treatment 2 and 5 respectively, while only 4/7 were positive in treatments 1 and 3. After pruning trees at 6 MPI for canopy management, sampling at 7 MPI showed a slight reduction in titer in the new flush of all treatments except treatment 1, where no Las was detected until 8 MPI. This suggests that treatments 2,3, and 5 may potentiate movement of Las to new flush where psyllids are most likely to feed and acquire Las. Sampling and fixing of plant material for microscopy of phloem and leaf blade tissue began at two months and continues monthly. Fixed samples were observed at 6 and 8 MPI and sectioning of 9 MPI samples is underway. At 6 MPI reduced phloem plugging and necrosis was observed in treatments 3 and 5, however these treatments had some symptomatic leaves without detectable Las. These leaves had abnormal starch buildup preferentially in phloem tissue instead of mesophyll cells. At 8 MPI no difference in phloem plugging or starch accumulation was observed among treatments. At 6 MPI disease symptom and tree health ratings were routinely taken. Initially no treatment differences were observed. By 8 MPI at least one tree in each treatment had begun to decline and treatment 2 showed the most severe symptoms. At 9 MPI all HLB inoculated trees in treatment 2 had significant decline, while there was no treatment effect in uninoculated trees. In the other three treatments symptom development was variable, but a strong decline of tree health was observed in HLB inoculated trees of all treatments. At 10 MPI most HLB inoculated trees were dead, so this replication was terminated and treatment of a second replication of the experiment was initiated. An additional full nutrient treatment without phosphite was added to test the nutrient treatment without phosphite.
The objective is to evaluate soil-applied neo-nicotinoids and other SAR inducers on HLB disease progress in newly planted citrus trees subjected to psyllid-mediated infection or graft-inoculation. One yr-old Hamlin trees were planted in May 2009 and treated as follows: 1) non-treated check (UTC), 2) foliar insecticide to control psyllids, 3) soil-applied imidacloprid/thiamethoxam (IMID/THIA) to induce SAR, 4) soil-applied IMID/THIA plus foliar insecticides, 5) graft-inoculated UTC, 6) graft-inoculated with IMID/THIA. There were 50 trees per treatment (5 blocks of 10 trees). In 2009, the effect of SAR inducers on HLB infection progress was inconclusive perhaps attributable to the interaction of IMID/THIA with psyllid control which may have an uncontrolled effect on psyllid transmission. In 2010, the SAR inducer acibenzolar-S-methyl (ASM, Actigard 50WP) which does not control psyllids was substituted in treatments 3, 4 and 6. At 24 months after treatments began, 105 trees were PCR+ (35%) in the trial. Higher number of PCR+ trees occurred in the UTC (20), the UTC with graft inoculation (22), and the IMID/THIA/ASM with graft-inoculation (28). A lower number of PCR+ trees occurred in the treatments with SAR inducers (11), foliar insecticides (12), and foliar insecticide plus SAR inducers (12). Two years after treatments were initiated, the effect of SAR on HLB disease progress has been minimal, which indicates a lack of promise for SAR inducers in HLB management. This information has been communicated to citrus growers in a recent Citrus Industry magazine article entitled “The ABCs of SAR” (May issue).
Under Objective 1 and 2 the following was submitted and published in Plant Disease: Soil applications of inducers of systemic acquired resistance (SAR), imidacloprid, thiamethoxam or acibenzolar-S-methyl, at various rates and application frequencies were evaluated for control of citrus canker caused by Xanthomonas citri subsp. citri in a field trial of 3- and 4-year-old ‘Ray Ruby’ grapefruit trees in southeastern Florida. Reduction of foliar incidence of canker produced by one, two or four soil applications of imidacloprid, thiamethoxam, and acibenzolar-S-methyl was compared with 11 foliar sprays of copper hydroxide and streptomycin applied at 21-day intervals. In 2008 and 2009 crop seasons, canker incidence on each set of vegetative flushes was assessed as the percentage of the total leaves with lesions. By the end of the 2008 season, despite above average rainfall and a tropical storm event, all treatments significantly reduced foliar incidence of citrus canker on the combined Spring-Summer-Fall flushes. Sprays of copper hydroxide and streptomycin were effective for reducing canker incidence on shoot flushes produced throughout the season compared to the untreated control, whereas soil applied SAR inducers reduced foliar disease depending on rate, frequency and timing of application. Except for the treatment of four applications of acibenzolar-S-methyl at 0.2 g a.i. per tree or two applications of imidacloprid, SAR inducers were ineffective for reducing foliar disease on the flushes that were present during the tropical storm. In 2009, all treatments significantly reduced the incidence of foliar canker on the combined Spring-Summer-Fall flushes but not all treatments of Spring-Summer flushes with SAR inducers were effective compared to the untreated control. Hence, depending on rate, frequency and timing of application, soil-applied SAR inducers reduced incidence of canker on foliar flushes of young grapefruit trees under epidemic conditions. Under Objective 2 Integration of soil applied SARs with foliar applications of copper sprays for control of canker, we are testing the efficacy of the inducers for control of citrus canker on young fruiting Red grapefruit and Hamlin orange trees. We previously demonstrated that trunk application of imidacloprid is effective for canker control on fruit as 21 day copper sprays. To keep the chemicals out of the soil, we are spraying the SAR inducers on the trunk. These trials involve testing of non-registered acibenzolar-S-methyl and the insecticides imidacloprid, thiamethoxam at elevated rates. Hence, fruit from the treated trees must be dropped and destroyed (called ‘crop destruct’ of nonregistered chemical treatments). Under Objective 3, In 2011 the complementary use of ASM, THIA and IMID soil applications to increase and/or extend canker control in 3-yr-old grapefruit and 2-yr-old Vernia orange trees. The highest incidence of disease trees and/or leaves is in the unreated check in each trial compared with a very low incidence of canker in the integrated SAR treatments. A field trial with soil applied neonicotinoids in Parana, Brazil was evaluated. IMID (Confidor) as a soil drench and IMID (Winner) applied to trunk gave comparable in disease control activity on 2-yr old Valencia orange trees, as well as, the other neonicotinoids tested, THIA and Clothianidin. Clothianidin is now registered for use on non-bearing citrus in Florida, hence all of neonicotinoids registered for non-bearing citrus in Florida have been shown to have SAR-inducing activity against canker as well.
This project includes experiments being conducted by USDA-ARS and University of Florida on protecting newly planted citrus trees from Asian citrus psyllid and huanglongbing. This progress report reviews research conducted by University of Florida in Southwest Florida. Research reviewed pertains to the efficacy of insecticides for psyllid control. One study compares three rates of cyazapyr (150, 225, and 350 g ai/ha) against Admire Pro (280 g ai/ha) and Platinum 75g (140 g ai/ha) applied on 19,20 Jul to newly planted Hamlin trees. Evaluations were conducted 10,23 ‘ Sep. and no psyllids were found. However, all products have been 100% effective for leafminer control. Psyllid nymphs were observed 19-Apr-2011 on new flush and evaluations were made by removing 10 shoots per plot and counting nymphs under a stereo microscope. A more uniform sample was later obtained by pruning back and caging a single branch on 11 Apr on two trees in each plot. On 27-Apr , 10 adult psyllids from a lab reared colony at SWFREC were placed in each of the cages which was then sealed with a 6 inch twist tie to keep out predators. On 11-May, one shoot from each cage and two additional shoots randomly selected from the same trees in each plot were removed and nymphs were counted with a stereo microscope. Counted shoots were sent to a laboratory for HPLC residue analysis. On 19 April, fewer nymphs compared to the control were seen from all treatments except Admire Pro, with no differences among rates of cyazpyr or thiamethoxam. On 11 May, only the two highest rates of cyazapyr resulted in fewer nymphs compared to the control. All other treatment trees were 100 % infested with psyllids compared to 40 and 80% for the high and medium rate of cyazapyr. Thus, we were able to detect activity against psyllids from these two treatments 10 months after application to young citrus trees. Another study is designed to continue for 3 years with rotations of cyazpyr at the low and medium rate with Platinum and Admire Pro at the same rates as above every three months. The objective is to assess the ability of these treatments to delay or prevent the onset of HLB in an area of high incidence. Initial treatments were made 29-Jul on newly planted Hamlin trees and evaluations conducted 2,22-Sep with no psyllids being observed although all treatments have been 100% effective in controlling leafminer. Applications were repeated on 24-Jan and 25-April and evaluations on 13-Apr and 18- May by removing 10 shoots per plot and counting nymphs under a stereo microscope. Untreated shoots were highly infested, but no nymphs were observed on shoots from treated trees except on the last sample date when 2.4 nymphs per shoot were seen with the rotation that began with Admire Pro and employed the low rate of cyazapyr, although this was not statistically different from the other 3 insecticide treatments. Leaf samples are being analyzed for residues of all insecticides. A review of the efficacy of different insecticide treatments for suppressing psyllids and consequently HLB will be presented in this project’s next annual report.
The intent of this study is to examine the effect of windbreaks, copper sprays to reduce infection, and leafminer treatments to determine there individual and combined effects on control of citrus canker in Brazilian commercial citrus and the applicability of this strategy to the US commercial citrus industry. A preliminary study was published in Crop Protection 27:807-813, that indicated that copper and insecticide applications significantly reduced canker infection but windbreaks did not have any effect. As described previously, a new series of plots with much more extensive windbreaks were established via a USDA/ARS specific cooperative agreement with the University of Sao Paulo, and the Brazilian cooperator at an IAPAR farm, in Xambr’, Parana state, using, 2-yr-old P’ra on Rangpur lime. Windbreaks were completed and plants were be established in Mid April 2010, but severe winds damaged the windbreaks during two storm events. These windbreaks have been reinforced and rebuilt. This delayed the experiment which is now scheduled to begin in March 2012. The following treatments will then be applied: 1) no sprays (control), 2) Cu++ sprays to reduce citrus canker incidence, and 3) insecticide sprays to inhibit infestations of Asian leafminer (secondary effects). Main effects are windbreak versus no windbreaks. Citrus canker incidence will be estimated on multiple branches on each tree treated as the number of leaves per branch infected. We anticipate running these plots for 2-3 more years to collect all necessary data. The development of the Programmable leaf wetness controller (PLWC) software was written, debugged, is complete, and the control program is working well. New leaf wetness sensors were designed and constructed and calibrated. An electronic glitch was determined in the leaf wetness sensors and new control circuitry was designed and constructed to overcome the glitch. New hydrophobic materials used as the sensor bridge allowing the detection of leaf moisture are being evaluated as well. The newly designed probes with various sensor bridge materials are currently being tested Publications: 167. Bock, C. H., Graham, J. H., Gottwald, T. R., Cook, A. Z., and Parker, P. E. 2010. Wind speed effects on the quantity of Xanthomonas citri subsp. citri dispersed downwind from canopies of grapefruit tree infected with citrus canker. Plant Di Bock C.H., Graham, J.H., Gottwald, T.R., Cook, A.Z., and Parker, P.E. 2010. Wind speed and wind-associated leaf injury affect severity of citrus canker on Swingle citrumelo. Eur J. Plant Path 128:21-38 Bock, CH, Parker, PE, Cook, AZ, Graham, JH and Gottwald, TR. 2001. Infection and decontamination of citrus canker and inoculated the surfaces. Crop Protection 30:259-264. Hall, D.G., Gottwald, T.R. and C.H. Bock. 2010. Exacerbation of citrus canker by citrus leafminer Phyllocnistis citrella in Florida. Florida Entomologist. Florida Entomologist 93:558-566. Bock, C.H., Gottwald, T.R. and Parker, P.E. 2011. Distribution of canker lesions on the surface of diseased grapefruit. Plant Pathology (Accepted).
The objectives of this study are: 1) to develop a series of flexible stochastic models to predict the temporal increase and spatial spread of citrus HLB and canker. They can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies both in plantations and State-wide. 2) Test various control methods under field conditions to evaluate effects and collect data to parameterize models. SEIDR model. Using Markov-chain Monte Carlo (MCMC) methods, and data from infected areas of South Florida for successive snapshots of the occurrence of symptomatic detected trees in known populations of susceptible trees, we continue to estimate the transmission rates and dispersal kernel for HLB. A working model has been developed and continues to be improved that focuses on the differential effects of host age on epidemiological parameters as well as variability. Both residential and commercial citrus scenarios are being tested with user selectable and changeable via sliding controls. We continue to explore various aspects of Baysian MCMC methods to infer posterior densities on the model parameters. The uncertainty will be incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. We are also considering a second modeling approach known as Approximate Bayesian Computation (ABC) and willcompare model outputs of the ABC versus the MCMC estimates to determine HLB dispersal distances be that we believe may improve control strategies. We have begun to develop a web based version of the model (known as a front end) and will soon go into validation testing. Both residential and commercial citrus scenarios are being tested with user selectable and changeable via sliding controls. We will use the front end to visualize the effect of these various control strategies. This should benefit researchers, growers and regulators to compare the results of thousands of simulations for practical management decision making and/or regulatory intervention/strategy building. Via this model we have begun to examine the effects of various controls such as using insecticide applications or not, removing infected trees or not, and the effect of HLB infection in young versus older trees. Model output confirms that controlling secondary infections by diseased tree removal and insecticide applications plus controlling primary infection from new insect immigrations can reduce disease increase to a manageable 2 to 5% increase per year.
Using an electrical penetration graph monitor, we examined the duration of feeding disruption provided by 5 insecticides commonly used in Florida citrus production. For chlorpyrifos, at 1DAT, while there was no significant difference in probing duration, psyllids on chlorpyrifos performed half of the probing durations when compared to untreated plants. Phloem related feeding behaviors were prevented up to 7 DAT on chlorpyrifos treated plants when chlorpyrifos residues on the leaf surface averaged 27.13 ppm. For both fenpropathrin and imidacloprid-treated plants, there was an immediate reduction 1 DAT in D. citri probe duration, number of probes, non-probing/walking activities, stylet penetration, phloem activities and xylem ingestion when compared to untreated plants. Over time, the duration of those behaviors increased as insecticide residue levels decreased. Fenpropathrin residues provided significant disruption of phloem related feeding behaviors by D. citri up to 21 DAT. Disruption of phloem feeding behaviors for psyllids on imidacloprid-treated plants lasted up to 28 DAT. At this time imidacloprid residue levels were 34.66 ppm, which was 83% lower than the concentration found at 1DAT. Psyllids on spinetoram-treated plants performed probes which were shorter in duration, but disruption of phloem feeding behaviors was not prevented, even at 1 DAT. The decrease in probing duration for psyllids on spinetoram-treated plants was only significant 7 DAT. Psyllids on spirotetramat-treated plants performed normal phloem activities throughout the entire experiment. The results from this study demonstrate that while some insecticides may cause relatively rapid mortality of adult psyllids, there is considerable variability that exists among these products in terms of the duration of feeding disruption provided. While some insecticides provided feeding disruption lasting 3-4 weeks (fenpropathrin and imidacloprid), protection provided by other products was much shorter. Overall, the results of this study can be used to help guide citrus growers in product selection and also determine when additional applications will be necessary.
In previous work, we reported that soil-applied neonicotinoid insecticides can significantly disrupt psyllid feeding behavior and thus reduce (if not totally prevent) treated young trees from becoming infected with the HLB bacterium. Since the majority of this work was done in a laboratory setting and we only have anecdotal evidence from the field to support this claim, a multi-year field trial was initiated to validate this claim. At the end of May, a 10-acre block of ‘Valencia’ orange was planted at the Mid-Florida Citrus Foundation Water ConservII grove site in Orange County. Within this planting, 60 24-tree (4 rows x 6 trees) plots were assigned to one of 6 experimental treatments. Each treatment is replicated 10 times across the 10-acre block. The treatments are as follows 1) soil-applied neonicotinoid applications every 6 weeks; 2) monthly foliar-applied broad spectrum insecticide applications only; 3) monthly applications of a selected psyllid deterrent material; 4) soil-applied neonicotinoid applications every 6 weeks plus monthly applications of foliar-applied broad spectrum insecticides; 5) soil-applied neonicotinoid applications every 6 weeks plus monthly applications of the selected psyllid deterrent material and 6) and untreated control plots for comparison. One day after planting, and just prior to application of the first treatments, leaf samples were collected from the two center rows of trees in each plot. These leaves were then analyzed in the laboratory, first for the presence of Las to confirm that our experiment began with HLB pathogen-free trees, and second, to quantify the level of neonicotinoid residues already present within the leaf tissues. Every two weeks following planting, psyllid and leafminer counts will be made in each plot as an assessment of the level of pest control provided by each treatment. On the same day these pest assessments are made, leaf samples will be collected from the two center rows of trees in each plot. These samples will be taken back to the lab for pesticide residue analysis. To determine the rate of HLB infection between treatments, leaf samples will be collected every 3 months from the two center rows of trees in each plot and analyzed for the presence of Las using pcr. Additionally, tree growth measurement will be made every three months to compare for treatment effects on plant growth rate. During the first two months of this particular objective, PCR analysis of all plant material has shown that none of our trees were HLB infected coming out of the nursery. Analysis of leaves collected showed they contained imidacloprid as a result from treatments applied prior to leaving the nursery. Despite the presence of imidacloprid in the leaf tissue, within the first week after planting, leafminer damage was as high as 5% in foliar insecticide and control treatments, and increased to 30-40% approximately 4 weeks after planting. Such results were anticipated since disruption of the root system at the time of planting has been shown to dramatically decrease the duration of control provided. Thus, these results provide further direct evidence for the need to quickly retreat young trees immediately following planting in order to provide maximum protection from both leafminer and asian citrus psyllid. This objective as described above will be continued over the course of the next 4 years to compare the success rate of bringing young trees into production using the different management strategies tested.
Following three freezes in southwest Florida during the 2010-11 winter in which we experienced temperatures of 25 degrees F for several hours resulting in leaf and fruit drop, and some terminal twig damage, spring growth was good and the trees in our trials receiving foliar nutrients rebounded with thick canopies, flowered and set a good crop. Yield from the 2010-11 Valencia crop in our trial gave the same results as the two previous years when comparing the treatments in the Boyd nutritional experiment. The treatments with the three highest yields were from the complete Boyd cocktail, the complete cocktail without SARs, and the complete cocktail without hydrogen peroxide. These treatments all contained the macro- and micro nutrients along with phosphite. Juice quality was affected by some drying in the Valencia fruit resulting from the freezes, and assuming all trees experienced the same amount of damage to the fruit the juice quality results follow. Percentage juice and juice per box was not different among treatments except lowest for the treatment with only SARs + phosphite, which was highest in acid. Acid, Brix, Ratio, and lbs solids/box were highest for treatments with Boyd cocktail, Boyd cocktail w/o SARs, and Boyd cocktail w/o hydrogen peroxide, macro nutrients only, and phosphite only, and lowest with SARs + phosphite. HLB bacteria as detected by real time PCR during the last 3 year shows the incidence at the test site increased from 41% to 79%, and is now at 100%. Furthermore, at the most recent sampling in 2011 regardless of the treatment the ct values were lowest indicating the the bacteria population in the tree was higher than in each of the previous years. We are entering the 4th year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test effects of micro-nutrients + systemic acquired resistance inducers, and Asian citrus psyllid (ACP) chemical control on ACP populations on Can. Libericacter asiaticus (CLas) titer, and plant yield. While we are continuing with the experiment, results from the first 3 years are being written up for publication. Psyllid populations in the insecticide and insecticide+nutritional plots reached the 0.20 threshold on 22Apr, 6May, and the insecticide+nutritional plot reached threshold on 4June. Insecticide and Insecticide+nutritional had significantly (P< 0.05) fewer psyllids than control or nutritional only on dates 8Apr, 22Apr, and 7May. All three treatments were significantly (P < 0.05) less than control on 21May, but no differences were found between treatments on 4Jun. Since our last report we have sprayed one application on the treated plots: Movento MPC at a rate of 16oz/ac + 2gal/ac of 435 horticultural oil. Three years of sticky card data are now being examined to assess movement of adult psyllids among treatment plots. Tap and flush sampling have been modified to include counts of nymphs (all stages) and eggs on flush. In Jan and May, we collected plant samples for HLB detection. Results of the fruit samples sent to the CREC fruit quality laboratory (Apr 11) for analysis of lbs. juice per box, acid, total brix, and ratio have not been received. An initial citrus leafminer damage assessment using a modified Horsfall Barratt scale (22 June) showed significant differences (p < 0.0001) between insecticide + nutritional and insecticide only treatments on the one hand and nutritional on the other with this latter showing most damage.
The goal of this project is to identify insecticidal peptides with efficacy against phloem-feeding citrus pests. Our first objective is to evaluate a range of known insecticidal peptides against the Asian Citrus Psyllid (ACP) in artifical feeding assays. We have procured candidate peptides for screening and are optimizing artificial bioassays for evaluation of their effect on ACP mortality. In a second objective, we have proposed to utilize Citrus tristeza virus (CTV) for delivery of insecticidal peptides into citrus plants for management of the ACP, Brown Citrus Aphid (BCA), and Citrus Leafminer (CLM). Development of CTV as a vector for foreign peptides was previously conducted in a separate project by Dawson et al. Using the CTV expression vector, we have elected to examine four peptides known to have insecticidal activity against phloem-feeding agricultural pests. To facilitate bioassays for screening individual insecticidal peptides, we have recently established laboratory colonies of BCA and CLM. BCA were obtained from a colony currently housed at the Citrus Research and Education Center in the laboratory of Dr. Bill Dawson. The CLM colony was established by collecting insects from citrus groves located in Polk County, Florida. In addition, we have procured plants for use in experiments evaluating CTV-expressed insecticidal peptides. Prior to the inception of the current project, one of the peptides proposed for evaluation, snow drop lectin, Galanthus nivalis agglutinin (GNA), was successfully cloned into the CTV-expression vector and inoculated into citrus plants. At the present time we are conducting experiments to evaluate the efficacy of CTV-vectored GNA against ACP and BCA. These bioassays will evaluate the effect of GNA on insect feeding, survival, development, and fecundity. Concurrently, we are conducting bioassays using synthetic GNA in an artificial feeding system to evaluate the effect of GNA concentration on ACP mortality. In addition to GNA, we have proposed to evaluate three CTV-vectored insecticidal peptides against ACP, BCA, and CLM. Indolicidin, which was previously cloned into the CTV vector, has been inoculated into citrus and should be available for screening in bioassays with the target insects in the next few months. Allium sativum leaf agglutinin (ASAL) and Pinellia ternate agglutinin (PTA) peptide genes have been sequenced and are currently being cloned into CTV-vectors. This process should be completed within two months, after which the CTV-peptide constructs will be bark flap inoculated into citrus. Following successful establishment of CTV vectors into initial citrus hosts, stems from plants exhibiting a high level of CTV will be harvested and graft-inoculated onto subsequent ‘Valencia’ sweet orange plants for use in bioassays. This process is expected to be completed in the next four months. To quantify peptide expression in CTV-inoculated plants, we are developing enzyme-linked immunosorbant (ELISA) assays specific to the peptides under investigation. At the present time, an ELISA assay for GNA is being optimized. Identifying the concentration of expressed peptides should facilitate comparisons between the efficacy observed in artificial bioassays with synthetic peptides and in planta assays with CTV-expressed peptides.
This project is focused on evaluating the impact of psyllid control programs on non target insects and mites. We are conducting replicated experiments at the SWFREC and commercial groves to test effects of insecticides, horticultural oils and nutritionals on psyllids and non-targets. Stem tap sampling, vacuum sampling, yellow sticky card and pheromone traps and shoot and trunk examinations are being used to monitor populations of psyllids and secondary pests such as citrus leafminer, citrus rust mites, citrus red mite, snow scale, Florida red scale, Chaff scale, citrus black and whiteflies, as well as their parasitoids and predators. For this report citrus rust mites (CRM) are compared under different psyllid control programs. The experiment in Collier county compares (1) Nutritional (2) Insecticides (3) Insecticides + Nutritionals and (4) Untreated control. Insecticides currently recommended for psyllid control are used when adults exceed 0.2 per tap sample and nutritional products and their application timing follow a program developed by McKinnon Corporation averaging three sprays per year. Psyllids averaged 0.6 adults per tap sample in the untreated control; significantly more than 0-0.07 per tap sample in the other three treatments which did not differ. Averages of 6 and 4 CRM per lens field were observed in the nutritional and untreated treatments compared to < 1 in the treatments using insecticides indicating that these particular insecticides which included spirotetramat (Movento) were providing control of both psyllids and mites. Each of the two experiments in Hendry county, one in a 35 acre block of 'Early Gold' and the other in a 16 acre block of 'Valencia' include 4 treatments: three spray programs for psyllid, one receiving monthly calendar sprays, two intended to maintain ACP populations below thresholds of 0.2 or 0.7 psyllids per tap sample and an untreated check. Calendar applications are being applied approximately every 1 month and consist of a rotation of insecticides recommended for managing this pest. Sprays in both experiments included carbaryl (Sevin XLR Plus ) at 0.75 gal/ac in April, spinetoram (Delegate WG) at 4.5 oz/ac in May and imidacloprid (Admire Pro) in June at 4.5 oz/acre. No sprays have been applied in threshold treatments because selected population levels have not been reached. No psyllid adults were observed in the tap sampling conducted in the calendar application and 0.01-0.19 per tap sample in the untreated control. CRM averaged 2 per lens field in the untreated 'Early Gold' and increased to 5 per lens field in the calendar application but did not differ between the two treatments in 'Valencia' (average 1.7 per lens field) indicating that calendar application either did not help or increased CRM populations, probably through negative impact on predators particularly phytoseids mites which were significantly lower in the calendar application treatment than in untreated plots. Natural mortality in cohorts of psyllid immatures averaged 82% in the untreated control compared to 69% in calendar application and provided additional evidence of the greater activity of natural enemies in the control. Another trial was conducted at the SWFREC to test some commonly used and experimental insecticides for effects on psyllids and non targets. Delegate, Movento and Danitol all provided significant reduction in psyllid populations compared to the untreated control for more than three weeks. However, CRM populations compared to untreated control were reduced only in Movento and Danitol treatments averaging 2 and 5 per lens field, respectively, compared to an average of 8 per lens field in control and Delegate plots which did not differ.
The goal of this project is to develop potential repellent formulations for Asian citrus psyllid (ACP) that originate from guava (and related) plant volatiles. We have made progress in identifying volatiles that repel ACP in the laboratory, but the hurdle has been how to effectively deliver these volatiles in the field so as to reduce ACP populations. The main obstacles have been: 1) that these repellent chemicals are highly volatile in nature and it is challenging to maintain sufficiently high concentrations in the field so as to affect psyllid population densities, and 2) that these chemicals are quite noxious and foul smelling. The most promising formulation to release DMDS and related psyllid repellent chemicals that we have evaluated to date is the flowable and wax-based SPLAT formulation. This is a proprietary and established release device for insect behavior modifying chemicals and is manufactured and distributed by ISCA Technologies in California. We have seen mixed results with DMDS released from SPLAT. Although some tests in the field verified laboratory tests, showing reduced psyllid population densities, other tests have shown no discernible effect of the treatment. Our release rate analyses with the initial SPLAT formulation indicated that the DMDS active ingredient was released rapidly from this formulation (approximately 80-95% loss within 5-10 days). More advanced slow release formulations were developed. Initial field testing with these formulations showed promising results–psyllid populations were reduced beyond three weeks as compared with control plots. However, a follow-up experiment in the early spring of 2011 under low psyllid population densities again resulted in no effect of the SPLAT-DMDS treatment with the advanced slow-release formulation that was determined as the best one based on 2010 results. It is possible that this test failed because of low psyllid population densities during the timing of the experiment; however, it has become evident that we need to be able to directly quantify DMDS airborne concentration levels in the field in order to directly establish how much DMDS is needed to affect psyllid population densities. Also, this information will allow us to understand how environmental factors influence how much DMDS is present in the air surrounding citrus trees treated with the SPLAT-DMDS formulation. Our objective is to use this DMDS-monitoring technique and the information it provides to figure out why the treatment appears to work during certain applications, but fails during others. Therefore, we recently developed a purge and trap procedure to directly quantify levels of DMDS in the field following application of the SPLAT-DMDS formulation. The purge and trap analytical procedures for the analysis of DMDS in air from the four compass points surrounding a test tree has been optimized. The newer, slow-release DMDS concentration formula was evaluated. Depending on wind direction, initial DMDS concentrations ranged from 0.4 to 1.5 ng/L. Maximum concentrations were observed 3-4 days after application. These maximum concentrations ranged from 1.6 to 2.6 ng/L. Concentrations decayed exponentially during the next 29 days. Final concentrations after 29 days ranged from 0.18 to 0.62 ng/L. We will be correlating how concentration of DMDS relates to behavioral activity on psyllids with the objective of figuring out how much DMDS is needed in the field to affect psyllids and what factors potentially affect concentration changes in the field. We hope that this will help us understand why the emerging technology appears to be effective in some experiments, while showing inconsistent results in others.
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. The production of greenhouse-grown citrus trees with nutrient deficiency symptoms for both zinc and boron continues. Early deficiency symptoms have begun to develop for boron and nutrient analysis shows low borderline deficient boron levels in the leaf. Zinc deficiency has yet to develop. Nutrient analysis show that zinc levels are falling, but not yet low. The high mobility of zinc within the plant allows redistribution of existing zinc to new flush, delaying the development of deficiency and the associated symptoms. Multiplication of our stocks of HLB infected citrus is also underway to provide a constant source of symptomatic tissue for experimentation once strong nutrient deficiency symptoms have developed. As soon as strong deficiency symptoms develop, then psyllid testing for objective 1 and 2 will commence and is expected to move rapidly. We are also interested to determine if strains of Las will have any effect on the attractiveness of trees to D. citri. It has been reported that Las strains have varying levels of virulence and symptomatology (Tsai et al. 2008). We have analyzed DNA samples from HLB positive trees from Polk and Highlands counties as well as the ‘Smoak Grove’ CREC greenhouse strain by PCR and sequencing. Three putative strains of Ca. Liberibacter asiaticus (Las) were found with 5 (CREC greenhouse isolate), 13, and 15 tandem repeats of DNA in the LAPGP locus described by Chen et al. 2010 and have identified sources of budwood. Cloning and sequencing of loci including the b-operon, OMP (outer membrane protein) gene and phage DNA polymerase to support the differentiation of the three strains is complete (Bastianel et al. 2005; Lin et al. 2008; Okuda et al. 2005; Tomimura et al. 2009). Results from sequence analysis clearly defines two strains based on conserved mutations in the b-operon sequence, matching strains from Japan and Vietnam and a strain from Vietnam for the northern and southern Florida strains, respectively.
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