Objectives of this project include 1) refinement of sampling methods, 2) testing the influence of adult density and shoot infestation on precision of estimated means and distribution of population within blocks, and 3) evaluation and integration of methods for assessing psyllid density, shoot density, and infestation rates into a user friendly system accessible to consultants and managers. Accomplishments and plans for further studies are described for each objective. 1) Data from an experiment conducted at a commercial grove showed that although sticky traps caught more adults over a two week period compared to one time tap sample at the end of that period conducted at both low and high psyllid densities, they were not only expensive but consumed more time and provided data after at least a 1-week delay compared to a tap sample that provided instant data with no more supplies than a laminated sheet of letter size paper or a smooth white surface such as a clipboard, and a one-foot piece of PVC tube. We have collected more data on comparison of these methods which is being entered and analyzed. Our commercial collaborators are now using tap sampling method and shoot examination to assess ACP populations in their groves and providing us data that is being entered for analysis. Comparison of the tap sampling and sweep net sampling methods at the SWFREC orchard showed that at low density tap method was more efficient in detecting ACP than sweep net although no significant difference was observed in numbers detected and sampling time at high density. However, density averaged less than 0.5 adults per sample with either method. Therefore, we have planned to conduct this comparison at a commercial grove where ACP density is much higher and maintained at two levels (low and high) through use of insecticides and untreated control. 2) Regression analysis using bootstrapping procedure indicated that the precision targets of 0.25 and 0.01 SEM:mean were reached at high psyllid density with 8 and 66 tap-samples, respectively, compared to 12 and 75 YCRW traps and 13 and 74 ACP traps. At low psyllid density, 267 and 1929 tap-samples, 16 and 130 YCRW traps, and 43 and 219 ACP traps were needed to attain these precision ratios, respectively. At low density 100 and 571 tap-samples and 160 and 1295 sweep net samples were needed at ratios of 0.25 and 0.1, respectively, compared to 35 and 196 tap samples and 37 and 198 sweep net samples at high density. Therefore, the sweep net would take more time to provide the same data and more work and difficulty in counting at high density. As indicated above we have included additional locations to conduct these comparisons at density above the one reported here. 3) A rapid system that includes tap samples, flush inspections and natural enemy evaluations along with forms and spreadsheets is available on our website swfrec.ifas.ufl.edu/entlab/. Just during this quarter 400 tap sampling kits were distributed among growers. Five presentations and 5 workshops on psyllid monitoring were conducted at SWFREC to train 250 attendants. A survey of 27 growers in SW Florida conducted by SWFREC and IFAS extension in 2009 covered 106,148 acres of commercial citrus (80% of productive area) reported that among 96% conducting sampling, 85% use the tap method, along with shoot examination (56%), sticky traps (7%), and sweep nets (4%). We are starting another survey to collect information for 2010. We are also working on the development of a website where growers will be able to enter data on the incidence of pest and disease in order to assess the need and recommendation of appropriate treatments. Findings from the above studies were published in citrus industry and being prepared for publication in a peer reviewed journal.
Objective 1 is to conduct a field evaluation 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 set 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. Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application, Hamlin sweet orange trees have been acquired and treated with two rounds of nutritional sprays and leaf samples have been taken for baseline nutrient content of the different treatments. Graft inoculation with HLB infected budwood was performed on half of the trees in each nutritional treatment group. Monitoring of macroscopic and microscopic symptom development is underway. Monitoring of Las titer will begin when one month has passed since inoculation.
Reducing excessive vegetative growth that is produced annually by citrus trees in Florida would reduce the opportunities for Asian citrus psyllid (Diaphorina citri Kuwayama) reproduction and thereby, the spread of Huanglongbing. Excess tree growth is routinely removed through hedging and branch re-growth can be reduced after hedging in the fall season under Florida conditions because of the onset of cool temperatures. Additionally, late-summer hedging may synchronize a final late-season flush and thus, reduce new flush leaves present during the winter to support over-wintering psyllids. We determined timing effects of fall and early winter hedging of ‘Hamlin’ orange trees on vegetative growth flush and subsequent yield during a two year period (2008-2010). None of the fall/winter (Oct – Jan) hedging times tested stimulated a growth flush in either year. Yield, in terms of fresh fruit weight, was only affected by late-Nov and early-Dec hedging. This suggests that although earlier hedging times reduced the number of fruit harvested, there was sufficient time left before harvest for the remaining fruit to increase in mass, thus compensating for the pieces of fruit removed by hedging. We also tested the effects of summer hedging times (late-Aug – Sept) on yield and flush management. Similar to early fall hedging, summer hedging reduced the number of fruit harvested, but not the total weight of fruit harvested. Summer hedging times did stimulate a uniform growth flush approximately 2 weeks after hedging on which psyllid control was needed. We have concluded that hedging practices can be modified to affect flushing patterns in a way that benefits ACP control strategies. However, because of environmental effects on flushing that are poorly understood in citrus the hedging effect cannot be consistently reproduced year-to-year and is likely to be adapted by growers. These data have been published in the proceedings of the Florida State Horticultural Society (Spann et al., 2009, Proc Fla State Hort Soc 122:161-165) and are being prepared for publication in a refereed journal. We hypothesized that plant growth regulators (PGRs) could be used to reduce vegetative growth of citrus, thus reducing ACP populations. Seedlings of Volkamer lemon were treated with six commercially available PGRs (10 replications each): Embark, Sumagic, Atrimmec, Apogee, Profile and Cycocel. Untreated seedlings served as controls. Ten days after PGR application, a pair of ACP was caged on five seedlings of each treatment. The five seedlings from each treatment without ACP were used for vegetative growth measurements. All seedlings were maintained in a greenhouse at 28 C and 70% relative humidity. Over the course of fifteen days, the number of eggs laid on each plant was counted daily. Survival of ACP nymphs to adults was assessed. Adults were collected and weighed to determine adult body weight. Due to severe phytotoxicity no data were collected from Atrimmec-treated plants. Only Sumagic and Apogee significantly reduced total shoot growth. The average number of eggs laid per plant was reduced by 85% on Embark and Sumagic-treated plants, and by 65% on Apogee and Profile-treated plants. In contrast, the number of eggs laid increased by almost 40% on Cycocel-treated plants. Survival of nymphs to adults was also significantly affected by PGR treatment. Survival was lowest for plants treated with Profile (7%), followed by Sumagic (36%), untreated control plants (45%) and Apogee (47%). The highest survival rates were observed on Cycocel (72%) and Embark (68%) treated plants. ACP adults that matured on seedlings treated with Sumagic weighed significantly less than those from Embark, Apogee, Profile and control plants, but were similar in weight to those from Cycocel-treated plants. These data indicate that the PGRs tested significantly effected the overall fitness of the ACP; however, contrary to our hypothesis, these effects were observed even in the absence of significant growth reduction. This indicates that the PGRs are affecting ACP fitness through plant biochemical changes, direct toxicity or some other undetermined mechanism. Results were presented at the Plant Growth Regulation Society of America (Aug 2009), the Entomological Society of America (Dec 2009) and the Western Plant Growth Regulator Society (Jan 2010). A manuscript based on the results is being prepared for submission to a scientific journal. Continuing research on this project will investigate the plant biochemical changes induced by PGR applications that are affecting psyllid fitness, field trials to replicate greenhouse results with native ACP populations, and duration of efficacy studies in both greenhouse and field trials.
From our microscopy imaging research, we discovered that excessive callose is deposited at the plasmodesmata in the phloem tissue of leaves infected Liberibacter. Callose is an important structural element of plasmodemata surrounding the desmotubules and it is well known that permeability of plasmodesmata is regulated by contolling synthesis of the callose. For instance, levels of plasmodesmata callose incresease in response to viral infection and the increase in callose reduces transport efficiency of solute through the plasmodesmata, which blocks passage of viral RNA molecules. HLB symptomatic leaves accumulate massive amount of starch and we have shown that phloem loading is inhibited in such leaves. These data suggest that the abnormal starch accumulation is caused by photosynthetate export inhibition. We examined export efficiency of HLB leaves by C14 labeling experiment. We labeled photosynthetate for 2 hrs with 14CO2 and monitored export of radioactive carbon over 4 days. Approximated 50% more radioactive carbon is retained in the HLB leaves than healthy leaves. Therefore, it is likely that callose overaccumulation in the phloem cells induced by Liberibacter leads to inhibition of carbohydrate export.
We previously cultured a small bacterium and showed it produced symptoms of Huanglongbing (HLB) under growth chamber conditions. This project has three major objectives: improving cultivation of the phloem-limited bacteria, Candidatus Liberibacter species, and confirming pathogenicity under field conditions; improving diagnostics of HLB; and providing DNA from cultured cells for genome sequencing. One mm size colonies are visible only after 10-14 days. To improve growth, we analyzed leaf petiole extracts and phloem cells dissected with a laser in cooperation with UC Davis (A. Ibanez and A. Dandekar) and Zeiss. Over 100 compounds were identified with 10 being dominant; alanine, asparagine, urea, ethanolamine, mucic acid, lauric acid, leucine, malic acid, citric acid, and sucrose. Metabolic analysis showed ethanolamine improved growth. We will continue to test new formulations of the medium. A workshop on cultivation will be held in July. To confirm pathogenicity under field conditions, trees have been inoculated in collaboration with Ft. Pierce, FL (E. Stover). Symptoms have yet to appear after six months. Plans are to inoculate additional trees. For diagnostics, we have improved extraction of HLB DNA from citrus and psyllids by combining pressure and temperature. We continue to work on real-time PCR and are aiding Bruce Cary, (Mesa Technology) in testing lateral flow strips by providing RNA of different strains of Liberibacter. Polyclonal antibodies to cultured Liberibacter cells have been developed. The IgG and IgY fractions of the sera have been purified for developing ELISA and immunofluorescence (IF) protocols for field assays for citrus and for psyllids in cooperation with V. Damsteegt. The chicken spleens have been sent to A. Danaker to prepare monoclonal antibodies. A major problem with sequencing has been growing enough cells of Liberibacter and extracting DNA. Genome sequencing of cultured cells has been completed using Illumina Solexa technology. However, results showed the sequenced bacterium had Rhizobiaceae-like 16s rDNA and did not match exactly our China1 HLB strain. A check of partial 16s rDNA sequence was done prior to submitting DNA for sequencing. The partial sequence was present in the sequenced genome and China1 but the full length 16s rDNA was not. Therefore, annotation was stopped. Plans are to repeat the sequencing making sure we have the correct DNA. Identity of third generation cells will be checked by phase contrast microscopy and IF. The extracted DNA will then be checked by real-time PCR and 16s and ITS sequences cloned to confirm bacterial identify. If sequencing is a match and the culture is pure (only ITS and 16s sequence present from the clones) and we have 2ug of DNA, we will proceed with sequencing. If not we will increase the amount of DNA using a DNA replication technique and confirm again by cloning and sequencing the 16s and ITS sequence. Unless 16s sequence evidence is an exact match, will not send for sequencing. Our plans are to sequence China1 and Florida1 strains previously showed to be pathogenic and a new strain from Mexico, if pathogenic. The latter will require 4-6 months.
The objective is to test the effect of soil applied neonicotinoid insecticds used for systemic psyllid control as inducers of SAR to determine if possible the direct effect of SAR on HLB disease progress in newly planted citrus trees subjected to psyllid mediated infection or budwood-inoculated infection. Hamlin trees were planted in May 2009 at the USDA-ARS, Picos Farm in Ft. Pierce FL and treated as follows: 1) untreated check, 2) foliar insecticide to control psyllids, 3) soil applied imidacloprid/thiamethoxam to induce SAR, 4) soil applied imidacloprid/thiamethoxam plus the foliar insecticide to induce SAR and control psyllids, 5) HLB bud graft-inoculated untreated check, 6) HLB bud graft-inoculated with soil applied imidacloprid/thiamethoxam to induce SAR. The experimental design is 50 trees per treatment in a completely randomized block design (5 blocks of 10 trees per block). Tree and insecticide treatments commenced in May and HLB budwood graft inoculations occurred in July. In December 2009 (6 months post-HLB inoculum challenge), 34 of 300 trees in the experiment are positive (11%). Most of the infected trees are located on the west side of the trial area which is proximal to the infected citrus, but few infected trees occur on the east side bordered by pine flatwoods. Based on the incidence of pysllid transmission versus graft-transmission, the gradient of infected trees from west to east is unlikely to persist given the preponderance of HLB-infected citrus and pysllids in the site. The highest number of HLB positives trees (8) is in the untreated checks with or without graft inoculation (treatments 1 and 5), followed by 6 positives in the graft-inoculated soil applied imidacloprid/thiamethoxam (Treatment 6), 5 positives in the foliar insecticide and soil applied imidacloprid/thiamethoxam (treatments 2 and 3), and 2 positives in foliar insecticide plus soil applied treatment (treatment 4). The effect of SAR induction on HLB infection progress is inconclusive because the soil applied imidacloprid/thiamethoxam control of the psyllid is causing an uncontrolled interaction with psyllid transmission. Although the best treatment to control psyllids, foliar insecticide plus soil applied (Treatment 4) has the lowest incidence of infection, this treatment is still not completely preventing pysllid infection. Hence even the most stringent psyllid control (foliar insecticdes applied every two weeks plus systemic activity) has not protected trees from transmission by infected psyllids. In a separate project on SAR-Canker control (see annual report for contract no. 72642) we have confirmed that soil applied acibenzolar-s-methyl (Actigard, Syngenta) induces SAR in the field and provides similar levels of canker control as the imidacloprid/thiamethoxam neonicotinoid insecticides but not interact with psyllid control. Henceforth, the trial will utilize soil-applied Actigard instead of imidacloprid/thiamethoxam in treatments 3,4 and 6. Two HLB-SAR experiments of similar design have been set up in Parana, Brazil in late 2009 (spring for southern hemisphere). One is HLB graft-inoculated and other proximal to an unmanaged farm with HLB. Each of these trials will utilize soil applied Actigard for the SAR treatments.
During the first year of growth under the ACPS, trees grew larger and needed less water and nutrients than under conventional cultural practices in the Auburndale Ridge experiment. Trees grown with drip OH received only 0.4x the water and 0.17x the nitrogen amounts compared to conventionally grown trees at the same planting density of 218 trees per acre. Even at the high density of 363 trees per acre, the drip OH method required only 0.67x the water and 0.28x the nitrogen of the conventionally grown trees. Canopy height and volume growth was consistently improved by ACPS, with the drip OH treatment at 363 trees per acre growing 2.74x more canopy volume than under conventional practices. Because fruit yield is strongly correlated to canopy volume, the improvement of 2.74x in canopy growth per acre bodes well for achieving an economic yield in half the time and/or twice the yield at a given age. In fact the trees in drip OH treatments have already set a crop of fruit in their second year, suggesting that juvenility traits may also disappear more quickly with ACPS. By 17.5 months age, the drip OH tree heights averaged 4.5 feet, well on their way to reaching 6 feet by year 3, which was the original objective of the ACPS experiment. It is noteworthy that only 0.8 to 1.3% of the soil surface area is wetted by the drippers in the ACPS to reduce water losses by evaporation. For comparing water or nutrient efficiencies, we divided tree canopy volume produced in year 1 by the amount of water or nitrogen applied in the same year. Efficiencies for the drip OH method were impressive, ranging from 4x more efficient for water, to nearly 10x more efficient for nitrogen, compared to the conventional practice. The microsprinkler OH method operating at a frequency of about every 2 to 3 days was intermediate in water and nitrogen efficiency (1.64x and 4.45x, respectively) at the standard 218 trees per acre density. The benefits of ACPS in conserving renewable and nonrenewable input resources in the first year are self-evident. In general, gains of efficiency in any production system leads to conservation of resources, reduction of pollution and byproducts (environmental protection), lower energy requirements from fossil fuels and other nonrenewable sources, and hence a dramatic reduction in carbon footprint. Historically we know from the citrus nitrogen BMP studies that the most important factors which regulate nitrate leaching to groundwater on the Ridge soils are the fertilizer rate and the vigor and productivity of the crop. We installed vacuum lysimeters in each of the main treatments in the Auburndale experiment to sample soil water leached to below five feet. The sampled water was analyzed for nitrate-N concentrations after major leaching events from rain or freeze protection. The results illustrate the strong reduction of nitrate concentrations in soil leachates destined for the groundwater when nutrient and water efficiencies are improved by ACPS. The lowest nitrate concentrations were measured in the drip OH lysimeters, where levels remained below 2 ppm for most of the samples except for the one taken immediately after the January freezes (5.92 ppm). In contrast for the conventional treatment nearly half of the samples were above 10 ppm nitrate-N, with a maximum of 26.5 ppm. The microsprinkler OH lysimeters showed intermediate nitrate concentrations as expected. Nitrate leaching was therefore inversely proportional to the nutrient efficiencies, which in turn were determined by fertilizer application rates and tree growth rates.
Irrigation and fertilizer practices in the Advanced Citrus Production System flatwoods site at SWFREC indicate potential for improved plant nutrition of three year old trees. Hamlin and Valencia trees were planted in 2006 at the flatwoods site at 151, 198 and 545 trees per acre on rootstocks Cleopatra mandarin (Hamlin) or Volkamer lemon (Valencia), Swingle citrumelo, and Flying Dragon; respectively. Irrigation/fertigation practices were daily drip fertigation (Drip OHS), daily irrigation and weekly fertigation with a strip shaped microsprinkler (Microsprinkler OHS) and a Grower Microsprinkler control with periodic irrigation based on soil depletion and monthly fertigation. Adequate tree nutrient status, root density in the irrigated zone and water conservation were three factors investigated in 2009. Leaf N , P and K content were similar for all treatments at the beginning of 2009 (January and March), however samples taken in June and September indicate that the Drip OHS and Microsprinkler OHS maintained leaf concentrations greater that those for the Grower Microsprinkler irrigation treatment. The exception to this is P, where no significant differences were found among treatment at any sample date. All treatments maintained average leaf nutrient concentrations in the optimum or high range. Root length density (RLD, root diameter <1mm) varied as a function of irrigation/fertilizer treatment (p<0.05), decreasing with depth (p<0.0001) and distance from the tree (p<0.0001). RLD was consistently higher for Drip OHS than the other two treatments. Prior to the start of the summer rainy season, Drip OHS significantly increased RLD by 13-90% for roots <3mm in diameter in both the irrigated and non-irrigated zones when compared with the conventional practice. After the rainy season, RLD in the Drip OHS plots were 19% and 112% higher than conventional practice in the non-irrigated and irrigated zones, respectively. These data indicate that RLD in the drip irrigation zone may not be greatly influenced by the rainy season as first speculated. Thus, irrigation methods such as drip which apply water and fertilizer frequently and in small pulses within a limited root zone offer a viable option for increasing root water and nutrient uptake efficiency compared with the microsprinkler based systems when the trees are small. Total water used was greatest for the Grower Microsprinkler treatments plots and increased with planting density ranging between 10 and 18 inches. Water use was lowest for Drip OHS irrigation treatments at low (5 in yr-1) and moderate (6 in yr-1) densities compared with the Grower Microsprinkler treatments at the same densities (10 and 14 in yr-1, respectively). Thus, overall water use efficiency for the Drip OHS and Grower Microsprinkler treatments were 0.03 and 0.07 inches per tree, respectively. However, the highest water user was the high density planting with drip irrigation (19 in.). The reason water use increased about 3 to 4 fold for the drip treatment at high tree density is that each tree had the same number of drippers and thus water use was a function of tree density (i.e. 545 trees at high density, 151 and 198 at low and moderate densities). The relationship of increased water use with increased tree density existed for the microsprinkler treatments but not at the same ratio as tree density because the emitter output and pattern sizes were selected to give similar application rates on a gal. per area basis with little overlap.
During the first year of growth under the ACPS, trees grew larger and needed less water and nutrients than under conventional cultural practices in the Auburndale Ridge experiment. Trees grown with drip OH received only 0.4x the water and 0.17x the nitrogen amounts compared to conventionally grown trees at the same planting density of 218 trees per acre. Even at the high density of 363 trees per acre, the drip OH method required only 0.67x the water and 0.28x the nitrogen of the conventionally grown trees. Canopy height and volume growth was consistently improved by ACPS, with the drip OH treatment at 363 trees per acre growing 2.74x more canopy volume than under conventional practices. Because fruit yield is strongly correlated to canopy volume, the improvement of 2.74x in canopy growth per acre bodes well for achieving an economic yield in half the time and/or twice the yield at a given age. In fact the trees in drip OH treatments have already set a crop of fruit in their second year, suggesting that juvenility traits may also disappear more quickly with ACPS. By 17.5 months age, the drip OH tree heights averaged 4.5 feet, well on their way to reaching 6 feet by year 3, which was the original objective of the ACPS experiment. It is noteworthy that only 0.8 to 1.3% of the soil surface area is wetted by the drippers in the ACPS to reduce water losses by evaporation. For comparing water or nutrient efficiencies, we divided tree canopy volume produced in year 1 by the amount of water or nitrogen applied in the same year. Efficiencies for the drip OH method were impressive, ranging from 4x more efficient for water, to nearly 10x more efficient for nitrogen, compared to the conventional practice. The microsprinkler OH method operating at a frequency of about every 2 to 3 days was intermediate in water and nitrogen efficiency (1.64x and 4.45x, respectively) at the standard 218 trees per acre density. The benefits of ACPS in conserving renewable and nonrenewable input resources in the first year are self-evident. In general, gains of efficiency in any production system leads to conservation of resources, reduction of pollution and byproducts (environmental protection), lower energy requirements from fossil fuels and other nonrenewable sources, and hence a dramatic reduction in carbon footprint. Historically we know from the citrus nitrogen BMP studies that the most important factors which regulate nitrate leaching to groundwater on the Ridge soils are the fertilizer rate and the vigor and productivity of the crop. We installed vacuum lysimeters in each of the main treatments in the Auburndale experiment to sample soil water leached to below five feet. The sampled water was analyzed for nitrate-N concentrations after major leaching events from rain or freeze protection. The results illustrate the strong reduction of nitrate concentrations in soil leachates destined for the groundwater when nutrient and water efficiencies are improved by ACPS. The lowest nitrate concentrations were measured in the drip OH lysimeters, where levels remained below 2 ppm for most of the samples except for the one taken immediately after the January freezes (5.92 ppm). In contrast for the conventional treatment nearly half of the samples were above 10 ppm nitrate-N, with a maximum of 26.5 ppm. The microsprinkler OH lysimeters showed intermediate nitrate concentrations as expected. Nitrate leaching was therefore inversely proportional to the nutrient efficiencies, which in turn were determined by fertilizer application rates and tree growth rates.
During the first year of growth under the ACPS, trees grew larger and needed less water and nutrients than under conventional cultural practices in the Auburndale Ridge experiment. Trees grown with drip OH received only 0.4x the water and 0.17x the nitrogen amounts compared to conventionally grown trees at the same planting density of 218 trees per acre. Even at the high density of 363 trees per acre, the drip OH method required only 0.67x the water and 0.28x the nitrogen of the conventionally grown trees. Canopy height and volume growth was consistently improved by ACPS, with the drip OH treatment at 363 trees per acre growing 2.74x more canopy volume than under conventional practices. Because fruit yield is strongly correlated to canopy volume, the improvement of 2.74x in canopy growth per acre bodes well for achieving an economic yield in half the time and/or twice the yield at a given age. In fact the trees in drip OH treatments have already set a crop of fruit in their second year, suggesting that juvenility traits may also disappear more quickly with ACPS. By 17.5 months age, the drip OH tree heights averaged 4.5 feet, well on their way to reaching 6 feet by year 3, which was the original objective of the ACPS experiment. It is noteworthy that only 0.8 to 1.3% of the soil surface area is wetted by the drippers in the ACPS to reduce water losses by evaporation. For comparing water or nutrient efficiencies, we divided tree canopy volume produced in year 1 by the amount of water or nitrogen applied in the same year. Efficiencies for the drip OH method were impressive, ranging from 4x more efficient for water, to nearly 10x more efficient for nitrogen, compared to the conventional practice. The microsprinkler OH method operating at a frequency of about every 2 to 3 days was intermediate in water and nitrogen efficiency (1.64x and 4.45x, respectively) at the standard 218 trees per acre density. The benefits of ACPS in conserving renewable and nonrenewable input resources in the first year are self-evident. In general, gains of efficiency in any production system leads to conservation of resources, reduction of pollution and byproducts (environmental protection), lower energy requirements from fossil fuels and other nonrenewable sources, and hence a dramatic reduction in carbon footprint. Historically we know from the citrus nitrogen BMP studies that the most important factors which regulate nitrate leaching to groundwater on the Ridge soils are the fertilizer rate and the vigor and productivity of the crop. We installed vacuum lysimeters in each of the main treatments in the Auburndale experiment to sample soil water leached to below five feet. The sampled water was analyzed for nitrate-N concentrations after major leaching events from rain or freeze protection. The results illustrate the strong reduction of nitrate concentrations in soil leachates destined for the groundwater when nutrient and water efficiencies are improved by ACPS. The lowest nitrate concentrations were measured in the drip OH lysimeters, where levels remained below 2 ppm for most of the samples except for the one taken immediately after the January freezes (5.92 ppm). In contrast for the conventional treatment nearly half of the samples were above 10 ppm nitrate-N, with a maximum of 26.5 ppm. The microsprinkler OH lysimeters showed intermediate nitrate concentrations as expected. Nitrate leaching was therefore inversely proportional to the nutrient efficiencies, which in turn were determined by fertilizer application rates and tree growth rates.
The new variable rate controller continues to perform well in the approximately 70 units currently deployed in Florida citrus groves. A newer controller prototype with color touchscreen liquid crystal display is being tested and will be the most advanced final version to be deployed in the 1.5 year project period. The elimination of all physical switches and pilot lights from the enclosure will increase the durability of the controller and make it easier to upgrade all of its functions by using virtual switches and status lights on the display. Other features in the new controller include the ability to collect and store tree canopy characteristics data measured by the sensors, for later downloading, viewing, analyzing and mapping in a GIS program. Growers will benefit from up-to-date information on tree sizes, and the locations of gaps and resets in their groves. Automatic spray buffers based on prevailing wind direction and speed could improve spray deposition on canopies while avoiding off-target drift. The accuracy and reliability of the VRT controller is our top design priority. The new controller is being designed to work with a range of different approved sensors because certain sensor technologies may work better than others in a given situation. For example the currently implemented optical sensors work well in detecting mature trees but occasionally miss a small reset tree target. The latest sealed weather-proof ultrasonic sensors could be more efficient than optical sensors in detecting small reset tree targets or partially defoliated canopies. Variable rate application technology in Florida citrus continues to evolve in order to offer the best possible permanent savings in agrochemicals, including fertilizers, by increasing application efficiency without adversely affecting product efficacy. The combined savings of comprehensive VRT precision agriculture can offset much of the increasing costs of citrus production and fits well into integrated pest management programs.
All experiments have been carried out under insect-proof conditions. After solve problems regarded to inoculum sources (Citrus and Murraya with CLas and CLam) and ACP rearing and transmission protocols, all experiments are already established and we are now waiting for symptoms appearance. Exp.1 – On the contrary of our thought, the species of Murraya we have in Brazil is not M. paniculata but M. exotica according to the description and illustration made by Huang 1959. Exp.1 began in April/08. The first diseased plants appeared in samples of October/08 for CLas from citrus source. Real time PCR of the samples collected in January/09 indicated that ACP had transmitted CLam from Murraya and from citrus inoculated with CLam to other citrus plants. Also, it was observed the predominance of diseased plants with positive conventional PCR for CLas in the two compartments where citrus plants served as primary source of inoculum of CLas and CLam. Although bacterial source plants infected with CLam showed more severe symptoms than plants infected with CLas, real-time PCR detected 14 and 5 plants with CLas in compartment 1 and 2, respectively, while no CLam infected plant was detected yet. Compartment 2, that received more sun light, had more nymphs and adults ACP than compartment 1 (19,088 against 4,033). Collected psyllids samples in compartment 1 and 2 were PCR positive only for CLas with infectivity varying each assessment from 0 to 50%.There were 8 plants with CLam (infected and symptomatic) in compartment 4, where Murraya served as source of inoculum and no plant with positive for CLam in compartment 3, where citrus served as inoculum source. The number of ACP (adults and nymphs) was higher in compartment 4 (20,357) than in compartment 3 (2,685) with higher rearing on infected Murraya than on infected Citrus). It was the first time that the transmission of CLam from M. exotica to citrus by ACP was observed and a note will be published in Journal of Plant Pathology. In December/09 we stopped to release new ACP and killed all psyllids present in the compartments. All plants were moved to other insect-proof screenhouse and will be there for symptoms observation and leaf sampling for PCR analysis. New inoculum source Citrus and Murraya plants are being prepared to repeat this experiment. At this time we want to include sources of Murraya infected with CLas. Exp. 2- The delay to start this experiment was caused because took so long to obtain inoculum source plants for CLam, but this problem was already solved. During January/10, ACP was reared on symptomatic inoculum sources for CLam and CLas. The emerging adults from nymphs reared on such inoculum sources were used for inoculation in March/10. After the inoculation, adults of ACP free of Liberibacters will be periodically feed on these inoculated plants to detect the moment when they can acquire the bacteria from inoculated plants. Exp. 3 – aims to quantify and compare the HLB symptoms progress in sweet orange cultivars (Hamlin, Pera and Valencia) infected with CLas or CLam by D. citri at different ages (planted in 1999, 2004 and 2006). Nine insect-proof screenhouses were built in a commercial citrus farm (6 in Dec/08 and 3 in Dec/09). Each screenhouse covered 15 trees and monthly assessments looking for the presence of HLB associated bacteria in each protected tree by real time PCR were done before inoculation and unfortunately in each screenhouse the CLas were detected in few trees. Plants from Hamlin and Valencia were already inoculated at the beginning of July/09 with Clas and Clam infected adults ACP and plants from Pera were not inoculated yet, because some of screenhouse suffered some wind damages. Monthly assessments for symptom severity have been done, but no HLB-symptoms were observed yet on inoculated trees. Also, leaf samples have been collected to detect the presence of Liberibacter species on inoculated shoot. To accelerate the results of this experiment, new trees showing very low symptoms severity will be selected in farms without removal of symptomatic trees but with strong ACP control program.
During this first year data were collected from this field experiment (Exp.1) and its results have been compared with the results of other similar experiment (Exp. 2). While Exp.1 have been carried out in a region with high external inoculum pressure because of its proximity with groves without HLB management, Exp.2 is in a region with lower external inoculum pressure, located in the middle of a large farm with regional disease management. In Exp.1 the disease epidemics started 13 months after planting, while in Exp.2 it started later, 22 months after planting. Also in Exp.1 the cumulative incidence of HLB-symptomatic trees and cumulative number of caught adults ACP in plots without vector control was 72.5% and 319, respectively, after 48 months after planting, against 4.2% and 99, respectively, for Exp.2. This preliminary results showed the great effect of regional management of HLB (In the case of Exp.2, at least 4 inspections/removal of symptomatic trees and 6-8 insecticide sprays per year in the area up to 2 km surrounded the experimental area). Differences on the time that a symptomatic tree remained exposed in the field before be eliminated were observed among different frequencies of local inoculum reduction. However, such differences did not result in significant differences on HLB progress rate and HLB incidence 48 months after planting among different frequencies of local inoculum reduction in each experiment. The tested program of ACP in Exp.1 has been efficient to reduce the number of adult psyllids captured on yellow stick traps in 81% and to reduce the number of eggs and nymphs observed on new shoots in 95%. However this vector control reduced HLB-incidence in only 40%. The disease progress rate (estimated by Gompertz model) in plots with ACP control program was significantly reduced in 27% compared to the disease progress rate in plots without ACP control (Average Gompertz rate of 1.35 for plots without vector control and 0.99 for plots with vector control). From December/09 to May/10 the disease incidence increased from 28.9% to 43.8% in plots with ACP control program and from 61.8% to 72.5% in plots without vector control. For Exp.1, all psyllids captured on yellow stick traps from the winter/07 to winter/09 were tested for the presence of Candidatus Liberibacter spp. by conventional PCR. PCR-positive ACPs have been observed during spring/07 (1.8% of tested samples), summer/08 (2.1%), spring/08 (4.5%), summer/09 (10.2%), fall/09 (48.1%), and winter/09 (28.6). Infective ACPs were found both in plots with and without vector control program, respectively 6.3 and 7.5%. Samples from spring/09 to fall/10 are being processed. However, no psyllid sample was positive for Candidatus Liberibacter spp. in Exp.2. This result could explain why even with similar population of adults ACP in plots with vector control, the incidence of HLB-symptomatic trees were much lower in vector controlled plots of Exp.2 (3.1% in Exp.2 and 43.8% in Exp.1). Annual maps of HLB-symptomatic trees were prepared for spatial analysis using stochastic models (MCMC) to verify the effects of each treatment on primary and secondary spread of HLB. This analysis is being done at USDA lab in Fort Pierce by Gottwald’s team. So far, it could be comment that recommended HLB management for new groves is much effective under low pressure of external inoculum and that only local disease management could be not enough to control HLB in a region with high external inoculum pressure, as is the case of Exp.1. The assessments on both experiments will continue at least for one more year to allow more detailed temporal and spatial analysis, as well economical analysis, and get final conclusions. These and others preliminary results were presented in four seminars given in CREC, Florida Citrus Show, Syngenta Experimental Station, and Florida Natural’s in the last week of January/10. Also, they were present in three Brazilian events: 3rd Huanglongbing Day (March/10), 2nd National Conference on Agriculture Protection (May/10), and 32nd Citrus Week (June/10).
The objectives of these studies were to determine the appropriate rates of Remedy Ultra (triclopyr) to apply to citrus stumps and if delaying herbicide application would have an impact on subsequent sprout development. Two studies have been conducted during 2008-10 to determine appropriate rates and timing of applications to control citrus stump sprouting. The first study examined various application rates (25, 50 and 75% Remedy mixed with diesel) consisting of 15 replications per treatment. The second study looked at the impact of delaying application of the herbicide on the control of sprout formation and utilized 7 replications per treatment. Studies were conducted using a randomized complete block design in groves near Lake Placid, a typical ridge site and Arcadia, a typical flatwoods site. Stumps were treated with a spray volume that consisted of approximately 1 to 2 fluid ounces of spray mixture per stump applied with a compression pump sprayer. Larger stumps received more spray volume than smaller stumps. Herbicide applications were direct the to the outer stump area to completely wet the cambium area while minimizing runoff to the soil. In all studies, observations were made on 30-day intervals over an 8-month period after clipping or shearing the tree as the removal method. During the first year of the study in the grove near Arcadia, 12 of the 15 (80%) untreated stumps sprouted. Sprouts were noted over the study period with 0, 5, 3, 1 and 3 of the 15 stumps sprouting at 29, 56, 85, 113 and 141 days after tree removal, respectively. The number of sprouts per untreated stump ranged from 2 to 26 and averaged 8 per stump at the end of the study. Ten stumps with sufficient sprouts were tested for HLB and 80% had one or more sprout testing positive. The percentage of HLB positive sprouts recovered from a positive stump ranged from 33% to 100%. All treated stumps, regardless of the treatment rate remained sprout free during the study period. During the second year, studies were conducted at Lake Placid and Arcadia. Treatments of Remedy Ultra were applied in a 25 or 50% solution mixed with diesel fuel at time of clipping, 24, 48 and 72 hours later. At the Lake Placid site, 2, 2, 1, 1, and 1 of the 7 (100%) untreated stumps sprouted at 30, 60, 90, 120 and 150 days after tree removal, respectively. At this site, 4 stumps within the 56 treated stumps had root sprouts but not on the treated stump. All of the root sprouts occurred on trees originally grown on rough lemon rootstock, whereas, none of the other rootstocks sprouted at this site. Remedy Ultra was slightly less effective in controlling sprouts when applied at the 25% Remedy/75% diesel than at the 50% rate, especially on rough lemon stumps. Delaying application using the lower rate also had a slight negative impact on sprout control. At the Arcadia site, 5 of the 7 untreated stumps sprouted at 60 days after removal with one additional stump sprouting by 90 days. At this site, 86% of the untreated stumps sprouted within 90 days of clipping. In addition to the untreated stumps, one additional stump developed sprouts which had been treated with a 50% spray solution at time of clipping. Only one stump exhibited root sprouts which occurred within 60 days after treatment regardless of the time of application of the herbicide. The number of sprouts per untreated stump ranged from 1 to 8. In this experiment, rootstock and trunk diameter had an impact on root and/or stump sprouting. Trees with larger trunk diameter supports greater incidence of sprouting compared to smaller trunks. It has been previously reported that rough lemon and Cleopatra mandarin are more prone to sprouting than other rootstocks. In these studies, no problems were associated with Remedy Ultra damaging adjacent trees. Remedy Ultra was effective in controlling citrus sprouts at 25% Remedy mixed with diesel fuel with larger stumps of rough lemon being more difficult to control sprouting.
Narrative Experiments ‘ From February to June 60 plants of sweet orange Hamlin were grafted into Rangpur lime and established at screen house. After reached 30 cm tall, they were grafted with budwoods infected with Ca. Liberibacter americanus (CLam) and budwoods infected with Ca. Liberibacter asiaticus (CLas). All budwoods were checked by conventional PCR and RT-qPCR by the presence of the bacteria. Healthy plants are the negative control. After the plants were confirmed bacteria positive (RT-qPCR) they were pruned and transferred to a growth chamber at 22 to 24 oC and photoperiod of 16/8 hours. Approximately 40 days after pruning 15 cm branches were were collected. Leaves and barks were separately grinding in liquid nitrogen. Concentration and integrity of total RNA were evaluated before hybridization experiments in microarray plataform (Roche Nimblegen). Hybridization experiments – The RNA samples were sent to Roche NimbleGen Systems, where cDNA synthesis and Cy3 labeling was performed. Hybridization, scanning, and image analysis of the arrays were performed according to the manufacturer’s recommendations. The oligo-array includes 32,000 genes of sweet orange with six replicas of each one with density of 340,000 spots. Preliminary results were presented in the previous report. Next steps ‘ Array hybridizations with plants infected with Ca. L. asiaticus, but symptomless. Array experiments with symptomatic plants infected with Ca. L. asiaticus and plants infected with Ca. L. americanus. Statistical analysis of the global gene expression experiments. Primers design for specific gene expression during the infection with both bacteria. First draft of the manuscript for publication. Expected results and practical application The main expected result is the knowledge on the gene expression pattern in sweet orange during the process of infection by Candidatus Liberibacter spp. Such knowledge will enlarge our understanding on the basic aspects of the disease and may be help to approach new technologies for control, especially technology based on gene silencing or gene over expression. Revised Budget for the Second Year The last part (15 %) of the approved budget will be used for the payment of the temporary Post doctor working in the project. 10 % will to be deducted for the Department, according with the contract (Considerations 6.2).
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