The goal of this project is to obtain the in vitro culture of the bacteria – Ca. Liberibacter asiaticus (LAS)- associated with the Citrus greening syndrome. The strategy consists in getting primo-cultures of the bacteria in insect cells cultures used as feeder cells. We fulfilled objectives 1 by setting up conditions of LAS inoculation in insect cell cultures. Objective 2 consisted in obtaining primo-cultures of LAS. Among various insect cell cultures in various culture media tested, two drosophila cell lines (D-mel2, DS2) and a mosquito cell line (C6/36) were selected. Those cell lines show a detection of LAS after at least 3 transfers, the minimum number of transfers after which we consider them as primo-cultures. Detection: LAS presence is detected by direct PCR. We designed new primers to avoid non specific amplification that occurred with A2/J5 (LAS .-ribosomal protein operon) and OI1/OI2c (LAS 16S rDNA), commonly used LAS specific primers. Those non-specific detections tended to occur when working with high insect cell density or when a close bacterium was present (ie: Bradyrhizobium). We had similar difficulties to quantify LAS in insect cell cultures with previously described qPCR conditions. LAS bacteria could be detected by qPCR but using the primers HLBr/HLBas/HLB probe lead to non specific signals in our cell culture conditions. New primers were designed, giving more reproductible and reliable LAS detections in our cell cultures. We are currently adjusting qPCR conditions to our material. We periodically sequence the PCR fragments obtained to validate the presence of LAS. For objective 2, We continue to test new insect cell lines. The Keyhani’s lab recently sent us Diaphorina citri cell lines. As soon as the lines are maintained and stable, we will test our inoculation protocol on D. citri cells to obtain LAS/ D. citri co-cultures. Our objectives 3 and 4 are aiming to improve culture conditions to get a higher bacterial titer and to clear the insect cells from the primo-cultures (axenization). Objective 3: Axenization. Some of our LAS/Aedes or Drosophila primo-cultures were obtained with high insect cell concentrations. In order to axenize those primo-cultures, we tried to progressively dilute the concentration of insect cells through each new passage. However, we lost LAS detection due to faster growth rate of the insect cells, meaning by diluting the insect cells, we diluted even more the bacteria cells. We are currently testing new culture medium and conditions to contain the insect cell growth itself and we are analyzing new LAS inoculations in these cell cultures. Objective 4: Medium optimization. To maintain the bacteria for a longer period of time, over more transfers and to reach higher bacterial concentrations, we started complementing the primo-cultures with various sugars, vitamins described in citrus/periwinkle phloem. In parallel we analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to define limiting factors and/or growth inhibitors. We are monitoring several LAS positive co-cultures with different additive to improve LAS concentration and decrease the insect cell ratio. Some of these cultures are positive after more than 10 transfers are we are continuously refining our culture media with new additives.
This is the first progress report for the second year of this study. No experiments have been conducted yet for the second season as this work is on fresh citrus and those experiments will begin with the fresh season in the fall (Sept. and Oct.). To summarize results from the first season: Between Oct. 5th and 30th, five experiments were conducted that included Fallglo (1 time), sunburst (2 times), red grapefruit (4 times), and navel oranges (1 time). Fruit were treated on a commercial packingline (3 experiments) or on the Indian River Research and Education Center research line (2 experiments). Treatments included 1) full wash (brush bed + high-pressure wash) + waxing (carnauba), 2) full wash, 3) brush bed only, 4) brush bed with brushes rotating half normal speed, 5) high-pressure wash (HPW) only, 6) HPW for 10 seconds, 7) HPW for 5 seconds, 8) running fruit only over PVC rollers, and 9) a control (not washed or waxed). On the commercial line, fruit remained on the brush bed for ~ 1 min. 10 seconds, and on the full HPW for ~35 seconds. Normal brush rotation speed was ~100 rpm. Fruit were also evaluated for how surface dirt obscured the ability to grade the fruit for canker and other grade defects. Fruit from all treatments were degreened under simulated commercial conditions (5 ppm ethylene, 85F, 95% RH) and color development and weight loss measured almost daily. Fruit were subsequently stored and evaluated for the development of decay and disorders during storage under ambient conditions (~70-75F). In general, all very early season Fallglo fruit were relatively clean and did not need washing for adequate grading. This changed somewhat by the end of October when grapefruit that received more extensive washing (i.e., full washing) was significantly easier to grade compared to unwashed fruit. HPW produced fruit with intermediate gradeability. However, even minimally washed fruit were sufficient for adequate grading. These experiments will be repeated this season to determine variability in initial fruit cleanliness from year to year. Washing and waxing the fruit gave the greatest inhibition of degreening, almost stopping color development completely. Compared to preliminary results in 2008, results again showed that full washing of fruit on both the brush washer and HPW, or washing on the brush bed along inhibited degreening significantly more than did washing fruit only as they passed over the HPW. Fruit that were not brushed at all, but only passed over rollers experience a slight, but significant delay in color development compared to the control, but the delay was relatively minor compared to the other washing treatments. As the season progresses, fruit exterior surfaces become more soiled with dirt and sooty mold that makes grading more difficult without washing. Experiments were conducted in July 2010 using late-season red grapefruit, harvested from a block with citrus canker to test how well the different washing methods allowed graders to detect canker and other peel blemishes. Unwashed fruit were run past commercial graders and the number of fruit with any canker or surface defects that would justify elimination based on export grade standards were counted. These fruit were then taken to a commercial packinghouse and either left un-washed (control), or washed over 1) the entire line (brush + HPW), 2) the brush line only, or 3) HPW only. Fruit were then evaluated by commercial graders again. Data is still being fully analyzed from these experiments, but it appears that commercial graders were able to detect canker lesions and other surface defects from even unwashed fruit.
The goal of this proposal is to investigate whether Ca. Las is transmitted between infected and uninfected ACP adults in a sex-related manner to better understand the mechanisms by which disease is spread in field. We carried out a series of experiments to evaluate if Ca. Las is transmitted from male to female psyllids during routine mating. Our preliminary investigations indicated that Ca. Las may be transmitted from male to female psyllids but not from females to males or among psyllids of the same sex. Pairs of Ca. Las infected male and healthy female, Ca. Las infected female and healthy male, Ca. Las infected female and healthy female, Ca. Las infected male and healthy male, Ca. Las infected female and Ca. Las infected male (positive control) healthy female and healthy male (negative control) adult psyllids were introduced separately in Petri dishes filled with agar medium. The insects were allowed to mate for 72 hrs. After 72 hrs, the insects were transferred to Ca. Las resistant Murraya koenigii plants for 12-14 days for multiplication of bacteria in recipient psyllids. DNA was prepared from each of the female and male psyllids separately and analyzed for Ca. Las presence utilizing a real time PCR assays. Our results indicated that Ca. Las was transmitted from Ca. Las infected male psyllids to roughly 15% of healthy females. Ca. Las was transmitted from Ca. Las infected males to healthy females but not from Ca. Las infected females to healthy males or among insects of the same sex. The females that acquired Ca. Las from males during mating were also able to transmit the bacteria transovarially. We were unable to detect transmission of Ca. Las when the recipient insects were analyzed for Ca. Las DNA after 72 hrs of mating. Similarly, no Ca. Las was detected in recipient insects when the recipient insects were maintained on M. koenigii plants for longer than 4 weeks. Currently, we are investigating for the presence of bacteria in genital organs of male and female psyllids with scanning and transmission electron microscopy to characterize the pathway of Ca. Las transmission from male to female psyllids. We also continue to evaluate if the Ca. Las recipient females are capable of infecting new citrus plants and to quantify the HLB transmission rate through sexual transmission.
DNA bar coding was initiated by developing and optimizing a pair of primers that amplify 1500 bp of the mitochondria COXI psyllid gene. A nested primer pair was designed to be specific to the Asian citrus COXI gene, and validated. FL field collections were obtained from cooperators that represent 2006 and onward. PCR amplification and sequencing of field collections is underway. We optimized qPCR to detect Ca. Liberibacter presence (or absence) in the psyllid colony cohorts over different AAPs, for immatures and adults (Roberts, SWFREC; Brown lab UA). Ca. Liberibacter-infected Asian citrus psyllids are now routinely maintained at SWFREC, and the potato psyllid study system is in place in AZ to explore as a more tractable system for optimizing parameters under study in Obj 3, 4 e.g. localization and time course feeding for FISH, SEM/TEM. Our FISH probes against Liberibacter support our earlier suggestion that the pathogen exists in multiple locations in the abdomen of D. citri. Distinguishing them from a potentially broader, extra-bacteriosome flora requires the bringing together of numerous visualization techniques. After fixing specimens in 2 different ways, coagulative and noncoagulative, 4′ paraffin through-sections of the same insect are mounted for three different techniques to give the best bouquet of results ‘ ISH/FISH, haematoxylin/eosin and SEM. ISH indicates areas of infection, H&E helps with anatomical interpretation, and SEM shows gross bacterial morphology as well as any possible mix of flora. These techniques are compared with SEM of exposed organs and/or extirpated organs of all life cycle stages, including the pharate adult and the teneral. These results are compared with ISH using a 10nm colloidal gold-labeled probe and TEM. TEM allows for ultrastructure of labeled bacteria. SEM of extirpated organs of infected potato psyllid has shown that one component of the flora multiplies inside and outside the gut of young adults to the extent that it overwhelms old adults and completely replaces epithelial cytoplasm. Such a scavenging species has not been found yet in uninfected psyllids. First views of the external salivary gland surface from these old adults do not show such tissue scavenging. The condition of the salivary gland internally is being investigated using SEM. The oral box, 0.06mm3 in size, delimited by the tentoria and their arms, is an extremely complex conglomerate of esophageal, pharyngeal, salivary ducal and stylet tissues that converge to allow passage of ingested, egested and secreted materials. Because of the potential ability of bacteria to proliferate in the hemocoel, and burrow through epithelia, this box has been given considerable attention, along with other, well defined, models of transmission through the gut and salivary glands, taking the study of their organization well beyond classical groundwork laid down using other model Hemiptera.
Objective: Determine if Carrizo rootstocks, either wild type or over-expressing the Arabidopsis NPR1 gene (with an enhanced, inducible defense response) have any effect on gene expression and/or the defense response of wild type (non transgenic) grapefruit scions to HLB. Some transgenic ‘Carrizo’ citrange lines (lines 854, 857, 859 and 884) transformed with the AtNPR1 were produced in Year 1 of this project. In this quarter we were able to start to propagate new transgenic lines from cuttings: 757, 761, 763, 775, 854, 857, 890, 896 and 897, all transformed with the AtNPR1 (the plants were now large enough to propagate). We have also identified sequences for several additional citrus genes that are associated with SAR, including AZI1, BLI, CHI, R13032, R20540, RAR1 and SGT1. These genes were preciously undescribed for citrus, however our microarray studies indicated that these sequences were differentially regulated by chemical and pathogen treatment. R13032 and R20540 belong to the NPR1/NPR3 family of genes in citrus and our experiments show they are all differentially expressed during SAR. Objective 1 of this project proposed to compare the response of AtNPR1 transgenic plants vs. wild type plants to the treatment of the SAR inducer salicylic acid (SA). This has been done with the first set of transgenic lines but we wish to repeat the experiment when the new plants have been propagated so we have more replications.
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.
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