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).
During the first year we have made progress in developing the non radioactive digexigenin labelled probes for the detaection of Candidatus Liberibacter asiaticus in citrus tissues and psyllid vector. We used both digoxigenin-labeled Plabeled PCR probes as well as RNA probes. we produced digoxigenin-labeled PCR products using specific primers for the OMP, RNA polymerase beta subunit, DNA polymerase region, the r-DNA region, and the 23S and 16S ribosomal RNA intergenic regions. initially dot blot hybridizations were carried out using the known amount of HLB specific DNA from the infected plants. A linear relationship was observed with amount of input DNA and the degree of hybridization. Citrus plants from the green house infected with HLB pathogen were used as the source plants for obtaining the needed tissue for the blots and to isolate nucleic acid template necessary for the amplification of CLas specific DNA. Primers were designed based on the CLas sequence information and amplified specific amplicons from the DNA isolated from HLB infected plants, and amplification was not observed from the healthy plants. In the initial tissue blot experiments we did not observe hybridization signals specific for HLB. We used healthy plants grown under similar conditions as controls. The reason probably is the low titer of pathogen and/or the non uniform distribution of the pathogen in the infected tissues. It is also possible that the PCR probes were not of sufficient specific activity so as to detect low titer of the pathogen. Therefore, the amplified regions of Las were also cloned in the transcription vector, and digoxigenin labeled strand specific RNA probes were generated by transcription. However, use of the high specific activity RNA probes did not improve hybridization of the tissue blot and occasionally non specific hybridization was also observed in healthy tissue blots. This suggested less than optimal hybridization regimen. We have subsequently focused on optimizing the hybridization conditions and use of multiple probes in an effort to increase the extent of signal. These changes have substantially improved detection of Las in citrus tissues. In addition to tissue blots of the stem sections, we have used the midrib and petiole region imprints from the leaves of infected citrus on nylon membranes. The hybridization observed with the midrib imprints showed much clear signals compared to stem imprints. In forthcoming experiments we will use imprints of the leaf midrib, petiole and stem from new flushes of infected citrus since the psyllids preferentially feed and acquire the pathogen from such tissues. We also imprint on the membrane the inside surface of the bark which contains phloem tissue in which the HLB pathogen is located. Although this procedure is not optimized, we believe this to be very useful since the distribution of Las in infected tissue could be easily documented. The second area of our focus is on the detection of Las in psyllid vector by tissue blots (squash blot) on nylon membranes. At the outset, a procedure for isolating the nucleic acid from single psyllid was optimized, and we have been able to amplify CLas specific amplicons from single infected psyllids using pairs of Las specific primers. Conditions of amplifications were optimized with different primer pairs and now we have been able to amplify HLB specific amplicons without non-specific bands in PCR. In initial studies of whole psyllid tissue blots, hybridization signal was also observed with healthy psyllids (psyllid colony from the healthy psyllid containment facility). However use of specific primer pair corresponding to the EFTU gene of CLas has been promising and we will use the probe generated for this gene in tissue blots of psyllids henceforth. The observed background was probably due to the extraneous tissues of the psyllids like wings that attach to the nylon membranes during psyllid squash and very hard to exclude during membrane wash. Therefore we intend to separate the head and the abdomen regions of the psyllids in tissue blots and exclude the wings region to reduce the background signal.
The project has two specific aims. We outlined below the progress made for each of them. Specific Aim 1: Identify sweet orange responses to Huanglongbing disease (HLB) through deep transcriptome profiling using new DNA sequencing technologies. We have performed the bioinformatic analysis of the deep sequencing runs (Illumina Genome Analyzer II, read lengths up to 85 bp pair-ended) for the four types of fruit samples of the experimental design: symptomatic, asymptomatic and apparently healthy from an infected orchard and healthy plants from a disease-free location. We have also completed the deep sequencing runs for the same samples of mature and young leaves: 32-36 million reads were obtained for the immature leaves (95-97% high quality reads after quality trimming) and 32-34 million reads (96-97% high quality reads) obtained for the mature leaves. The bioinformatic analysis of these data is ongoing. In regards with the immature fruit set, the RNA have been extracted and cDNA libraries have been constructed. The runs for these samples are scheduled to take place in the next weeks. For the validation of the mature fruit transcriptome analysis, Taqman Real Time assays have been performed for genes involved in important differentially regulated pathways such as hormone signaling and biosynthesis (gibberellin, ethylene, cytokinin), photosynthesis, transcription factors, terpenoid metabolism. Other assays have been designed to validate the transcriptome results of the mature fruits and related to energy metabolism, hormone, sugar and starch metabolism. In anticipation of the leaf analysis we have initiated the designed of 13 Taqman Real Time assays based on previously published leaf responses to HLB obtained with microarrays. Other Taqman assays will be designed once the leaf samples will be analyzed to validate the transcriptome profile obtained by the deep sequencing. Specific Aim 2: Define and validate gene networks and identify host (sweet orange) response biomarkers regulated by HLB at different stages of infection. We have validated more than 10 Taqman Real Time assays for early HLB-regulated genes developed as biomarkers to distinguish infections at asymptomatic stage in the fruit. These genes belong to sucrose metabolism, photosynthesis, cell wall, lipid, volatile pathways, gibberellin signaling, ethylene response, transcription factors. These biomarkers provided a valuable insight into fruit peel responses to HLB. A significant number of regulated genes in the symptomatic fruits were involved in transport functions. An upregulation of light reactions was observed with the corresponding increased regulation of sucrose and starch metabolism. Genes encoding heat shock proteins appear to be major hubs in a protein-protein network analysis we conducted. The downregulation of these genes would stimulate protein misfolding processes and disrupt source-sink communications. Pathways encoding plant growth regulators like gibberellin and cytokinin were downregulated, whereas ethylene, jasmonate and salicylic acid metabolism and signaling were induced and these could affect fruit metabolism. WRKY, AP2-EREBP and bZIP transcription factors were highly regulated at different stages of disease. Several receptor kinase genes (LRR protein) seemed to be involved in reception of the signal triggering the host response. While cell cycle and organization were downregulated, protein modifications and RNA processing were induced in symptomatic fruits. Interestingly, genes involved in ATP synthesis were upregulated in mature fruits and agreed with previous findings of the presence of an ATP translocase gene in the pathogen genome (Duan et al., 2009). Taqman assays have been designed and analysis are on going to confirm these preliminary results.
This report covers the period January 1, 2010 through March 31, 2010. This project was funded July 1, 2009. Seven coordinating meeting were held during this period. The field personnel, in particular, closely coordinated their activities with the personnel analyzing data. Coordination with the aerial application program through Southeastern Air and the Indian River Citrus league continued as did the mapping of data for subsequent time and space analyses of Asian citrus psyllid populations. Due to the massive amount of data being collected in the field, four Garmin GPS units and five laptop computers were purchased to enable higher staff productivity and more efficient data handling. The extensive trapping program carried out the following activities. 1. Total trees surveyed: Indian River County–2,460; St Lucie County–2, 700; Martin County–900. 2. Traps by county: Indian River 205; St. Lucie, 225; Martin 72. 3. Total traps set and Retrieved: 6,060. 4. Total Psyllids caught: 3,013. By county: Indian River–1,690; St. Lucie 729; Martin 594. To give some perspective on the size of this program 17,000 miles were driven by field personnel during this period. The data analyses from the first aerial application were inconclusive. It was the judgement of the participants that the data from the second application will allow a conclusion as to the efficacy of the aerial program. The data from the second aerial application are now being analyzed. These analyses will be available for the next quarterly report.
Salicylic acid (SA) is the key defense signaling molecule in plants. Increases in SA levels and/signaling lead to enhanced disease resistance against viral, bacterial, and fungal pathogens in many plants. In order to introduce broad resistance in citrus against HLB, citrus canker, and other diseases, we proposed in this study to identify SA-related genes in citrus and manipulate the expression of these genes genetically to elevate SA levels and/or signaling in citrus. Towards this goal, we have three specific objectives in the proposal and have made the following progress with the first-year funding. We believe that this progress has met the expectations in our proposal. Objective 1: Identify genes positively regulating SA-mediated defense in citrus Bioinformatics analysis of the HarvEST:Citrus database and citrus genome database available in the Joint Genome Institute revealed that citrus and the model plant Arabidopsis share significant similarity in their genomic sequences. Thus, most SA regulators that have been characterized in Arabidopsis also exist in citrus. The availability of EST and genome sequences of citrus genes makes it easy for us to clone citrus SA homologs. So far using the RT-PCR approach, we have obtained full-length cDNA sequences for four citrus SA genes, ctNPR1, ctEDS5, ctNDR1 and ctPAD4. We are also in the middle of cloning another four citrus SA genes, ctEDS1, ctSID2, ctALD1, and ctWIN3. Homologs of these SA genes are known to play critical roles in Arabidopsis defense. To provide a more detailed analysis of expression of citrus SA regulators, we collected citrus tissues infected with Ca. L. asiaticus at several time points for RNA extraction. Quantitative RT-PCR will be performed to study expression of these citrus SA genes and other important defense genes in citrus afflicted with the HLB disease. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues We cloned ctNPR1, ctEDS5, ctNDR1 and ctPAD4 to the binary vector pBINplus/ARS and transformed corresponding Arabidopsis mutants and wild type with each of these constructs. We are in the process of selecting T1 Arabidopsis transformants expressing citrus SA homologs and testing some T2 transgenic plants for disease resistance. So far, preliminary data obtained from Arabidopsis expressing ctNDR1 are promising. The Arabidopsis ndr1-1 mutant was previously shown to lack a hypersensitive response and have enhanced disease susceptibility with infection of the Pseudomonas syringae avrRpt2 strain. Overexpressing ctNDR1 in the ndr1-1 mutant rescued these phenotypes conferred by ndr1-1, suggesting that Arabidopsis produces a functional ctNDR1. Summary for other constructs: Plants expressing CtNPR1/pBINplusARS did not show a drastic change of disease resistance with P. syringae infection. We will isolate homozygous lines and plant them side by side with WT and the npr1-1 mutant for a resistance test in the next generation. For plants expressing ctPAD4/pBINplusARS, we obtained T2 seeds and will infect the T2 plants soon with P. syringae. For the ctEDS5/pBINplusARS construct, we were unable to obtain any transgenic lines. This is possibly due to the toxicity of overexpressing ctEDS5 and/or the failure of transformation. We will repeat the transformation process to rule out the latter possibility. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in Citrus Plants of the citrus rootstock US-812 have been transformed with ctNPR1/pBINplusARS and are being grown in the greenhouse in Ft. Pierce in preparation for testing with HLB and citrus canker. The construct ctNDR1/pBINplusARS is in the pipeline for citrus transformation in the next two months, and transgenic citrus plants generated will also be tested with the HLB disease later in the year.
The new microscopes with their superior optics and ergonomic designs have increased productivity. The Leica teaching microscope is used more frequently than expected as each variety that is cleaned up looks a little different. Teaching is a long term and on-going process. By frequent examination of each developing STG, the reasons for success or lack thereof can be determined. This is knowledge is applied to improve subsequent STGing. The new spectrophotometer, a Nanodrop 8000, is used to quantify nucleic acid (NA) content of all samples. An excel spreadsheet with formulas is used to determine what quantity of water needs to be added, if any, to standardize the NA content. Each sample then has the requisite amount of water added to dilute it to the pre-determined concentration in the master plate. Plates for all assays are drawn from this master plate so that the precise amount of NA determined to be optimal for that set of primers is used. Chiefland Foundation source trees were tested for Citrus tatter leaf virus (CTLV) for the first time during the 2009 testing season. Samples were adjusted to the proper titer using the Nanodrop 8000. A SYBR green real-time PCR assay showed that three trees of one little-used variety had CTLV. Several nearby trees had been infected by root grafting. Implementation of this important testing allowed us to immediately remove the diseased trees for STGing and has increased the disease-free state of this valuable resource. During the 2010 testing season we will determine if all CTLV has been removed from the Chiefland Foundation trees. The list of people and the varieties they want to enter into the budwood parent tree indexing and shoot-tip grafting program continues to be long as does the list of varieties that we are working to clean up for the Chiefland Foundation, Florida citrus breeders, and citrus producers. However the improvements to the program are allowing us to get shoot-tip grafted and tested material back to the owner in less than two years in most cases. With improved testing, the Florida citrus industry is provided with budwood tested repeatedly for Citrus greening, Citrus tristeza virus, Citrus leaf blotch virus, Citrus tatter leaf virus (Apple-stem grooving virus), and six viroids. Our Budwood program remains strong and well provided with up-to-date equipment to provide these services thanks to the support of the Florida Citrus Production Research Advisory Council.
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 producing fruit with intermediate gradeability. However, even minimally washed fruit were sufficient for adequate grading. These experiments need to be repeated next 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 are currently underway using late-season red grapefruit, harvested from a block with citrus canker. 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 collected and analyzed from these experiments.
Research results from the first (2007-08) season were successfully completed, the results reported at the Florida State Horticultural Society (FSHS) meetings and at Packinghouse Day and the Indian River Postharvest Workshop in Sept. 2008, a manuscript published in the FSHS proceedings (http://www.fshs.org/; Proc. Fla. State Hort. Soc. 2008. 121:322’325), and subsequent results reported in FCPRAC progress reports. To summarize all experiments and results, preharvest treatments included 1) foliar K (8lb K2O), 2) foliar Mg (6% solution), 3) foliar K + Mg, 4) foliar Vapor Gard’ (1% or 2%), and 5) induced tree water stress by discontinuing irrigation and covering the ground with large plastic sheets. Treatments were conducted in commercial groves on White grapefruit three times, Flame red grapefruit twice, and Valencia oranges once. Fruit were harvested at various times after foliar applications (sometimes weekly), held for 3 to 4 days at ~70F with ~60% RH, washed & sometimes waxed (carnauba), and stored under ambient conditions. Fruit were evaluated at regular intervals for peel breakdown and decay. While foliar treatments did not always significantly reduce peel breakdown, even when the differences were not significant, such applications tended to reduce peel breakdown. In general, foliar K or Mg treatments significantly reduced peel breakdown once each. In one experiment, foliar K actually increased peel breakdown, but the reason for this is unclear. However, a combination of foliar K + Mg or use of an antitranspirant (Vapor Gard’) significantly reduced postharvest peel breakdown more than the control or K or Mg alone. Blocking irrigation and rainfall for 49 days before harvest increased peel breakdown in the first year. In the second year, while differences in peel breakdown were not significantly different, trees under water stress tended to develop twice as much peel breakdown compared to the control. In postharvest experiments, fruit were harvested, held for 3 to 4 days, washed and waxed (carnauba) on a research packingline at the IRREC, and then stored at ambient conditions. Postharvest treatments including 1) holding the fruit for 3 or 4 days after harvest at 30%, 60%, or 100% RH at ~70F, 2) including a fungicide (TBZ or Imazalil) in the wax, 3) not waxing the fruit, or 4) washing, but not rinsing the detergent from the fruit before storage. Exposing fruit to low (30%) RH after harvest dramatically increased peel breakdown and often significantly increased postharvest decay as well. Exposure to intermediate RH occasionally increased peel breakdown compared to the control (100% RH), but results were not consistent and sometimes were not significantly different from the control. Not waxing fruit or rinsing detergent from the fruit usually had no effect on peel breakdown. Interestingly, postharvest treatments with Imazalil occasionally reduced peel breakdown. The reason for this is unclear but worth further investigation.
Funding for this project has enabled us to develop a comprehensive program for ACP management. We are building programs targeting ACP adults using dormant sprays of broad-spectrum insecticides and treatments during the growing season based on scouting. Dormant sprays have evolved into a successful annual area-wide cooperative effort in SW Florida. For the growing season, we have developed a wide range of choices such as (1) recurrent low-volume applications of horticultural oil, (2) wide selection of tested selective and broad spectrum insecticides, and (3) strategies to conserve and augment biological control. A survey of local managers covering 106,148 acres of commercial citrus in SW Florida showed widespread adoption of these management strategies. Our first objective was to assist growers to plan and implement effective strategies for ACP control. All growers surveyed used dormant sprays during the 2008-2009 dormant season in cooperation with a voluntary area-wide program initiated and promoted in cooperation with Gulf Citrus Growers Association and Cooperative Extension. 71,916 acres were sprayed by air in 08/09 and 73,180 in the 09/10 seasons with an additional estimated 30,000 sprayed by ground, virtually all the present commercial acreage in SW Florida. Field counts obtained from DPI-CHRP and growers’ personnel trained by us indicated that populations by air were only 1.5X winter numbers three months into the growing season compared to a 28X increase in untreated blocks. Results of the post application survey will be evaluated this May. Our 2nd objective was to assist in development of efficient monitoring methods. The “tap” sampling method we developed has proved to be an effective and efficient tool for monitoring ACP adults, as well as other pests and beneficial insects. We integrated this and a technique for assessing immature stages into a rapid and easy to use system promoted in Citrus Industry Magazine, fully explained on our website http://swfrec.ifas.ufl.edu/ and used by 95% of surveyed growers. Our 3rd objective was to accelerate testing of new chemical tools for psyllid suppression. We completed four field evaluations of insecticides for ACP control in 2009 and submitted reports for publication in the ESA journal Arthropod Management Tests. These join 17 previously published reports on insecticidal control of ACP available online. We found that foliar applications of spirotetramat, fenproximate + 435 Oil, spirotetramat, or 435 Oil alone during bloom suppressed ACP populations for two months. Our 4th objective was to evaluate the economic feasibility of psyllid management strategies. Two aerial dormant sprays could cost less than $25/ac. The cost of monitoring psyllids using the tap method is about $25 per block up to 100 acres so could be done 20 times per year for as little as $5/ac, and would avoid unnecessary sprays during the growing season that could cost up to $50/ac. Unnecessary applications also cause secondary pest outbreaks of mites, scales and leafminers, reported by 70% of surveyed growers. Our 5th objective was to provide an information bridge between growers and researchers. Experimental results and recommended management programs have been presented at 18 extension meetings across Florida including two production managers meetings and two CCA trainings, as well as the Entomological Society of America, the Florida State Horticultural Society, the Florida Entomological Society, Citrus HLB & Potato Zebra Chip (ZC) in Mc Allen, TX, and the XIII Simposio Internacional de Citricultura. CD Victoria, Mexico. 91% percent of surveyed growers were satisfied at some level by their present management programs and 85% of respondents were monitoring ACP more than 3 times per year. We are working on emphasizing the use of this information to make informed decisions to use insecticides only when needed. (1)Stansly, PA, JA Qureshi, and HA Arevalo. 2009. Why, when and how to monitor and manage Asian citrus psyllid. Citrus Industry 90(3):24-26. (2) Stansly, PA, HA Arevalo, M Zekri, and R Hamel. 2009. Cooperative dormant spray program against Asian citrus psyllid in SW Florida. Citrus Industry 90(10):14-15.
Obj. 1.DNA bar coding in progress using mtCOI primers to amplify a 780 bp fragment from psyllid field and colony collections from various U.S. states and off-shore locations. The purpose of this objective is to examine diversity within and between varied populations in relation to the associated Liberibacter spp. based on 16S rDNA. Over 50 collections are in hand. Obj. 2. qPCR has been applied to detect Ca. Liberibacter presence (or absence) in the psyllid colony cohorts used for light and TEM studies, and by FISH (over different AAPs), for immatures and adults (Roberts, SWFREC, HLB diagnostics lab). The FL psyllid rearing system is fully functional and Ca. Liberibacter-infected and bacterium-free colonies are being maintained routinely. About 200 field-collected adult psyllids testing 10% PCR positive were placed into 2 more cages each with 6 ungrafted plants and 100 psyllids. Grafted plants were held in an air conditioned greenhouse at (28 ‘C 64% RH) and psyllid inoculated plants were held in an air conditioned insectary under similar conditions and 14 h light. All plants tested negative for HLB before they were infected with either method. After about one to one and a half month 85-90% grafted plants tested HLB positive based on PCR conducted on dark green mature leaves, whereas, plants exposed to infected psyllids still tested negative. When grafted plants were pruned to induce new shoots, 31-35% tested HLB positive by PCR. Two weeks later when PCR was conducted on leaves from young shoots 90-100% were positive. Four weeks later 97% tested positive by (dark green mature leaves). Two months after psyllid exposure to infected (PCR +) plants, 83% of the psyllid infested plants tested HLB positive by PCR of the dark green mature leaves. Adult psyllids collected on 8/12/2009 and tested at the SWFREC HLB lab were 61% and 67% HLB positive from budded and psyllid infected plants, respectively. Adult psyllids collected on 8/25/2009 and PCR tested at the USDA-ARS Riverside lab were 36% and 13% HLB positive from budded and psyllid infected plants, respectively. Obj. 3. Because it can be reared year-round and because of quarantine constraints we have adopted Bactericera cockerelli as a surrogate species for Diaphorina citri. All three sections, V1,V2 & V3, of the B. cockerelli ventriculus support bacterial growth and replication that advances into huge colonies in the epithelia and on the outside surfaces as well. The esophagus and external hindgut harbor small numbers on their outside surfaces. Thus focus on transmission is now shifted to the oral region. The salivary glands apparently do not support massive, organ-destroying cultures, however, high titers of bacteria occur in the hemolymph surrounding them. Their role in the terminal stages of transmission is still under study. FISH technique, involving coagulant fixation, adopted from prior works by various authors, have proven unsatisfactory for this species. New techniques are under development, including the rendering of individual organs on slides, and the de-shelling of aldehyde fixed whole bodies for acceptable in-situ localization. Obj. 4. We are investigating whether bacteria gain lateral access to the salivary canal without invading the glands. The most likely site would be near the precibarium, where the maxillary stylets converge (Ullman and McLean 1989) and glandular secretions are present. Studies are underway to determine the configuration of the oral area to aid in interpretations of liberibacter-anatomical TEM visualization.
Our initial efforts in using canines to detect canker started in 2000 but were thwarted by the September 11 attack on the US, which permanently diverted the canine we had been training (it was included in the much needed efforts to detect explosives). However, prior to this dog’s reassignment, our initial research with canines for canker detection demonstrated their ability to detect citrus and citrus canker volatiles/aromas/essences, i.e., minute concentrations of volatile/aromatic compounds, deposited on cloth and other materials as well as directly from the air. A second attempt with USDA, APHIS agriculture detector dogs was cut short by decreasing budgets combined by increasing needs of detection of contraband agricultural products at points of entry into the US. However, our third and ongoing attempt has been much more successful. This most recent canine, ‘Juice’, has demonstrated an ability to detect canker infected citrus fruit with high reliability. The initial success of this project with regards to canker detection, suggests that the same technique may be useful for detection of HLB, perhaps presymptomatic while the disease is still latent. Such a tool would be very useful and is much needed, as it would allow both detection and early removal of presymptomatic (latently infected) trees, which also may be below the titer required for psyllids transmission. Two dogs have been trained to recognize the scent of citrus canker. Dogs were trained on infected versus non-infected trees and fruit. Training of the canines for the detection of canker infected citrus trees was accomplished via a commercial canine training facility in North Florida. As indicated above, ‘Juice’, demonstrated an ability to detect canker infected citrus fruit with high reliability (> 99.3%). Juice was capable of discriminating canker infected from non-infected fruit in 30 runs of 5 blind stations each, with only one false positive and no false negatives (Peruyero and Gottwald, unpublished). Results indicate that canines can detect and differentiate citrus fruit infected with canker from non-infected tissues. The canine was then trained on infected versus non-infected Duncan grapefruit seedlings. Preliminary tests at the canine training facility are very promising. Field trials were conduced in early April at the USHRL farm. Trees (75) were placed in the field in a 3 row by 25 tree/row design. Ten replications were conducted in which the proportion of disease trees ranged from 2-10%. The conditions were sunny but with a 15-20 MPH breeze. Even so, detection highly reliable. Data are presently under analyses. Short movie footage was also taken to document the dogs search and detection behavior. More field trials are scheduled over the next few months to validate the canine’s performance. At the conclusion of the canker field testing (proof of concept) the dog will the be cross-trained for HLB detection and field tests will be conducted to assess performance and detection ability to HLB. This will go beyond the time frame of this grant, but we will pursue it to conclusion utilizing USDA, ARS funding internal.
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