The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. The specific objectives of this proposal are: transcriptional and microscopic analyses of citrus varieties which are either susceptible or tolerant to Ca. L. asiaticus infection at different infection stages in greenhouse and citrus grove; and transcriptional and microscopic analyses of host response to Ca. L. asiaticus infection with a model system tobacco. Microarray analysis and/or suppressive subtractive hybridization libraries approaches will be used to study the host response to the HLB pathogen infection followed by confirmation with Northern blot or quantitative reverse transcriptional PCR. Anatomical study will be performed with light microscopy using different staining methods. Major achievements: One refereed journal article was published. Kim et al. 2009 Response of sweet orange (Citrus sinensis) to ‘Candidatus Liberibacter asiaticus’ infection: microscopy and microarray analyses. Phytopathology. 99:50-7. Investigation of the host response was examined with citrus microarray. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes related to plant pathogenesis/stress, anthocyanin biosynthesis, cell wall metabolism, cell division, detoxification, lipid metabolism, metabolite transport, metal transport, nucleotide metabolism,phenylpropanoid / flavonoid / terpenoid metabolism, phytohormones, protein kinase, protein metabolism, protein-protein interaction, signal transduction, sugar metabolism, transcription/translation factors and unknown/hypothetical genes. The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since ‘Ca. Liberibacter asiaticus’ does not form aggregate in citrus. The upregulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants. The cDNA sequence of PP2 has been requested by four different research groups to use as the target to suppress the HLB symptom development. To further expand our current understanding of Ca. L. asiaticus-host interaction, we are currently comparing two susceptible and two resistant/tolerant cultivars grown in greenhouse. To understand the genetic mechanism of resistance/tolerance will help the breeding program in the long run. Suppression Subtractive Hybridization is being used to study grapefruit since microarray is not available for grapefruit. Currently, subtractive cDNA libraries were constructed. We are collecting samples We are collecting samples from the resistant/tolerant cultivars due to initial problems of inoculation to those plants. We are further investigating host response of sweet orange (Valencia) in the field conditions. Both leaf and root samples have been collected. Microarray data is available from one experiment (three biological repeats) using the leaf samples. More comparisons have been planned and are being conducted. Finding key genes involved in HLB symptom development will reveal potential management strategy and lead to innovative research to control HLB.
Comparison of the microbiomes associated with HLB pathogen positive and negative citrus will illuminate the causal agent of citrus greening. Potential beneficial endophytic microorganisms could be identified from escape plants which survived in heavily infected citrus groove with HLB. Beneficial microorganisms have been shown in previous studies to have the capacity to control plant diseases by accelerating seedling emergence, promoting plant growth and development, and preventing the invasion of plant pathogens. The investigation of the microbiomes associated with different hosts will help understand the transmission of microorganisms between different hosts. Major achievements: This research has resulted in three referred publications (two published, one submitted). 1. A comprehensive study of the bacterial diversity associated with healthy and HLB diseased citrus indicated that Candidatus Liberibacter asiaticus as the pathogen responsible for HLB disease in Florida. Phytoplasma was not found in any of the samples collected from Florida. Publication: Sagaram et al. 2009 Bacterial diversity analysis of Huanglongbing pathogen-infected citrus using PhyloChips and 16S rDNA clone library sequencing. Applied and Environmental Microbiology (AEM). 2. We characterized the effect of HLB on the bacterial community associated with citrus roots. This research has been summarized in the following manuscript ‘Huanglongbing, a systemic disease, restructures the bacterial community associated with citrus roots’ which has been submitted to AEM for publication. In this study, 16S rDNA clone library analysis of the citrus roots revealed shifts in the microbial diversity in response to pathogen infection. The clone library of the uninfected root samples have a majority of phylotypes showing similarity to well known plant growth promoting bacteria including Burkholderia, Pseudomonas, Stenotrophomonas, Bacillus and Paenibacillus. Infection by Ca. L. asiaticus restructured the native microbial community associated with citrus roots and led to the loss of detection of most phylotypes while promoting the growth of bacteria such as Methylobacterium and Sphingobacterium. The relative proportions of different groups of bacteria changed significantly after pathogen infection. These data indicate that infection of citrus by Ca. L. asiaticus has a profound effect on the structure and composition of the bacterial community associated with citrus roots. 3.A method was developed to screen antagonistic bacteria against uncultured HLB pathogen. The method uses the discrimination of live-dead cells by EMA and speed and sensitivity of QPCR. This finding is published on European Journal of Plant Pathology which has attracted tremendous interest. 4. Isolation of plant growth promoting bacteria from potential escape citrus. Isolation of bacteria with the potential of plant growth promoting and biological control potential might reveal innovative ways controlling the HLB disease. Fifty-four morphologically distinct isolates were obtained from surface sterilized roots of symptomatic and asymptomatic (potential escape trees) citrus plants from a citrus grove with a HLB infection rate of more than 60% and an infection history of approximate five years. Qualitative screening showed that for all of these criteria, asymptomatic plants harbor a significant greater diversity of potentially beneficial bacterial strains. Six different bacteria have been shown to be able to reduce the population of Ca. L. asiaticus when tested in vitro. We are further characterizing the potential mechanism to utilize it to control HLB. Furthermore, we have also located tress which have survived HLB for more than 15 years in China. We plan to investigate whether beneficial bacteria could be isolated from those trees to inhibit the population of the HLB bacteria in citrus. 5.Samples (leaf and root) from different varieties including grapefruit, Murcott, and Hamlin were collected from Florida. Samples (leaf and psyllid) from three different locations were collected. The bacterial diversity of those samples are being studied.
In this third quarter, we have primarily focused on the first two objectives of our proposal as planned in our timeline. Objective 1: Identify genes positively regulating SA-mediated defense in citrus So far, we cloned four citrus SA homologues, ctNPR1, ctEDS5, ctNDR1 and ctPAD4 via the RT-PCR approach. We are currently working on cloning the fifth gene, ctEDS1. We have also finished collecting citrus tissues infected with Ca. L. asiaticus in a time course. Samples will be used to study the expression profile of salicylic acid (SA) homologues and SA analysis. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues We cloned all four citrus homologues, ctNPR1, ctEDS5, ctNDR1 and ctPAD4 into the binary vector pBINplusARS and already transformed Arabidopsis with each construct. We obtained T0 seeds for all these constructs and selected some T0 seeds for T1 transgenic plants. Our transgenic lines and expected outcomes are summarized below: 1. CtNPR1/pBINplusARS to Col or the npr1-1 mutant: we have obtained T1 seeds and will test defense phenotypes in the next generation (T2). If the ctNPR1 gene is functional in Col, we expect that the transgene confers enhanced disease resistance in Col background and complement the npr1-1 mutant. 2. CtNDR1/pBINplusARS to Col or the ndr1-1 mutant: we have obtained T0 seeds and will select T1 seeds on Kanamycin containing plates and test T2 transgenic plants for defense responses in both Col and ndr1-1 background. 3. CtEDS5/pBINplusARS to Col or the eds5-1 mutant: progress is similar to the ctNDR1 construct. 4. CtPAD4/pBINplusARS to Col or the pad4-1 mutant: progress is similar to the ctNDR1 construct. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in Citrus A ctNPR1/pBINplus/ARS construct has been in the pipeline of transforming citrus.
We have examined callose accumulation in the Liberibacter-infected citrus phloem tissues by epifluorescence microscopy since the last quarterly report. Although it is a low-resolution technique, epifluorescence microscopy has an advantage over transmission electron microscopy (TEM) in that it enables callose imaging across larger leaf areas, facilitating extensive and thorough callose measurement. In healthy leaves, no callose was detected in the phloem. In contrast, callose deposition in sieve pores and plasmodesmata pore units (PPUs) was observed in huanglongbin (HLB) symptomatic leaf phloem cells. It was shown that callose accumulates in the phloem cells of senescing citrus leaves (1), raising a question as to whether the callose deposition is a secondary phenomenon following leaf deterioration by Liberibacter infection. To address this question, we examined callose deposition in citrus leaves that are positive for Liberibacter infection (assayed by PCR) but display no HLB signs yet. Callose was detected in the sieve pores and PPUs of the asymptomatic leaves almost as much as those in the symptomatic leaves. This demonstrates that callose synthesis in the Liberibacter-infected phloem precedes development of HLP symptoms. Callose deposition in the plasmodesmata is a natural plant defense reaction but it reduces solute transport efficiency through the plasmodesmata (2). This led us to hypothesize that the callose synthesis in the PPU accompanying Liberibacter infection may affect phloem loading in the citrus leaves. To test the possibility, we injected carboxyfluorescein into intercellular spaces of healthy leaves, Liberibacter-infected asymptomatic leaves, and Liberibacter-infected symptomatic leaves. When we imaged carboxyfluorescein at 24 hours after injection, the dye was seen to concentrate in the vein and spread through the vein in healthy leaves. However, dye remained at the injection site in the Liberibacter-infected leaves, indicating that phloem loading is inhibited in the infected leaves. The massive starch accumulation in the parenchyma cells of HLB symptomatic leaves suggests a blockage in photosynthetate export and inhibition in phloem loading of the infected leaves provides an explanation for the blockage. Our results also suggest that cell death in the HLB citrus trees is caused by inappropriate plant response against Liberibacter rather than damages actively done by the bacterial cells, which is consistent with the fact that this phloem-limited bacterium devastates the entire tree at a very low titer. As stated in the second report, we have identified phloem cells containing bacterial cells in HLB symptomatic leaves. We examined their bacterial cell shapes by serial section 3D reconstruction. (An image from our 3D models is shown at http://news.ifas.ufl.edu/2009/12/09/uf-researchers-find-lone-culprit-behind-greening/) We found two morphologically distinct bacteria cells in periwinkle leaf phloem cells. (Please see the first progress report.) But all the bacterial cells in the citrus leaf phloem cells displayed uniform morphological features including diameter, length, cytoplamic staining and cell wall staining, and so on. This observation suggests that Liberibacter vastly outnumbers other bacteria, if there is any, in the HLB-infected citrus phloem tissue and agrees with a recent metagenomic sequencing result by (3). 1. M. J. Jaffe, R. Goren, Physiologia Plantarum 72, 329 (Feb, 1988). 2. Y. L. Ruan, S. M. Xu, R. White, R. T. Furbank, Plant Physiol 136, 4104 (Dec, 2004). 3. H. L. Tyler, L. F. W. Roesch, S. Gowda, W. O. Dawson, E. W. Triplett, MPMI 22, 1624 (Dec, 2009).
We have examined callose accumulation in the Liberibacter-infected citrus phloem tissues by epifluorescence microscopy since the last quarterly report. Although it is a low-resolution technique, epifluorescence microscopy has an advantage over transmission electron microscopy (TEM) in that it enables callose imaging across larger leaf areas, facilitating extensive and thorough callose measurement. In healthy leaves, no callose was detected in the phloem. In contrast, callose deposition in sieve pores and plasmodesmata pore units (PPUs) was observed in huanglongbin (HLB) symptomatic leaf phloem cells. It was shown that callose accumulates in the phloem cells of senescing citrus leaves (1), raising a question as to whether the callose deposition is a secondary phenomenon following leaf deterioration by Liberibacter infection. To address this question, we examined callose deposition in citrus leaves that are positive for Liberibacter infection (assayed by PCR) but display no HLB signs yet. Callose was detected in the sieve pores and PPUs of the asymptomatic leaves almost as much as those in the symptomatic leaves. This demonstrates that callose synthesis in the Liberibacter-infected phloem precedes development of HLP symptoms. Callose deposition in the plasmodesmata is a natural plant defense reaction but it reduces solute transport efficiency through the plasmodesmata (2). This led us to hypothesize that the callose synthesis in the PPU accompanying Liberibacter infection may affect phloem loading in the citrus leaves. To test the possibility, we injected carboxyfluorescein into intercellular spaces of healthy leaves, Liberibacter-infected asymptomatic leaves, and Liberibacter-infected symptomatic leaves. When we imaged carboxyfluorescein at 24 hours after injection, the dye was seen to concentrate in the vein and spread through the vein in healthy leaves. However, dye remained at the injection site in the Liberibacter-infected leaves, indicating that phloem loading is inhibited in the infected leaves. The massive starch accumulation in the parenchyma cells of HLB symptomatic leaves suggests a blockage in photosynthetate export and inhibition in phloem loading of the infected leaves provides an explanation for the blockage. Our results also suggest that cell death in the HLB citrus trees is caused by inappropriate plant response against Liberibacter rather than damages actively done by the bacterial cells, which is consistent with the fact that this phloem-limited bacterium devastates the entire tree at a very low titer. As stated in the second report, we have identified phloem cells containing bacterial cells in HLB symptomatic leaves. We examined their bacterial cell shapes by serial section 3D reconstruction. (An image from our 3D models is shown at http://news.ifas.ufl.edu/2009/12/09/uf-researchers-find-lone-culprit-behind-greening/) We found two morphologically distinct bacteria cells in periwinkle leaf phloem cells. (Please see the first progress report.) But all the bacterial cells in the citrus leaf phloem cells displayed uniform morphological features including diameter, length, cytoplamic staining and cell wall staining, and so on. This observation suggests that Liberibacter vastly outnumbers other bacteria, if there is any, in the HLB-infected citrus phloem tissue and agrees with a recent metagenomic sequencing result by (3). 1. M. J. Jaffe, R. Goren, Physiologia Plantarum 72, 329 (Feb, 1988). 2. Y. L. Ruan, S. M. Xu, R. White, R. T. Furbank, Plant Physiol 136, 4104 (Dec, 2004). 3. H. L. Tyler, L. F. W. Roesch, S. Gowda, W. O. Dawson, E. W. Triplett, MPMI 22, 1624 (Dec, 2009).
First year objectives have been completed or exceeded 8 months after funds were released on 1 April 2009. Objective 1: Compare aerial and conventional ground application of insecticides for psyllid control. Results from a comparison of low volume (LV) aerial applications (10 Gallons per acre -GPA-) versus conventional ground-based airblast (125 GPA) applications during the 2008 summer indicated that broad-spectrum insecticides work well by air and ground although the selective insecticides spinetoram and imidacloprid provided much better control with ground application. This spring, we followed up with a two selective insecticides (spinetoram and spirotetramat) compared with the broad spectrum organo- phosphate phosmet, all applied at the same volumes as above by air and ground in replicated (N=4) 24-acre plots of mature orange trees. This time, the distinction between broad spectrum and selective insecticides was less clear: all materials worked better when applied by ground than by air and phosmet provided the best control with either method. Nevertheless, the voluntary area wide dormant spray program we initiated with Gulf Citrus Growers association resulting in over 100,000 acres sprayed by air has demonstrated that aerial applications during winter are efficient and effective (see report on FDACS contract number 000). We also conducted a study on a highly infested, 38-acre block of ‘pineapple’ orange to compare LV applications conducted with a modified London Fogger 18-20 provided by Chemical Containers @ 2GPA applied to bed tops only (typical application) with conventional application using an airblast sprayer that treated both tops and swales @ 116GPA. Applying every other row weighted the odds against the LV, but we wanted to test a typical application made many growers are using LV. Spinetoram (4oz/ac) and dimethoate (24oz/ac.) were both applied with 2GPA of horticultural mineral oil (HMO). Conventional ground applications resulted in fewer ACP for the duration of the one month trial compared with the control, whereas LV applications resulted in fewer ACP for only the last two weeks. So again, conventional application proved superior in terms of control, at least with the products tested. Objective 2: Assess the effects of frequent nocturnal LV applications of HMO on psyllids populations. In a preliminary replicated trial testing LV @1GPA HMO applied neat every two weeks for 4 months in summer, we found 14.1’3.8% of the flush was infested in untreated plots, compared with 2.1’1.2% for the HMO-treated plots. In 2009, we compared the modified London Fogger and the Proptec rotary atomizer P400D @ 2GPA, applied every 2 to 4 weeks depending on ACP populations, and monitored ACP every two weeks. High frequency LV applications of HMO maintained lower populations of ACP when compared with untreated trees: (Proptec rotary atomizer 1.4 ‘ 0.75, London Fogger: 4.4 ‘ 1.9, and the untreated control 6.3 ‘ 3.9 ACP adults x days). In 2010, we will conduct similar experiments in a block with a history of high ACP populations using LV-HMO as a control method after ACP populations are present rather than as a preventative (before populations are present) to better test the efficacy of this tactic. We are also collaborating with CREC and a private company in Gainesville, FL to assess the deposition of oil on citrus foliage by LV application using protocols we are developing to using gas chromatography to quantify residues eluted from leaves with a hexane wash. Objective 3: extend results to the citrus industry. Results from these experiments have been presented at no less than 18 extension meetings across the state in 2009 including two Production Managers meetings and two CCA trainings, the Entomological Society of America (ESA), and the Florida State Horticultural Society (FSHS). Arevalo, H. A. and P. A. Stansly. 2009. Comparison of Ground and Aerial Applications for Control of Adult Asian Citrus Psyllid, Diaphorina citri Kuwayama. Proc. Fla. State Hort. Soc. 122: (in press)
We have collected and germinated seeds from several accessions from within the candidate categories listed in the proposal, including pummelos, intergeneric hybrids with Poncirus, and others; seedlings are being grown in DPI-certified greenhouses at the CREC to provide budwood for topworking and young trees to plant directly in the field. In the current 2009-10 fruiting season we have continued seed collection to increase the number and diversity of accessions that can be tested; some of these have been planted already. We have a tentative agreement with one grower on the east coast of Florida to plant out the range of genetic diversity we hoped to test, both as seedlings and as top-worked trees, including some apparently tolerant lines we have identified in an HLB-devastated grove in Florida. We are currently exploring other options within Florida, and have several promising leads, to be followed up with agreements to move ahead; these represent locations where growers have decided not to remove HLB-infected trees, so we expect there to be opportunities to challenge our replicated materials. We are working on the list of accessions that can be sent to our collaborators in China, without compromising UF-IFAS intellectual property rights.
The project has two specific aims. We outlined below the progresses 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 are analyzing both leaves and fruits at two different developmental stage (immature and mature) comparing control plants (healthy plants in healthy location) with other three types of plants (apparently healthy, asymptomatic, symptomatic) in an infected location. We just completed the sampling of the immature fruits sampling a disease-free orchard in Lake Alfred (Polk County, FL) and an infected orchard close to South Apopka (Orange County, FL). The RNA isolations from the entire peel tissue (albedo and flavedo) are currently under way using the method developed by Albrecht and Bowman (Plant Sciences, 2008, 175:191-206) pooling four different fruits of the five biological replicates (different trees) for each treatment. We have started the transcriptomic analysis from the entire set of the four types of mature fruits combining same amounts of of RNA from the five biological replicates. The cDNA libraries were constructed following the mRNA-sequencing sample preparation protocol (Illumina Inc.). The complexity and quality of each library was determined using a high sensitivity chip from Agilent Bioanalyzer. We have run the 4 mature fruit cDNA libraries on the Solexa Genome Analyzer II to obtain read lengths up to 125 bp pair-ended (one sample for each lane). The raw Illumina (Solexa) reads/sample ranged between 25 and 31 million. Trimming was carried out using Velvet assembling into contigs (Zerbino and Birney, Genome Research, 2008, 18:821-829). Alignment of the individual reads and contigs to the Citrus sinensis unigene set (15,808 sequences; NCBI Unigene Build #11, 4/20/09) was performed using BWA (Li and Durbin, Bioinformatics, 2009, 25:1754-1760). We further processed the obtained datasets with the SAM Tools utility to efficiently align short sequencing reads against a large reference sequence (Li et al., Bioinformatics, 2009, 25:2078-9). An optimization of the read length/run is underway to define the optimal conditions for the next runs. We are currently in the process of defining the number of reads mapping to each unigene. We have developed a Taqman Real Time PCR gene expression assay to validate these results and to confirm specific biomarkers that strongly correlate with early disease detection. 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 re-analyzed published data on gene expression in HLB infected leaves (Albrecht and Bowman, Plant Sciences, 2008, 175:191-206; Kim et al., Phytopathology, 2009, 99(1):50-57) using functional enrichment and gene set analysis employing several bioinformatic tools (blast2go, pathexpress, mapman and cytoscape). These analyses have identified HLB-regulated pathways such as sucrose and starch metabolism, phenylpropanoids, hormone-related pathways (jasmonate, ethylene and gibberellins). We compared these results with other stress-induced responses such as citrus bacterial canker disease (Cernadas et al., 2008, 9(5):609-631) and ‘puff’ physiological disorder (unpublished data). We have a predicted protein-protein interaction network inferred from an Arabidopisis knowledgebase that identified the most interactive proteins encoded by HLB-responsive genes such as Heat Shock Protein 81-1 and fatty acid hydrolase 1. HLB regulation of other ‘hub’ proteins such as plant-glycogenin like starch initiation protein and carbohydrate transmembrane transporter reflect the transcriptional regulation observed in sugar metabolism that in turn cause the unbalance in source-sink communications.
We currently have in hand all IgYs that will be necessary to perform pCMAT. IgYs were raised in chickens against diseased plants at 3 months, 1 year and 2 years post-infection. The material from each time point was injected to two hens for IgY production. One of the two hens used for the 3 months time point unfortunately entered molting and had to be discarded. The last batch of antibodies was shipped to Oragenics on October 7th. We have demonstrated by Western analysis that IgYs raised in hens are broadly immunogenic. To perform that assay, plant extracts obtained by sonication of healthy plants were separated by electrophoresis and transferred onto nitrocellulose filters. The filters were blocked and probed with the IgYs raised against individual hens. All seven hens had a strong reactivity against the healthy plant extracts using IgYs at a concentration of 1:5,000, and a secondary antibody (peroxydase-conjugated rabbit anti chicken IgY) at a concentration of 1:20,000. Several hundred bands were observed as well as a smear, which indicates a measurable reactivity against a large number of plant (and pathogen) proteins. We are currently developing an ELISA to further measure the exact titer of each hen. In the absence of a convenient source of DNA or protein extracts from the infecting bacterial pathogen, we will perform all subsequent steps of IgY adsorbtion with healthy plant material. Plant fractions are currently being prepared for this step. We are currently optimizing the conditions necessary to bind healthy plant extracts to latex beads, which will be necessary for the specific adsorption of the IgYs. Adsorbed IgYs should be available by the end of the month. We are also currently optimizing the conditions for proteomic identification of those proteins of the plant induced upon infection, and of the proteins of the pathogen expressed during the infection of the plant. In summary, all material required to perform pCMAT are currently in hand and most quality control steps have been performed. As planned in the grant application, the next few month will be spent on optimizing the adsorption conditions and isolate in vivo induced proteins from diseased material using proteomics. Our work on the etiology of citrus greening disease is now published: Tyler, H.L., R.F.W. Roesch, S. Gowda, W.O. Dawson, and E.W. Triplett. 2009. “Candidatus Liberibacter asiaticus” and assessment of microbial diversity in Huanglongbing-infected citrus phloem using a metagenomic approach. Molecular Plant-Microbe Interactions 22:1624-1634.
During this period of the project, we continued evaluating the effects of hedging date and nitrogen fertilization on D. citri populations in a sweet orange grove. Densities of adults found feeding on flush shoots were independent of either hedging treatment or nitrogen application to citrus trees. In contrast, populations of immatures, eggs and nymphs significantly varied with hedging and the interaction between nitrogen and hedging. Late hedging (done between April and May) dramatically increased population of D. citri immatures because of the abundant flush shoot production following the pruning of trees coincides with high flight activity of D. citri. Although early hedging initially increased D. citri densities in spring, a rapid decline in the psyllid numbers was observed in this treatment from late summer to fall. One possible explanation of this late season decline may be the lower densities of new flush shoots produced later in the season when trees are pruned before spring in February. Early pruned trees may have utilize most of their resources in producing profuse flush in spring, thus only producing fewer flush shoots in summer and fall. Based on these data, early pruning can be recommended to grower as a major cultural practice to naturally reduce psyllid populations in citrus groves. Several phytohormones were also tested to evaluate their effects on suppressing citrus flush shoot production. Both greenhouse and field tests were conducted. In the greenhouse, potted Mexican lime trees (ca. 1 year-old) were pruned and each phytohormone was immediately sprayed following the label recommended rate. A group of 10 pots was used for each treatment including the untreated control that received tap water treatment. Three phytohormones namely Apogee (0.7g/L), Sumagic PGR (100mL/L), and NAA-1-Naphthaleneacetic acid (0.5µL/L), significantly reduced the number of new flush shoots produced by lime trees. A minimum of 50% reduction in flush shoot production was recorded with these growth regulators relative to the untreated control. In addition to reducing the number of new flush shoots produced, Apogee and Sumagic PGR also significantly delayed their growth. Field tests are ongoing to evaluate the effects of these phytohormones on flush production and D. citri populations.
These studies looking at the impact of delaying the treatment of citrus tree stumps with herbicide (Remedy’) for up to 72 hours is continuing and has reached 90 days after treatment (DAT) at the Lake Placid site and 60 DAT at the Arcadia location. At both locations, none of the citrus stumps have sprouted from any of the treatment times (at time of clipping, 24, 48 or 72 hours) or rates (Remedy’ at 25 or 50% solution). In both studies, 6 of the 7 untreated control citrus stumps have sprouted indicating that Remedy’ is effectively providing sprout control, at least to this point in the study. The study will continue until at least 180 DAT.
The new variable rate controller is a major enhancement and upgrade of existing commercial controllers using real-time “sensor eye” actuation in the Florida citrus industry. The addition of a liquid crystal display was successfully tested in a prototype, and will eliminate the need for a separate wireless hand-held computer for changing task configurations. All 50+ prototype units are performing well in a fully deployed commercial environment and no failures have been reported in 14 months. Exceptional reliability was an important criterion when we designed and fabricated the controller’s computer motherboard. Prototype controller testing at the CREC and in commercial groves initially included both split-chain granular fertilizer spreaders and air blast sprayers, but this year we also successfully tested controller performance on a hoop sprayer. The controller performance can be seen on a short video (allow time to load): http://128.227.177.113/pa/Video.html Hoop sprayers are ideal for profitable caretaking of young solid-set citrus plantings such as in new ACPS / OHS blocks because the agrochemical savings could be greater than 75% compared to a conventional air blast sprayer. Additionally because the application is not low- or ultralow-volume, nutrient supplements can be included in the spray as part of a comprehensive nutrition IPM against HLB or in an ACPS. We are adapting and testing the controller for variable rate or spot spraying of weeds, which will be the fourth application in citrus production. Efficient weed sensing is crucial to the success of this application, and initial results from using a small color digital camera look promising when green color pixels are extracted to identify weed positions on the ground. Comprehensive testing is continuing this fall. The camera sensor system was successfully tested to recognize trees with special colored flags in the grove, such as for identifying trees marked as diseased. Fertilizer and spray applications to those trees could then be omitted or modified according to the management plan of the particular grove, thus increasing agrochemical use efficiency and profitability. Two simple software programs have been developed and posted on-line to assist growers when setting up their variable rate fertilizer spreaders: http://128.227.177.113/pa/Software.html The sensor angle program will calculate the best angle to use when adjusting the sensor eyes so that they detect the required canopy size of the trees. The gate height calculator program is useful for obtaining the best gate height of a variable rate fertilizer spreader when used at a given ground speed, fertilizer rate and density, and row spacing. Inappropriate gate heights will decrease performance by causing controller errors during operation which wastes time or causes inaccurate application.
(1) Continue screening of chemical compounds that eliminate or suppress the Las bacteria in periwinkle using the optimized regeneration system. The other chemicals, Metronidazole and ortho-phenylphenol , were tested for their ability to eliminate or suppress Las bacteria and to promote the growth of severe HLB-affected cuttings using the optimized regeneration system. The results showed that more than 2.0% of Metronidazole and ortho-phenylphenol significantly improved periwinkle cutting regeneration. The cuttings treated with less than 1% of metroidazole also regenerated very well. The Las-titers of the regenerated plants will be tested by quantitative real-time PCR. (2) When PS was foliar-sprayed once a week for three consecutive weeks on the Las-infected periwinkle plants at different rates (1x, 5x and 10x), no significant differences were found among treatment rates, but each treatment reduced the Las-bacteria to undetectable levels in the infected plants as compared to the water control. DMSO or Silwet L-7 as emulsifiers had no effect on Las-infected periwinkle plants. (3) HLB-affected citrus was foliar-sprayed or soaked in PS solutions once a week for three consecutive weeks in the greenhouse. The dynamics of Las bacteria were tested by PCR and Q-PCR monthly for a period of six months to determine if the PS affects the bacteria in citrus. After six months the Las-infected, PS treated citrus tested negative for Las by nested PCR, and undetected by real-time Q-PCR, but oxytetracycline and water control treatments were positive for Las. (4) More than 100 citrus grove trees were tested by quantitative real-time PCR and conventional PCR at the Pico farm of the Horticultural Lab, USDA-ARS. The Las-infected citrus groves were used to evaluate the efficacy of screened chemicals in controlling HLB in the field. The experimental design was a randomized block design with three replications. The treatments were three rates of 1g/tree, 5g/tree and 10g/tree in combination with three the different injection methods; Direct-Inject QC (ArborSystems), Smart-Shot Injector (Treelogic) and Syringe-Injector. Citrus leaf samples will be collected every month for one year, and tested for Las by PCR, PS phytotoxicity and residues.
The three main components of an Advanced Citrus Production System (ACPS) are 1) intensive fertigation, facilitated by computer controlled pulse irrigation and liquid fertilizer injection together with monitoring equipment, 2) balanced complete nutrition achieved with traditional hydroponics nutrient formulation, and 3) high density planting with suitable rootstocks to achieve rapid bearing canopy development. During the first nine months of growth under ACPS drip fertigation, the ‘Hamlin’ block on the Ridge at a planting density of 363 trees per acre received 78% less fertilizer and 77% less irrigation water than under a conventional production practice of granular fertilizer and microsprinkler irrigation. Despite these significant savings of input costs, the tree growth rates measured in ACPS plots were approximately double the growth rates measured in conventional plots and tree height after nine months was increased by up to 20% in the ACPS plots. Measurements of soil water, transpiration and photosynthesis suggest that the young trees growing on an ACPS are subject to less short-term water and nutrient stress than conventionally grown trees. Leaf transpiration rates were 45% higher in ACPS plots than in conventional plots after several rain-free days during which only the ACPS plots received daily drip fertigation. Photosynthesis rate measurements in the leaves were 39% higher in ACPS plots than in conventional plots at the end of the same drying period. No wilting was visible at any time, suggesting that these short-term drought periods would be common in many grower blocks but remain undetected. Transpiration and photosynthesis measured after a subsequent rainy period were the same in ACPS and conventional plots, suggesting that recovery from stress was rapid. Short-term drought stress reduces transpiration, and consequently uptake of water and nutrients from the soil, leads to premature stomatal closure, reduced carbon dioxide absorption by the leaves, and therefore lower rates of photosynthesis. Because carbohydrates derived from photosynthesis are the primary energy sources for plants, transient reductions in photosynthesis will slow growth and productivity of the trees on average, as our measurements have shown. The main goals of ACPS are early, high production, early return on investment, and possibly disease avoidance and improved tree longevity. Built-in redundancy from the high planting density is designed to also compensate for removal of HLB- or canker-infected trees. Our projections from the first nine months of growth in the Ridge experiment suggest that the ACPS trees could reach a productive size of about 5.5 feet tall in as few as 2.25 years. Conventionally grown trees would normally reach a similar size in five years. Coupled with the rapid growth, the high planting density of 363 trees per acre (double the conventional density of about 150 trees per acre) should ensure that an economically viable sustainable production level can be reached sooner and therefore avoid some of the early losses by HLB infection. Adequate pest control is vital for the success of ACPS, especially against asian citrus psyllids and citrus leaf miners. The frequent, vigorous leaf flushes stimulated by the ACPS attract herbivorous insects, thus requiring more intensive pest control measures. Integrated pest management which involves multiple control methods such as pesticides (systemic and contact), biocontrol or biopesticides, and new repellant and pheromone chemicals may be the best method for limiting insect populations in an ACPS. The additional pest control required per acre per year in an ACPS may seem uneconomical but in reality due to the compression in space (more trees per acre) and time (higher growth rates) achieved, the costs of pest control required to bring a new ACPS grove into production in half the normal time may even be lower than in a conventional production system. In summary, ACPS is used to grow citrus trees quicker to ‘beat the disease cycle’, and with fewer nonrenewable resources than conventional production methods.
We proposed to identify and evaluate potential candidate genes for RNAi-induced lethality of sap-sucking Hemipteran insects using both in vitro and in planta dsRNA feeding assays. Since July 9th weÕve continued to make strides and have nearly completed Objective 1: the identification of target genes for oral delivery of RNAi-inducing dsRNA to Diaphorina citri and our model organism, Myzus persicae To date, we have developed several RNAi constructs for candidate targets. These include constructs targeting six Hemipteran specific mRNAs for essential proteins (e.g. actin, .-tubulin, .-tubulin, L19e ribosomal protein, S4e ribosomal protein and ATP synthase) , and a construct targeting a vacuolar-ATPase subunit G , which is higly expressed in midgut tissues (encoding vacuolar-ATPase subunit G). We have also developed RNAi constructs for supplementary targets exhibiting preferential expression in the midgut (e.g. glutathione-S-transferase S1 and hexose transporter 1) and salivary glands (Coo2). These were developed to allow the examination of possible effects resulting from tissue-specificity of the dsRNA on RNAi efficiency. All resulting cloned sequences are in the process of being submitted to genbank. Since it has been shown that dsRNAs longer than 134bp have a high success rate we are continually working to create longer (400bp) dsRNAs for all of candidate targets. We are nearing the completion of our first objective as we have just established a working dsRNA feeding system for our sap sucking insect modelr, Myzus persicae, and will begin feeding assays with completed dsRNA constructs in November and December. New scientific evidence has come to light suggesting that insect RNAi may only work effectively in planta, so we are proceeding with our in planta assay for RNAi induced resistance to sap-sucking insects using our Arabidopsis thaliana and Prunus domestica model systems (Objective 2). Previously, we mentioned making progress on cloning of phloem-specific promoters (sucrose synthase 1 and sucrose H+ symporter 2) from citrus. Indeed we have cloned SUS1 homologues from Citrus sinensis, which we have used to selectively localize transcripts (GUS) to phloem tissue in our Arabidopsis model system. This will allow us to ensure the localization of dsRNA to the feeding site of Hemipteran insect pests in any RNAi system we develop in the future. With this development we will begin to run in planta feeding assays (although slower) alongside the in vitro assay (comparably quick) allowing us to develop the ideal RNAi system more quickly. In summary, we have successfully developed several RNAi candidate constructs for use D. citri (objective 1), and are beginning to evaluate those constructs on sap-sucking insects in vitro and in planta (objective 2) in a phloem specific expression system.
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