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.
Sensory impacts in unpasteurized and pasteurized juice from HLB-affected fruit: Valencia fruit samples from April and May 2009 were evaluated in a consumer sensory panel. Objectives were to assess consumer acceptability of symptomatic, asymptomatic, and healthy fruit. Three treatment groups for each harvest sample date were hand-extracted to yield 6 total juice treatments, which were further split into thermal treatment types. Consumer sensory testing indicated that symptomatic samples produced juice that was less accepted, less sweet and less flavorful that either control or asymptomatic products. Pasteurization does not appear to affect acceptance in any sample except for the symptomatic product from the April Valencia harvest date, where pasteurization impacted the overall acceptance, sweetness and flavor. Flavor and aroma changes in HLB fruit: Analysis of juice samples from the previous season continued. Esters responsible for the fruity, sweet aroma in OJ were significantly lower in symptomatic juice. Esters such as ethyl butyrate and ethyl-2-methyl butyrate were 20-50% lower in symptomatic compared to control juice. Important aldehydes such as hexanal were 50% lower in symptomatic fruit. Alcohols were generally higher in symptomatic fruit. Linalool, responsible for the fruity, citrus aroma in OJ, was 45% higher in symptomatic fruit. Although there was little difference in flavor volatiles between juice from healthy or asymptomatic fruit, aroma volatiles in juice from symptomatic fruit were out of balance compared to that of healthy juice. Phytohormone changes in HLB fruit: Ethylene production, ACC content and expression of ethylene biosynthesis genes were analyzed in isolated juice vesicle tissue from early season Valencia fruit. In contrast lower biosynthesis-related gene expression and lower production in symptomatic whole fruit, ethylene production increased in juice vesicles when compared with asymptomatic or healthy ones. To examine the spatial role of phytohormones in HLB disease symptom development, IAA and ABA contents were evaluated in flavedo removed from the stem-end, mid-section, and stylar-end of healthy, asymptomatic, and symptomatic fruit. IAA content was highest in the mid-section, lower in the stylar end, and lowest in the stem-end in all fruit. However, IAA was 2-fold higher in mid-section but 2-fold lower in the stylar-end of symptomatic fruit. Although little difference in ABA content was measured in whole fruit, spatial analysis indicated that flavedo from symptomatic fruit had 3-fold higher ABA content in the mid-section and 3-fold lower content in the stylar-end. Results indicate increased stress in HLB-affected fruit and suggest a role for temporal and spatial redistribution of ethylene production, IAA and ABA contents in premature fruit abscission, fruit shape and seed abortion. Yield, peel color, fruit size and seed abortion in HLB fruit: Accumulation of chlorophyll a and b and total chlorophyll were 3-fold higher, while b-carotenoid content was 2-fold lower in flavedo of symptomatic fruit compared with healthy tissue. Pigment accumulation in asymptomatic flavedo was less compared with symptomatic flavedo but higher when compared with healthy fruit. Starch content was reduced in symptomatic and asymptomatic flavedo compared with healthy tissue. In symptomatic and asymptomatic flavedo, iodine-stained starch grains were sparse or absent when viewed with light microscopy, however, starch grains were abundant throughout the flavedo of healthy fruit. Iodine staining confirmed light microscopy observations. The use of iodine staining as a tool for early detection of HLB-affected in asymptomatic fruit in the field will be explored this coming season. Extension and education: Four members of our group made oral presentations at the International Citrus and Beverage Conference. A tabletop extension display was also prepared and presented, and written materials distributed. Similar displays are planned at upcoming industry meetings.
Sensory impacts in unpasteurized and pasteurized juice from HLB-affected fruit: Valencia fruit samples from April and May 2009 were evaluated in a consumer sensory panel. Objectives were to assess consumer acceptability of symptomatic, asymptomatic, and healthy fruit. Three treatment groups for each harvest sample date were hand-extracted to yield 6 total juice treatments, which were further split into thermal treatment types. Consumer sensory testing indicated that symptomatic samples produced juice that was less accepted, less sweet and less flavorful that either control or asymptomatic products. Pasteurization does not appear to affect acceptance in any sample except for the symptomatic product from the April Valencia harvest date, where pasteurization impacted the overall acceptance, sweetness and flavor. Flavor and aroma changes in HLB fruit: Analysis of juice samples from the previous season continued. Esters responsible for the fruity, sweet aroma in OJ were significantly lower in symptomatic juice. Esters such as ethyl butyrate and ethyl-2-methyl butyrate were 20-50% lower in symptomatic compared to control juice. Important aldehydes such as hexanal were 50% lower in symptomatic fruit. Alcohols were generally higher in symptomatic fruit. Linalool, responsible for the fruity, citrus aroma in OJ, was 45% higher in symptomatic fruit. Although there was little difference in flavor volatiles between juice from healthy or asymptomatic fruit, aroma volatiles in juice from symptomatic fruit were out of balance compared to that of healthy juice. Phytohormone changes in HLB fruit: Ethylene production, ACC content and expression of ethylene biosynthesis genes were analyzed in isolated juice vesicle tissue from early season Valencia fruit. In contrast lower biosynthesis-related gene expression and lower production in symptomatic whole fruit, ethylene production increased in juice vesicles when compared with asymptomatic or healthy ones. To examine the spatial role of phytohormones in HLB disease symptom development, IAA and ABA contents were evaluated in flavedo removed from the stem-end, mid-section, and stylar-end of healthy, asymptomatic, and symptomatic fruit. IAA content was highest in the mid-section, lower in the stylar end, and lowest in the stem-end in all fruit. However, IAA was 2-fold higher in mid-section but 2-fold lower in the stylar-end of symptomatic fruit. Although little difference in ABA content was measured in whole fruit, spatial analysis indicated that flavedo from symptomatic fruit had 3-fold higher ABA content in the mid-section and 3-fold lower content in the stylar-end. Results indicate increased stress in HLB-affected fruit and suggest a role for temporal and spatial redistribution of ethylene production, IAA and ABA contents in premature fruit abscission, fruit shape and seed abortion. Yield, peel color, fruit size and seed abortion in HLB fruit: Accumulation of chlorophyll a and b and total chlorophyll were 3-fold higher, while b-carotenoid content was 2-fold lower in flavedo of symptomatic fruit compared with healthy tissue. Pigment accumulation in asymptomatic flavedo was less compared with symptomatic flavedo but higher when compared with healthy fruit. Starch content was reduced in symptomatic and asymptomatic flavedo compared with healthy tissue. In symptomatic and asymptomatic flavedo, iodine-stained starch grains were sparse or absent when viewed with light microscopy, however, starch grains were abundant throughout the flavedo of healthy fruit. Iodine staining confirmed light microscopy observations. The use of iodine staining as a tool for early detection of HLB-affected in asymptomatic fruit in the field will be explored this coming season. Extension and education: Four members of our group made oral presentations at the International Citrus and Beverage Conference. A tabletop extension display was also prepared and presented, and written materials distributed. Similar displays are planned at upcoming industry meetings.
A manuscript reporting the sequence of symptom development and the identification of callose and phloem protein 2 (PP2) as the amorphous and filamentous plugging materials, respectively, has been prepared and is in the final stages of review for submission to Plant Physiology. The upset of normal phloem translocation of carbohydrates to other parts of the plant and ultimately the starvation of the root system (Etxeberria et al, 2009, accepted) may be the main reason for tree decline in response to HLB infection. >Field samples were collected and prepared to determine the relative amounts of amorphous versus filamentous plugs in order to evaluate if one type is more important than the other in disrupting phloem sap flow. Samples include sweet orange and grapefruit collected in the Indian River District or southern flatwoods. Additionally, samples from sweet orange were collected near Lake Placid and Murcott in the southern region. Examination of these samples has begun. >Earlier work by a colleague has indicated that two of our common rootstocks do not express HLB symptoms when grown as seedlings in a greenhouse or growth chamber and infected with the HLB organism. These are being examined for possible suppression of the phloem plugging mechanism. More importantly, field grown seed source trees have been found with + PCR reactions for HLB. These will be tested for phloem plugging and HLB tolerance. >Work to understand the mechanism by which the bacterial infection leads to this phloem plugging is underway as insufficient bacteria are present in the phloem to directly plug the phloem. To determine how the causal bacteria elicits the over expression of phloem plugging materials, one Agilent microarray has been designed based on the genome sequence of Candidatus Liberibacter asiaticus. Bioinformatics analysis was performed to identify potential virulence factors. Six potential virulence factors were cloned into pGEMT-easy vector. The insertion was confirmed using PCR with gene specific primers, and will be sequenced for further confirmation. Those virulence factors are being expressed in tobacco and citrus to study their potential roles in virulence. >Transgenic approaches to achieve over-expression of the citrus §-1,3-glucanase gene using different promoters, though protoplast/GFP co-transformation and Agrobacterium-mediated transformation are underway in order to minimize HLB associated callose-plugging. The citrus §-1,3-glucanases gene from Valencia embryogenic callus and young leaves (McCollum et al., 1999) was cloned. Citrus .-1,3 glucanase cDNA (GenBank accession number AJ000081) was synthesized from Valencia leaf and embryogenic callus. A 1011 bp citrus .-1,3 glucanase gene fragment was amplified. To obtain the suitable restriction site and additional cMyc tag (to facilitate subsequent western analysis) on the cDNA, another PCR reaction was performed using a new primer set with a different restriction site including the cMyc tag. The final PCR product (1059bp, designated BG3) was purified, cloned into the pGEM T-Easy vector (Promega) and sequenced. The cloned BG3 fragment was ligated into a vector named pUCLON between the 35S promoter and 35S terminator, and was transformed into host E coli DH5. cells. Plasmid DNA was isolated using 5prime kit and checked through restriction digestion and also PCR analysis. The re-ligated plasmid was named pUBG3. The HindIII fragment from pUBG3 was excised and ligated into the HindIII site on another vector pCIT101 holding GFP/NPTII fusion gene on it. The final vector was designated as pCITBG3 and was transformed into Agrobacterium. It is now ready for co-transformation experiments. Plasmid vector pARS108 with the ER-targeted GFP gene was also used to make a new construct for efficient protoplast transformation. Another vector, pGASS, was constructed to target BG3 expression in the phloem tissues only. Two new sweet orange callus lines are available to carry out these and other transformations.
Primary: Cultural Practices to prolong productive Life of HLB Infected Trees The first objective of the project has been accomplished. This objective was to duplicate at another site the positive results using nutrient/SAR foliar sprays to rejuvenate HLB infected trees seen in citrus grower Maury Boyd grove. We have seen a rejuvenation of heavily infected HLB trees using the foliar spray mixture of micronutrients (Mg, Zn, Mn, Mo), SARs (Salicylic acid, Serenade Max WP), Phosphorous acid, Hydrogen peroxide, and other macronutrients applied with citrus spray oil. These results have been seen in three randomized & replicated trial sites of 6 to 7-yr-old Valencia orange trees. Initial infection rates among the trials ranged from 100% infection at IFAS Immokalee site, to 50-60% infection in one commercial grove, and 15-20% infection in another commercial grove. In 2009 all possible combinations of the component groups were applied to the spring, summer, and fall vegetative flushes at each site with similar positive results. After applications to the 3 flushes in 2008, and during the drought in the spring of 2009, the trees produced vigorous flushes with normal internodes and large leaves. Flowering resulted in set fruit with a normal to large crop on trees that did not produce a harvestable crop in 2008. To date the crop has held on the trees with very little fruit drop. Although the trees are symptomatic of HLB they have continued to flush vigorously through the summer. New growth has regenerate from below the dieback on twigs with dieback. A dramatic recovery has been apparent in many of the treatments. Most treatments improved tree condition and the best results are apparent with the complete mixture. The macro and micro nutrients seem to be the most important. Fruiting appears to be enhanced with the addition of the Phosphorous acid and SARs. Yield from the 2008 Valencia crop from 7-yr-old trees harvested in summer 2009 at the 30-acre-commercial showed yield highest in the treatment containing macro & micro nutrients, Phosphorus acid, and hydrogen peroxide (0.80 box/tree). Yield was least in the treatment containing macro nutrients and hydrogen peroxide (0.43 box/tree). A spreadsheet has been developed and will be posted on the SWFREC website that calculates the net present value of grove returns over 10 years assuming production and price assumptions that the grower can input. The spreadsheet allows a grower to determine the threshold rate of tree removal after which an alternative nutrient management program for greening infected trees would return a higher net present value. Subcontract: Evaluation of Systemic Acquired Resistance Inducers Combined with Psyllid Control to Manage Greening in Infected groves In a 12-acre commercial block of Valencia oranges 7-years-old trees we evaluated the effect of micronutrients + systemic acquired resistance (SAR) inducers, and their interaction with psyllid chemical control. With 4 replicates in an RCB design for a 2×2 factorial experiment with two main effects: 1) 3 applications a year of micro-nutrients + SAR at two levels (Yes or No) and 2) insecticide applications based on scouting at two levels (Yes or No). Plant tissue, ACP adults, and nymphs (when available) are collected for PCR analysis every 4 months and results plotted and evaluated using quantitative geostatistical analysis. Incidence of positive trees was 33% in November 2008 and April 2009 and their distribution did not change over the two dates. With respect to the ACP, our results indicate significant differences in populations between insecticide-treated and untreated trees throughout the year, which maintained differences up to 26X despite close proximity. In contrast, the micro+SAR treatments are not having an effect on psyllid populations.
Specific hybridization technology using non-radioactive digoxigenin-labeled probes specific for HLB pathogen to detect the onset of HLB infection and its progression in citrus trees in the field is being developed. As outlined in the previous quarterly report, several regions of the HLB pathogen were identified, based on the sequence information of the HLB pathogen and were used to design specific primer pairs for the detection of HLB in infected citrus trees and psyllids. The amplified products were highly specific for the HLB DNA from HLB infected citrus plants. Probes were designed to these regions. Digoxigenin labeled single stranded RNA probes and PCR based DNA probes were generated and used in dot blot hybridization against nucleic acids isolated from the HLB infected citrus and Psyllids carrying the HLB pathogen. A linear relationship of the signal was observed in the dot blots with increasing amounts of DNA used. However, in the tissue blots from HLB positive citrus plants (from the green house) the hybridizations are not consistent. Tissues blots from some sections were positive but some were not. This probably is because of the uneven distribution of Las that is normally observed with HLB infection and the low titer of the pathogen. We are currently optimizing the tissue blot hybridization using high specific activity probes and multiple probes if necessary. The procedure for isolating the nucleic acid from single psyllids were optimized, and we have been able to amplify Liberibacter specific amplicons from single Infected psyllids using pairs of Las specific primers. The primer pair designed based on the tufB region of the Las genome was observed to be very specific and gave consistent amplification from the psyllids. We plan to use this primer pair to generate non-radioactive probes for the detection of Las in psyllid populations under laboratory and field conditions. Current work: Optimization of the citrus tissue blot hybridization on nylon membranes using Las specific non-radioactive DNA and RNA probes. Develop similar technology with psyllids.
The first year summer tests are concluded with mixed results. Over 300 trees were treated with the herbicide formulas Arsenal (Powerline) or Clearstand using 1 to 6 or 1 to 7.5 dilutions. These rates had a very poor percentage kill and required retreatment. For summer use, trunk applications of these herbicides will have to be in the 1 to 1 or at most 1 to 3 dilution rates. Next summer 1to 1, 1 to 2 and 1 to 3 dilutions will be tested. Fall tests are showing good results again. Alternatives to 1 1/2 inch blade cuts are being tested. Manual power saw blade cuts to different depths with two different herbicide dilutions have been made for evaluation. An electro-hydraulic mechanized tree trunk incision clamp system was developed and tested on a 4 X 4 ATV. Three problems were identified in the initial tests. First, the systemÕs gross weight proved too much for the ATVÕs front end suspension. Second, the systemÕs hydraulic motor was damaged, therefore resulting in erratic actuator operating pressures. Third, the clamping mechanism design was too vulnerable to operating stresses: The main actuator was not able to produce enough compression pressure. The incision blades and their corresponding compression angles were not adequate to reach the required trunk incision depth. To help resolve these issues, the entire system was transferred onto a utility tractor, which its front end suspension is capable of enduring the systemÕs gross weight. The system shares the same hydraulic pump as the tractor uses for its end loader operations, therefore eliminating erratic actuator operating pressures. A larger bore cylinder and a revised clamping mechanism design, including incision blade gaps and reduced compression angles, improved the trunk incision depth, which should be adequate for the herbicide spray application. This system should be completed and tested with herbicide sprays in November. Aerial sprays applied by the South Florida Water Management District to 600 acres of abandoned citrus were evaluated about 60 days after treatment this summer. Many trees were successfully killed, but in two or three blocks where scion and rootstocks differed between different ages of trees not all of the trees were killed. There small plot spray tests were not as successful as their earlier test last winter. Again, it appears that sprays during active growth are not as successful. There are 40 acres remaining in this test area and plans call for applying at least 4 treatments with 2 replications (5 acre plots) in order to evaluate some surfactants to improve efficacy. Also, additional chemicals will be tested.
Objective (2) Employ qPCR to detect Ca. Liberibacter presence (or absence) in the experimental adult psyllid cohorts given a range of acquisition-access periods as immatures or adults. Objective (3) Employ qPCR to quantify Liberibacter titer in psyllids and dissected organs, and in subsamples of the ÔtiteredÕ cohort, define the gross association of Ca. Liberibacter in thick sections by fluorescent in situ hybridization to develop a Ôgross anatomical road mapÕ of Ca. Liberibacter accumulation in key organs, tissues, and cells. With the completion of our new quarantine greenhouse, we are now maintaining 12 colonies of infected psyllids, 8 on budded Citrus, and 4 on Citrus infected by psyllids collected in the field. These colonies allow for a baseline of high HLB titer when sampling all instars as needed. New information from a recent publication (Inoue et al 2009) indicates that the best approach for determining organ-to-organ movement of C. Liberibacter in the host body is to work with 4th and 5th instars transplanted from noninfected plants to infected plants. To accommodate this finding, single branches were selected with enough leaves to allow for removing some for qPCR while at the same time hosting adjacent, transplanted larvae. These larvae were confined to the branches in such a way as to maintain control and vigilance during their AAP. Cohorts were given AAP on post-flush leaves for 1 hr to 24 hrs, then processed for qPCR and both flourescence and electron microscopy. Accompanying this approach, cohorts of 2nd, 3rd, 4th, 5th and adult instars were sampled directly off of different portions of infected plants and processed the same way. This series of samples will best represent actual proliferation profiles, since, in the field, larval mobility is negligible, and late instars are theoretically preinoculated from their early instar ontogeny. This inherited inoculum would build up to high titers along with the inoculum they imbibe themselves. We expect that amidst such a broad series of confined, transplanted larvae, incipient infiltration of bacteria into the digestive system will be seen, and will allow us to determine the organ-to-organ, or organ section, sequence of proliferation. We expect that in samples coming directly off of infected plants, the bacteria will have had time to build up to high titers inside the host, and the routes bacteria took during proliferation will be obfuscated using the current FISH labeling system. Twenty specimens of leaves and psyllids were sampled for qPCR from areas of the host plants from which the study specimen lots came. Eleven lots of psyllids were collected for microscopy, with each lot divided into two sublots, one for FISH, the other for TEM. The first TEM investigation of guts and glands of infected 5th instar larvae is in progress at this time. Inoue, H., Ohnishi, J., Ito, T., Tomimura, K., Miyata, S., Iwanami, T. and Ashihara, W. 2009. Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage. Annals of Applied Biology 155:29-36.
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