Valencia harvests were completed and fruit/tree, fruit drop and leaf drop data collected. Juice analyses data is being analyzed. Fruit drop data was 22 % and 19 %, respectively, for Hamlin and Valencia trials at Babson Park. Drop % were 22 % and 20 % at Sebring and 18 % and 15 % at Ft. Meade, respectively, for Hamlins and Valencias. No differences were found between the low concentration PGR treatments and Controls at any of the sites for either healthier or more declined trees. The reduced fruit drop in treated trees after the first year did not occur in the second year. Leaf drop was heavier in September than later in the fall or winter for Hamlin trees and leaf loss was heavier for Valencias in December than later in the winter or spring. No differences were found between PGR-treated and Control trees in the amopunt of leaf drop. At each site there was no difference in the fruit per tree between the Control and PGR treatments in the second year after one year of treatments.
The low concentrations (1/4 rate) of Citrus Fix (2, 4-D) and MaxCel (Cytokinin) with or without ProGibb (GA) every 45 days to Valencia orange trees in central Florida for the 2015-16 growing season did not reduce fall leaf drop nor preharvest fruit drop of HLB infected trees. Total leaf drop per tree averaged 400 leaves for the Control trees and the treated trees had 7 to 17 % more for the 3 treatents (MaxCel + 2, 4-D, GA treatments and combined). Most of the difference was in the accumulated summer leaf drop, August count. A similar result occured with the Hamlin trial. Percentage fruit drop was near 15 % for the Control and all the treatments.. The fruit per tree in the second year after one year of treatments was plus or minus 4 % of the Control for all the treatments and not significant. Fruit quality data was collected and will be presented in the next report..
The evaluation of the up-graded on-line ‘Citrus Flowering Monitor System’ continued with extensive grower and extension use and in making advisories to growers. No adverse comments were received and the system worked very well in collecting data for grower advisories. The totally abnormal weather pattern this past fall and early winter, essentially no cold induction before January 1st, has made advising and deciding on flower bud enhancement and spray scheduling a large challenge for the Florida citrus industry. Although we only have one year of data, it looks like 5 to 10 % open flowers is reached about 21 to 27 days before full bloom. Therefore, open flowers for PFD invasion was about March 3 to 9 for the first wave. This was the same date for stopping psyllid sprays that were not bee friendly. From May 3 to 9 flowers with petals were available to sustain PFD innoculum. The first estimates of vegetative flush were 7 to 10 days earlier than the 5-10 % open flower date suggesting a one week window for flush spray coverage with more effective chemicals for psyllid control.
April 2016 The objectives of this proposal are 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. In the large field trial, there was a greater amount of G. citricarpa DNA found in 2015 leaf litter so that while there was more G. mangiferae than G. citricarpa, it was less than 10 times. In 2014, there was no pattern in the number of leaves with Guignardia structures over time in any treatment but in 2015, the % leaves with structures increased until the third collection date and the started to decline. There was greater G. citricarpa DNA in the control whereas for G. mangiferae there was more DNA in the soilset treatment. The soilset treatment had the lowest disease incidence in 2015 (1st year trt) and 2016 (2nd year). The third year treatment was applied and will be assessed next spring. The bagasse field trials confirmed the laboratory experiments that bagasse increased the leaf decomposition rate compared to nothing or urea. Greater soil moisture also accelerate leaf decomposition. The manuscript preparation is still continuing. Collection of leaf samples from the grove in Immokalee has continued biweekly. Each batch of samples contained 40 samples of 25 leaves collected below 40 trees. Leaves were examined under microscope to check for fructification of Phyllosticta spp. Leaf portions without fructification were discarded and the remainder were immersed in 0.02% tween20 to collect conidia and ascospores. Conidia and ascospores produced in leaf litter were quantified, weather data were collected from FAWN. Data collection is continuing and some of the qPCR data is being processed. In 2014, very little G. citricarpa DNA was found overall while G. mangiferea was high but, substantially more G. citricarpa DNA was detected in the 2015 collections. In general, conidia are always present but ascospores are related to the level of leaf decay. Because there is an increase in pathogen presence in 2015, we have decided to continue sampling since levels were very low in 2014 In Australia, confirmation of the ascospore and conidia production results continues. They continue to sample leaf litter in two groves in Queensland mandarin growing region. Inoculations of fruit are underway in the field but no symptoms from this year. Confirmatory inoculations of promising germplasm accessions are incubating and will be ready soon. They are repeating the fungicide work to confirm previous results. As in 2015, mulch was the best treatment to reduce the amount of leaf litter under trees. The high volume fungicide applications did slightly reduce decomposition of the leaf litter but may not be significant. Our manuscript for the mating type work has been fully accepted and is currently available as a first look at http://apsjournals.apsnet.org/doi/pdf/10.1094/PHYTO-12-15-0338-R
April 2105 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: Manuscript preparation continues for the DMI fungicides. The spore germination experiments for Cannonball and Vanguard are near completion and the data will be analyzed soon. The assays for the SDHI fungicides have been completed and the data are being analyzed and manuscript prepared. Molecular characterization of the SDHB, C, D is underway. Objective 2: CBS was assessed by two methods. On 21 Dec 2015, 21 Jan 2016, and 18 Feb 2016, fruit on trees were assessed by holding a m^2 frame to both sides of three center trees in plots and counting the total number of fruit within. The number of symptomatic fruit with CBS lesions was counted within the same the same square meter. Percentage of fruit with CBS was calculated from the number of symptomatic fruit divided by the total number of fruit. The number of fruit on ground was assessed on 29 Jan 2016. Dropped fruit was counted under the canopy on the ground of the middle three trees within plot and scored for presence or absence of CBS lesions. The number of fruit with CBS lesions were added to the number of fruit without CBS lesions to get a total fruit drop and then the percent fruit with CBS was calculated. Objective 3: No update for this objective was given by Co-PI in charge despite requests. Objective 4: The volatiles from ClO2 in liquid or crystal form were tested to see if they inhibited mycelial growth of G. citricarpa. Both liquid ClO2 and powder ClO2 volatiles at 100 L/L inhibited mycelial growth of G. citricarpa. Liquid ClO2 inhibited mycelial growth by 73%, and powder ClO2 inhibited mycelial growth by 79%. Liquid ClO2 or powder ClO2 volatiles at 50 L/L did not inhibit mycelial growth of G. citricarpa. Another essential oil containing product, Genysis (0.4%v/v) was added to media and the effect on mycelial growth was observed. The product Genysis inhibited mycelial growth of G. citricarpa by 81% after 14 d incubation. We are making preparations for the final report.
April 2016 The objectives of this proposal are 1) To determine the temperature and relative humidity optima for Guignardia citricarpa pycnidiospore infection and production on citrus twigs, leaf litter, and fruit; 2) To determine the relative potential of Guignardia citricarpa to form pycnidiospores on citrus twigs, leaf litter, and fruit; 3) To determine whether Guignardia citricarpa can survive and reproduce on citrus debris on grove equipment. Experiments to confirm initial relative humidity findings continue. After the inconclusive results of the second experiment, we started a third experiment. We used fresh cultures to ensure better pycnidia performance. It is currently incubating Planning of further experiments is underway. A site has tentatively been found to conduct field experiments of inoculum potential and some preliminary work is underway. We have rebuilt a spore tower and have been testing with Diaporthe citri to ensure everything is working. Twigs from a P. citricarpa infested grove were collected and will be tested soon. Studies were conducted to examine P. citricarpa conidiospore survival under conditions existing in the grove and associated equipment. We found that when spores are maintined on inert surface with free moisture incubation periods of up to 3h do not affect the survival of the pycnidiospores at temperatures of 15-30. Spore survival under these moisture and temperature conditions persisted for at least 48 hours. Above 30 C, we saw a rapid decrease in spore survival. At 40 C spore survival of 1 to 2% was observed at 12 and 24 hours and no spores were able to survive for 48 hours. Similarly, for 50 C spore survival dropped to 1 to 2% by 3h and no spores survived for 12 hours. Spores deposited in free moisture but allowed to dry exhibited very poor survival regardless of temperature treatment. Upon rehydration, no spores survived beyond 3 hours.
April 2016 The objectives of this proposal are 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. In the large field trial, there was a greater amount of G. citricarpa DNA found in 2015 leaf litter so that while there was more G. mangiferae than G. citricarpa, it was less than 10 times. In 2014, there was no pattern in the number of leaves with Guignardia structures over time in any treatment but in 2015, the % leaves with structures increased until the third collection date and the started to decline. There was greater G. citricarpa DNA in the control whereas for G. mangiferae there was more DNA in the soilset treatment. The soilset treatment had the lowest disease incidence in 2015 (1st year trt) and 2016 (2nd year). The third year treatment was applied and will be assessed next spring. The bagasse field trials confirmed the laboratory experiments that bagasse increased the leaf decomposition rate compared to nothing or urea. Greater soil moisture also accelerate leaf decomposition. The manuscript preparation is still continuing. Collection of leaf samples from the grove in Immokalee has continued biweekly. Each batch of samples contained 40 samples of 25 leaves collected below 40 trees. Leaves were examined under microscope to check for fructification of Phyllosticta spp. Leaf portions without fructification were discarded and the remainder were immersed in 0.02% tween20 to collect conidia and ascospores. Conidia and ascospores produced in leaf litter were quantified, weather data were collected from FAWN. Data collection is continuing and some of the qPCR data is being processed. In 2014, very little G. citricarpa DNA was found overall while G. mangiferea was high but, substantially more G. citricarpa DNA was detected in the 2015 collections. In general, conidia are always present but ascospores are related to the level of leaf decay. Because there is an increase in pathogen presence in 2015, we have decided to continue sampling since levels were very low in 2014 In Australia, confirmation of the ascospore and conidia production results continues. They continue to sample leaf litter in two groves in Queensland mandarin growing region. Inoculations of fruit are underway in the field but no symptoms from this year. Confirmatory inoculations of promising germplasm accessions are incubating and will be ready soon. They are repeating the fungicide work to confirm previous results. As in 2015, mulch was the best treatment to reduce the amount of leaf litter under trees. The high volume fungicide applications did slightly reduce decomposition of the leaf litter but may not be significant.
Two citrus groves, one – 20 year-old Hamlin sweet orange trees predominately on Swingle rootstock and a second consisting of three year old Hamlin sweet orange trees on Swingle rootstock have received acid injection to selected blocks with and without sulfur applications for twenty-four months. Irrigation water was acidified at one of four target water pH (7.5, 6.0, 5.0, and 4.0). A controlled released form of elemental sulfur (Tiger 90) was allied at a rate of 500 pounds per treated acre to plots receiving either acidified irrigation water or control plots receiving irrigation water that was not acidified in December. Significant increases in nutrient concentrations of leaves collected in March 2016 were found in plots at both sites with reduced water and/or soil pH. Average mature leaf calcium concentrations have increased by 13.6% from an average of 2.32% when the study began in 2013 to 3.37% in the spring 2016 samples. Similar increases in leaf Mg (5.7%), Zn (16.9%), Fe (11.1%), Mn (8.6%), and B (7.8%) were found compared with no substantial increase in leaf nutrient concentrations for the control trees with no irrigation water or soil pH moderation. These results may indicate increased nutrient uptake from soils with soil solutions below 6.5 and is presumed to be because of lower soil pH levels (4.0 to 6.0). Average water uptake by trees affected with HLB continued to be 20%-25% lower than healthy trees in the greenhouse lysimeters. Water uptake for trees receiving water supplemented with calcium bicarbonate was significantly reduced (10-15%) compared with health trees but not to the extent found in the HLB affected trees. For the first time, tree root densities were significantly different for healthy trees irrigated with water supplemented with calcium carbonate (1.2 cm/cm3) when compared with healthy trees irrigated without supplemental calcium carbonate(2.1 cm/cm3). Tree size were similar for HLB affected and healthy trees irrigated with calcium carbonate but significantly smaller than healthy trees not receiving modified irrigation water.
The objective of this research project is to develop and study a potential non-phytotoxic, environmentally-friendly film-forming ACP repellent material for preventing HLB infection. In the last reporting period, OS-SG 11, 12 and 13 were studied for plant safety and rainfastness and these formulations were delivered to our collaborators for ACP infection trials. In this reporting period, a new series of formulations OS-SG 15 were developed using an EPA approved polymer, dispersant (EPA approved “for food use” only) and clay source (EPK clay, local supplier). This version of material is intentionally developed to have a fair comparison with an existing commercial product (Surround WP; as proposed to be used as control). Optimization process involved adjusting ratios of different components (dispersant, polymer and silica-alumina) to have best combination of formulations to achieve high colloidal stability in aqueous solution and high leaf surface coverage for foliar application. The material characterization was done using Fourier Transform Infra-Red (FT-IR) spectroscopy. FT-IR spectra suggested interaction between the EPK particulate with other ingredients (polymer and dispersant materials). The colloidal dispersity in aqueous solution of formulations was measured using UV-VIS transmittance technique and the measurements were done at different time intervals. High colloidal dispersity was achieved up to 4 hours which was comparable/improved compared to Surround WP control. Safety analysis and plant leaf surface coverage of OS-SG 15 formulations were conducted using Cleopatra orange (common citrus variety) as a model plant. The formulations were sprayed at the application rate of 0.5 lbs/gallon (recommended rate for the commercial control). Phytotoxicity studies were conducted using a Panasonic Environmental Test Chamber (Model MLR- 352H) to control light intensity, humidity and temperature cycling to simulate summer conditions (85% RH, 34 degree C). The formulations revealed high plant leaf surface coverage at the application rate which was comparable to the commercial control. OS-SG 15 series did not cause any plant tissue damage at the applied rates, neither the commercial control. Temperature testing was conducted to determine if the presence of polymer posed a risk of increasing the heat buildup on the plant surface. The OS-SG 15 series did not exhibit any significant increase in temperature over the commercial control. Film adherence (rainfastness) will be studied using AAS and atomic force microscopy (AFM). From the OS-SG 15 series, one or two best performing formulations will be delivered for the ACP trial.
Background information The objective of this project is to quantify the relative effect of copper (Cu), windbreak (Wb) and leafminer control (Lc) on the spatial and temporal progress of Asiatic citrus canker (ACC) under conducive conditions for epidemics and disease loss. The experiment is set up in a 10 ha plot planted with Valencia sweet orange grafted on Rangpur lime located in the municipality of Xambre, Parana, Brazil. The different treatments are the combination of up to three control measures (Cu, Wb, Lc) or none. The presence or absence of windbreak represents a plot. The presence or absence of copper sprays and leafminer control represents a subplot. Each subplot is composed of 112 trees. Each of the eight treatments has three replicates. Cu treated plots are being sprayed with Kocide (35% metallic copper) at 1 kg metallic copper/ha every 21 days. Lc is being performed with application of abamectin at 150 ml/ha every 21 days. Casuarina is used as a natural Wb around the plots. Disease evaluations started in December 2013 and include percentage of trees, leaves and fruits with ACC symptoms, and fruit yield. In the second season, the assessment of fruit drop was included. The present CRDF funding will cover the period of November 2015 to October 2016. Previous results Trees and leaves: Up to November 2015 (22 after ACC epidemics begun), incidences of diseased trees in the plots with complete management and no management reached 43.8 and 97.9%, respectively. The incidence of leaves with ACC in the canopy of affected trees dropped for all treatments and reached 0.6 and 2.5%. Fruits and yield: In the first harvest (September 2015), the incidences of symptomatic fruits from trees treated with the tree measures and none were 3.8 and 58.5%, respectively. Production of trees revealed the same trend observed for other assessments. Fruit yield of trees under complete management (40 kg/tree) was 186% higher than control trees (14 kg/tree). Latest results (February 2016 – 25 months of epidemics) Trees and leaves: Incidence of trees with citrus canker reached near 100%, except for trees with complete management (85%) or with the combination of copper sprays and leaf miner control (76%). Incidence of leaves increased since November 2015 and reached 5.6% for unmanaged trees and 1.1% for trees under complete management. Fruits and yield: Incidence of fruits with citrus canker on the trees reached 50.1% for the unmanaged trees and 5.8% for trees under complete management. Likewise, fruit drop per tree is, up to the present report, at 3.9 and 22.2 for unmanaged and managed trees, respectively. Harvest is planned for August-September 2016. All other treatments are showing intermediate results. Copper is more important for retarding disease dissemination among trees, whereas windbreaks are more important for reducing incidence of leaves with citrus canker on affected trees.
This project was initiated in 2014 and is focused on understanding the effect of nutrients applied through foliar fertilization programs (FFP) on HLB-affected trees in the Indian River marketing district. Two research trials have been established in commercial mature grapefruit groves in St. Lucie County and a young tree trial is being conducted at the UF-IRREC grove. Grove 1 has ~25 years old of ‘Flame’ grapefruit on Swingle rootstock. Grove 2 utilizes ~7-year-old ‘Ruby Red’ on Sour orange trees. Trial 3 is looking at the effect(s) that foliar fertilizers have on young tree growth and their ability to protract HLB disease symptoms in 2-year-old ‘Ray Ruby’ grapefruit on Kuharske rootstock. Combinations of macro and micronutrient treatments initiated on all three trials in February 2014 and applications have been made quarterly since. During the 2014 and 2015 production seasons, the population of the Asian Citrus Psyllid present in the groves were recorded using sticky traps. The monitoring shows a consistent decrease in the psyllid populations from 2014 until the recent counts in March 2016. The latest data had an average of only 0.02 psyllids per trap. However, the decrease in the psyllid population has not had an effect in HLB titer in the trees. The real time PCR analysis in trees exposed under each treatment has revealed a constant status of tree infection. The latest results (Jan 2016) showed a 100% presence of HLB in trees used in the experiments at the two commercial groves. There have not been any significant differences in the tree growth among treatments durig the 2 seasons in terms of tree volume or canopy density (a>0.05). Nonetheless, the canopy density (evaluated through Leaf Area Index (LAI) measurements) showed a trend of lower LAI in the control treatment compared to the rest of the treatments (receiving foliar applications) in one of the commercial groves. After two harvests, there has been no difference in juice quality parameters among treatments. There has, however, been a significant increrease in the percentage of medium size fruit (sizes 36 and 40) of trees treated with urea, di-potassium polyphosphate, and potassium phosphite compared to the control. This increment is reflected in an increase of 37% in the Gross Packed Value (GPV). (The GPV is the value of fruit when sold from a packinghouse and calculated by the number of fruit produced in each pack size and value of each size of fruit at the time of harvest). It is important to note that, although the other foliar treatments did not have significantly higher GPV compared with control (a>0.05), the GPV of these treatments were all 13%-33% higher and ranged from $85 to $103 per tree, compared to an average GPV of $75 in the untreated control trees.
The objective of this project is first to identify a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian citrus psyllid (ACP). The toxicity of the selected toxin will then be enhanced by addition of a peptide that binds to the gut of ACP. This peptide addition to the toxin is expected to enhance both binding and toxicity against ACP. The identification of Bacillus thuringiensis strains with basal toxicity against ACP was conducted by means of a series of bioassays using trypsin-activated toxin as described in previous reports. A new logistic regression analysis indicated that seven isolates showed promise with ACP mortality at 500ug/ml relative to control treatments. A single strain was selected for further analysis and individual toxins expressed by this strain were identified by LC-MS/MS analysis. Two individual toxins shown to have toxicity to ACP in bioassays were selected for modification with gut binding peptide 18. These modifications are now underway. Honeydew production data collected during the course of ACP bioassays both with toxin mixtures from Bt strains and with individual toxins, were analyzed to determine whether ACP died from starvation. Toxin-mediated damage to the gut epithelium can result in an anti-feedant effect. Honeydew excretion was monitored on the third, seventh and eleventh day of ACP feeding with the relative quantity of ACP excretions present in each feeding chamber recorded. Based on these data, toxin mixtures derived from four of the Bt strains had an anti-feedant effect on ACP, resulting in mortality from starvation.
Using choice and no-choice probing behavioral assays, we studied the orientation of adult Asian citrus psyllids (ACP) to chemical odorants from a distance and the interaction between visual attraction to yellow and olfaction or gustation. We confirmed that the 3-component blend of 3.5 formic:1.6 acetic acid:1.0 p-cymene is the optimal blend of those compounds to maximize ACP probing behavior. The yellow color acts as a long range cue that attracts the psyllids to the point of alighting on a yellow wax substrate. Probing is thought to be a reflexive action that is initiated upon landing on any substrate. Once the psyllids initiated probing on wax beads containing odorant blends, the 3-component blend had a stimulatory effect that increased the number of probes along with the size and complexity of the salivary sheaths left behind in the wax substrate. To support the hypothesis that increased probing was due to phagostimulants perceived by mouthparts and not olfactory cues perceived by antennal receptors, a no-choice experiment was performed to study ACP probing behavior through plastic film into wax beads thereby isolating antennal receptors from the odorant or tastant cues. Yellow w wax beads were prepared without tastants or with the 3-component blend (3.5:1.6:1 formic acid: acetic acid: p-cymene). Wax beads were covered with a polyethylene/polybutylene plastic film (Saran Cling Plus Wrap, S. C. Johnson & Son, Inc., Racine, WI, USA) to minimize the potential for olfaction or contact chemoreception associated with antennal tapping behavior. The film is impermeable to water and we assume to the larger molecules of the phagostimulant blend. Fifty D. citri adults were placed in each of four cages for 21 h. Each cage contained 5 cover slips with yellow wax beads prepared with or without tastants and with or without a covering of plastic film. The wax beads were collected, stained with Commassie Blue and scored for salivary sheaths. Stained salivary sheaths were removed from the wax substrate by gently lifting the plastic film and separating it from the substrate. The length of the stained salivary sheaths adhering to the plastic film was measured under a stereo microscope. Ten salivary sheaths were measured for each treatment (plastic film over wax beads without tastants and plastic film over wax beads with the 3-component tastant blend). The number of salivary sheaths produced on the plastic film were counted. The length of salivary sheaths were measured using a Cell Sens Dimension software (Olympus Corporation, Tokyo, Japan). ACP adults probed more on yellow wax beads containing the 3 component blend compared with wax beads without tastants. There was no effect of plastic film on the number of salivary sheaths observed in the wax beads. The mean number (+ SEM, n = 10) of salivary sheaths observed on yellow beads without tastants was 47.5 + 5.3 and on yellow wax beads containing the 3-component blend was 94.5 + 8.5 sheaths. Microphotography of the salivary sheaths that adhered to the plastic film revealed differences between sheaths recovered from beads containing the 3-component tastant blend and sheaths recovered from beads without tastants. Salivary sheaths recovered from beads containing tastants were 4.5 times longer (208 + 22 m) compared with sheaths recovered from beads without tastants (46 + 7 m). Sheaths recovered from beads with tastants were longer with filamentous portions extending beyond the thicker initial sheath. We conclude that the blend of 3 tastants is a phagostimulant that acts through perception by chemoreceptors located on the psyllids mouthparts. George, J., P. S. Robbins, R. T. Alessandro, L. L. Stelinski and S. L. Lapointe. 2016. Formic and acetic acids in degradation products of plant volatiles elicit olfactory and behavioral responses from an insect vector. Chem. Senses doi: 10.1093/chemse/bjw005
Objective 1, investigating the specificity and efficacy of immune priming response in ACP to pathogenic bacteria, is nearing completion and will be analyzed during the next quarter. Subsequent experiments to address the effects of non-pathogen immune priming to pathogenic bacteria are underway, and should be nearly complete by this summer. The second project objective, to determine whether double strand RNA (dsRNA) uptake can result in immune priming in D. citri, we will be conducting RNAi experiments on D. citri adults of a CLas-free laboratory colony. dsRNA of genes that exist (e.g., V-type proton ATPase) or do not exist in D. citri (a bacterial GFP gene) will be used for this experiment. Briefly, primers targeting GFP fragments in plasmid pCMV-GFP (Matsuda and Cepko 2004) or a vATPase gene fragment of D. citri [based on sequences determined in a published work (Vyas et al. 2015)] wereover time. Insect mortality will be recorded every 24 h. Depending on the results, we may also attempt to determine the effects of RNAi-treatments on D. citri transmission of CLas in the coming months. designed. The primers were synthesized with T7 promoters added to the 5′ ends (Table 2). Amplification products obtained by using these primers and their template DNA will be purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA). Purified amplicons will be used for dsRNA synthesis using the MEGAscript RNAi kit (Ambion, Austin, TX). RNAi treatments will be conducted by feeding D. citri with 30% sucrose solutions including 100 ng/ul of each type of dsRNA, using previously described methods (Wuriyanghan, Rosa, and Falk 2011) with slight modification. For the negative control, products of a non-template RNAi synthesis reaction will be used. The insects will be kept on the diets for 24 h. RNAi-treated insects will be moved to caged citrus plants (var. “Swingle”) for one week. The insects will then be challenged with Serratia marcescens by microinjection of bacterial suspensions (~107 cells/ml) using a FemtoJet Microinjector (Eppendorf, Inc., Fremont. CA). Effects of RNAi-treatments on Serratia marcescens growth will be determined by qPCR using primers specific to this bacteria (Joyner et al. 2014). Insects will be sampled every 48 h, over two weeks. A separate cohort of the challenged insects will be kept on the citrus plant and monitored for their survivorship
A second thermal treatment was applied to trees in our field assay during late November 2015. Adults and nymphs were enclosed in mesh sleeves on trees for acquisition feeding approximately 5 weeks following treatments during early January and March 2016. Insect and leaf samples were collected after 10d of acquisition feeding or upon adult emergence to assess adult and nymph acquisition, respectively. These samples are currently undergoing nucleic acid extraction and PCR to determine CLas titers. A second field site, consisting of a commercial grove in Lake Wales, was added to our project this quarter. We collected leaf samples this site for quantification of CLas titers. Trees are expected to be treated with thermal therapy in the next couple of weeks, and we’ll do an adult acquisition trial on those in about 2 months. led environmental chamber will be used to apply heat treatments to trees for use in subsequent acquisition experiments For the lab assay, we evaluated a fungicide treatment to improve the survival of the potted trees. Trees in the greenhouse tend to get secondary infection following thermal therapy, so once we have a method for improving the tree lifespan post treatment, laboratory thermal therapy assays will begin. Trees were inoculated with CLas three months ago, and will be tested using PCR during late March to confirm that they are positive prior to use in the lab assay.
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