The objective of this project is to determine if various combinations of ground and foliar applied fertilizer nutrients, coupled with psyllid control, can rejuvenate young 3-year old HLB-symptomatic trees in a grove with 14% HLB incidence, allowing them to grow and bear an economically viable yield of quality fruit for juice processing. The field experiment was established in a commercial 58 acre-grove of 3-year-old ‘Valencia’ / Kuharske Carrizo trees using a combination of three different foliar and three different ground applied nutritional programs. Factorial AxB treatments consist of A) ground-applied: 1) Liquid/dry+Ca (‘BHG standard’), 2) Liquid+Ca, 3) Liquid/dry-Ca B) foliar-applied: 1) ‘BHG standard’-Ca, 2) ‘BHG standard’+Ca, 3) ‘Prescription'(+Ca). The prescription treatment was designed to be dynamic, customized for optimization, with feedback for development based on frequent leaf tissue analyses, visual symptoms, and the growth of the tree canopies and yield. There are six replications of the treatments, with two being pure replications. By the time the project was approved and we began treatments, the HLB incidence was already approximately 30%, and all HLB-symptomatic trees showed severe canopy decline. In the three following years, HLB incidence rapidly increased to near 100% by the third year of the experiment, and yet the severity of HLB symptoms decreased steadily so that by year two, HLB symptoms were difficult to identify in the grove. At the end of the project period, all treatments continued to perform equally well, which was quite difficult to comprehend given that the grove is practically 100% HLB-affected. Yields per tree have increased from year to year, averaging 1.14 boxes/tree for the 2014/15 season, and passing the juice quality requirements for harvest. Unfortunately the commercial block was set widely at only 151 trees/acre, resulting in only 172 boxes/acre at age 6 years. Future higher density plantings (300-500 trees/acre) of similar HLB-affected trees responding favorably in the same way could potentially realize a respectable 342-570 boxes/acre. The lack of experimental treatment response in this project was unexpected and disappointing. However it did serve a very important purpose in demonstrating that the reason for the inconsistent performance of HLB-affected young groves in Florida lies more in the soil and water of the site or the basal management level used in the grove than in the multiple applied enhanced nutritional programs being used. The most advanced nutritional treatments often will not elicit a response on HLB-affected trees on one (unresponsive) site and yet the simplest standard grove fertilization will be sufficient on another (responsive) site. Obviously complete absence of grove fertilization will cause rapid decline and collapse of HLB-affected trees on any site. In this study site we could not identify any special soil amendment, pesticide or fertilizer that had been used before or during the experiment, that could explain the complete recovery of HLB-affected trees in the Valencia block. It is noteworthy that the grove is irrigated primarily with surface (pond) water, but that does not account for the initial poor condition of HLB-affected trees in year 1 of the project, and in the three preceding years before the project began. This experiment site and others with similar rehabilitation success of HLB-affected trees may contain important information waiting to be discovered, specifically the site conditions (soil and /or water) that permitted the sustainable recovery and reversal of HLB symptoms, putting the grove back on track to economic production. The Blumberg grove will be one of the 24 survey sites to be used in subsequent studies. By spreading the survey over a wide range of responsive and unresponsive grove sites, we aim to identify the key components responsible and then home in on developing a remedy that can be replicated in any HLB-affected grove in Florida.
The low concentrations (1/4 rate) of Citrus Fix (2, 4-D) and MaxCel (Cytokinin) with or without ProGibb (GA) every 45 days to Hamlin 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 249 leaves for the Control trees and the treated trees had 3 % more for the MaxCel + 2, 4-D or GA treatments and the combined treatment of all three had 4 % less loss of leaves. Percentage fruit drop was equal to the Control (22.5 %) or higher by one or four %. Two additional tests on Hamlin trees of 2, 4-D + GA compared top Headline, Priaxor or a Control did not affect fruit drop at either site. Average fruit drop for both 1 and 3 (decline) rated trees for all treatments ranged from 27.1 to 29.6 % at one site and 22.5 to 24 % at the other site. Highest rates of fruit loss occurred in October (2 week periods) and decreased to about 1/3rd to 1/4 of these after November 4th at both sites. The test comparing GA rates on root growth of HLB infected Valencia trees was completed and had less effect on root growth than the test on healthy trees. New growth of roots from the bottom third of the pots averaged 25 and 35 cm at 4 and 8 weeks for the highest GA level (.1 gm) while the controls averaged 14 and 20 cm. For the middle of the pot the best growth was for the lower concentration (0.00125) but only at 8 weeks, 18/27 versus 23/23 for the control. For the top third of the pot, there was little differnce between GA concentrations and the control (22/30 versus 20/36). At the end of 8 weeks the healthy trees has 160 gm of roots while the HLB control plants had 100. The best GA treatment (.0025 gm) had 132 gm versus 100 for the control. For dry wt the 0.00125 gm/plant was best and had 22, 18 and 26 % more root, stem and leaf weigth, respectively, than the control. Results were not as consistent as the previous test with healthy plants, but variability due to level of HLB infection probably is responsible. It does appear that there was a tendency for GA to improve root growth within the first 4 to 8 weeks of treatment.
The up-graded on-line ‘Citrus Flowering Monitor System’ has been tested extensively by grower and extension use, plus in making advisories to growers. Five advisories were prepared based on the models predictions by the end of December 2015. 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 a large challenge for the Florida citrus industry. The fist estimates of vegetative flush will be added to advisories in February along with 5-10 % open flower estimates. In analyzing the data from last years bloom, some deficiencies in data collection were discovered that will be corrected in this springs data collection.
Collection of trap catch and leafminer infestation data continued apace in citrus groves at three locations that received a second year of application of DCEPT CLM . Groves were monitored for disruption of trap catch and for citrus leafminer damage and populations. Citrus flush was sampled twice during 2015 and exhaustively evaluated for the number of mines present and the developmental stage of the leafminer larvae present within the mines. We also tabulated the degree of infestation by leaf and shoot. Analysis of these data was delayed due to hiring difficulties at USDA headquarters. Results will appear in the next quarterly report for this project. In general, it is clear from field inspections that biological control of the citrus leafminer by the introduced parasitoid Ageniaspis citricola has been eliminated from the three test locations . We assume this is due to the elevated number of insecticide applications made to control the Asian citrus psyllid, vector of the citrus greening pathogen. It is now rare to observe parasitized leafminer larvae at those locations. The unfortunate result for growers is that populations of citrus leafminers have increased dramatically, making the successful implementation of mating disruption more difficult. Nonetheless, tat least one grove manager in our test study has been impressed with the suppression of leafminer populations in blocks receiving the DCEPT CLM product and has expressed interest in continuing to participate in future trials if the cost of the product is reasonable. At another location, the decline of mature groves due to greening is seen to eclipse the benefit of CLM control.
1) As in previous years, ACP egg lays decreased substantially in cooler weather resulting in fewer embryos for injection, a fewer adults to maintain the colony. 2) During this quarter 524 Asian citrus psyllid (ACP) control uninjected eggs were set-up yielding a 36.3% nymphal hatch rate, resulting in an approx. increase of 11% above the previous quarter. 1,314 eggs were micro-injected with transformation vector/helper plasmids having a 4.2% hatch frequency, approx. 1% greater the previous quarter. Of the 55 newly hatched nymphs from injected eggs, 22 survived to adulthood and were backcrossed to wild (uninjected) ACP adults. Though none of the 63 G1 offspring exhibited DsRed fluorescence, a significant improvement in ACP viability post-injection from nymph to adulthood was achieved. Further improvement may be possible after completion of the environmental chamber retro-fit for temperature, humidity and lighting control. 3) To further improve embryo injections, nymphal hatching and survival to adulthood tests have been initiated to determine optimal heat shock temperature and duration. 722 eggs were collected and placed on tape, but not injected. After 18 hours 505 were heat shocked at 37 C for 45 min of which 20.2% hatched as nymphs, while 217 eggs that were not heat shocked (maintained at 25 C) had a hatch rate of 18.4% indicating that typical heat shock conditions did not have a negative effect on nymphal hatching.. 4) Reverse-transcriptase PCR (RT-PCR) was continued to test DsRed marker expression in ACP injected with vector plasmid (containing the IE1-DsRed marker) to determine if the IE1 baculovirus promoter functions normally in this species (as it does in caribfly, A. suspensa). For the A. suspensa controls and ACP samples, 400 embryos from each species were injected with pBac[IE1hr5-DsRed] vector plasmid and incubated for 24 hr before RNA extraction, and subsequent RT for cDNA production. cDNA from each sample and uninjected ACP and a water control were then subjected to PCR using DsRed primers which yielded positive DsRed expression in both A. suspensa and ACP, though approximately 2-fold higher in A. suspensa. This indicates IE1 promoter does function in ACP, though possibly less functional compared to A. suspensa. Tests will be continued to verify and quantify this activity, and similar tests will be performed to quantify piggyBac transposase expression in ACP.
Host plant resistance can be expected to provide the foundation for successful management of Asian citrus psyllid (ACP) in the future. We performed choice assays to assess antixenosis and no-choice assays to identify antibiosis effects of Poncirus trifoliata on host selection behavior of ACP to preferred and non-preferred hosts and studied how the chemical constituents of host plants affect oviposition and nymphal development. Some trifoliate accessions exhibited reduced larval and adult development (antibiosis) and reduced colonization (antixenosis). Experiments were performed to explore these aspects of decreased oviposition and nymphal development in trifoliates and the concurrent reduced feeding by ACP. Electrical penetration graph (EPG) studies were performed on resistant and susceptible accessions to characterize feeding from xylem and phloem and to provide insight into physical barriers in plant vascular elements that reduce phloem ingestion. EPG is used to characterize feeding behavior of piercing-sucking insects such as aphids, psyllids, whiteflies etc. While the insect probes, ingests, and salivates within the food source, characteristic voltage waveforms are produced that, in conjunction with histological studies, allow researchers to determine feeding patterns associated with acquisition and inoculation of pathogens. A primary limitation to this method is data analysis. Recordings of multiple insects for >3 hours generate gigabytes of data. Classification of data into insect feeding states representing ingestion, salivation, or other activities is typically accomplished through visual inspection of waveforms and manual annotation by comparison to published standards. Analysis is time consuming, requires expert training and manual annotation that precludes high-throughput analysis. We removed the data analysis bottleneck through application of machine learning algorithms designed to teach a computer to recognize and learn from minimal human coding. Machine learning algorithms can transform raw EPG data to feeding states with little or no human input. We used supervised and semi-supervised machine learning models to annotate ACP feeding waveforms and discovered previously unrecognized feeding states. With minimal (5%) human annotation of EPG recordings, machine learning models classified the remainder with >95% accuracy. A manuscript has been submitted for publication. Further analysis of feeding states should provide insight into the nature of pathogen transmission and allow identification of characteristics that render certain plant varieties more resistant to pathogen infection.
Volatile compounds often serve as cues for orientation by phytophagous insects to their host plants. However, all citrus volatiles studied to date in Y-tube olfactometer tests or field tests have elicited little to no orientation from the Asian citrus psyllid (ACP). We devised a probing choice assay to measure short range orientation and subsequent probing behavior by ACP on a wax substrate containing odorants. ACP probing increased with addition of a blend of formic and acetic acids compared with a control. A 2:1 blend of formic:acetic acids received the highest number of probes compared to single odorants including formic acid, acetic acid, ocimene and citral. Our results demonstrated that acetic and formic acids (and perhaps others compounds) act as phagostimulants for ACP. These novel results have been accepted for publication in the journal Chemical Senses and will be more fully described in the next report. We also studied the effect of incorporating additional compounds into multiple component blends to increase the stimulatory effect of acetic and formic acids on probing behavior of adult ACP. Compounds identified as stimulatory to olfactory receptors by single-cell recordings from neurons in ACP antennae were selected as candidate compounds. Experiments were performed using geometric mixture designs and response surface methods to identify primary drivers in 5- and 3-component mixtures to identify an optimal blend of odorants to maximize probing by ACP. We also studied the interaction between visual attraction to yellow and olfaction or gustation. Test compounds were incorporated into a slow-release wax matrix (SPLAT , ISCA Technologies Inc., Riverside, CA) and offered to caged ACP adults. Treatments (white or yellow SPLAT with or without odorants) were applied as 1 ml wide strips or beads to glass cover slips and arrayed on the floor of a cubical cage. Cohorts of ACP adults were starved for 6 h and released into each cage and allowed to move freely and probe on beads for 21 h. The beads were stained with Coomassie blue dye to visualize stylet sheaths produced by feeding attempts on the wax beads. There was a strong interaction between color and odorant. The yellow beads always received more probes compared with white beads. Addition of 1 (formic acid), 2 (formic and acetic acids) or 3 (formic acid, acetic acid and para-cymene) resulted in progressively more probing behavior on yellow beads, but not on white beads. Ethyl-butyrate and myrcene had no effect or a negative effect on probing behavior. The optimal 3-component blend will be reported as a phagostimulant blend in a manuscript under preparation. This blend will be further tested in greenhouse and field trials. This phagostimulant may be used in an attract-and-kill strategy for ACP control.
The large-scale validation of citrus leafminer (CLM) disruption with the ISCA DCEPT CLM technology came to an end for the 2015 season and we are analyzing and processing data currently. We are currently in the process of facilitating data analysis to determine how much better the technology worked on a larger scale as compared with previous seasons. We have continued to collaborate and support Dr. Stephen Lapointe’s laboratory for this research. This has required counting traps, measuring damage levels by CLM, and also performing advanced statistical analyses to determine efficacy of the products that we were are testing. One of the factors we have continued to investigate was dormant season applications of pheromones for management of CLM. After winter treatment, dispensers provided >85% disruption of male moth catch in traps for 37 weeks, and after spring treatment >92% for 26 weeks, but there was only a 12% reduction in leaf infestation in spring. Two applications were not better than only a single application in spring. Disruption of moth catch was weaker in treated plots where traps were placed high (3.1 m) rather than low (1.6 m) in the tree canopy. Dispensers provided effective and persistent disruption of male catch in pheromone-baited monitoring traps but were minimally effective in reducing leaf infestation by ACP. Winter application of pheromone did not reduce leaf mining in spring compared with spring application alone. Tops of trees may have provided a refuge for mating. Mining of leaves in treated plots may have derived from inadequate disruption of mating in the upper canopy of trees or influx of mated females from neighboring areas, or because of dissipation of pheromone near edges of treated areas. Border row application of pheromone requires further investigation, as well as, other application methods of the pheromone. The possibility of fly-in mated females requires significantly more investigation as well, as we have begun this research. Inadequate disruption in tops of trees may be fixed by placing pheromone dispensers at the very top of tree canopies as is for other lepidopteran species. We have also begun investigating this hypothesis. Dissipation of pheromone due to edge effects may be decreased by increasing size of the treated area even beyond our current efforts or by increasing density of dispensers near borders of treated areas. Insecticide applications near borders might also improve control of this species within pheromone-treated areas. Our results corroborate previous investigations and suggest that management of CLM with mating disruption on a small scale may be ineffectual, despite the fact that this was the largest trial ever realized for CLM. It appears that the size should be of ever larger scale. Our results point to the likely need for even larger-scale area wide treatments for effective mating disruption of ACP than we had previously expected. However, we were able to effectively reduce CLM damage with these treatments.
Although harvests are not complete for the Hamlin tests at all three of the sites, leaf drop and fruit drop counts are completed through different dates in December for each site. Leaf drop was not greatly different between the sites, nor were large differences found between the low concentration treatment and the control trees. Total leaf drop for Treated vs Control at each site was 370 vs 340; 243 vs 254, and 231 vs 233 total leaves for the fall count period. There was very little difference between 1 rated and 3 rated (decline) trees. Leaf drop was much heavier during September and early October than later. In one case almost 70 % of the leaf loss occurred by October 8th. This late summer, early fall leaf drop did not occur prior to HLB. Fruit drop was more uniform from late September until December. Per site drop rates for Treated vs Control trees was 19 vs 15.5, 18.8 vs 19.4 and 24.5 vs 25.1. There was usually only 1 or 2 % difference between 1 and 3 rated trees. Although fruit drop was reduced by the low concentration sprays of PGRs a year ago, there does not appear to be any effect on this year’s drop in the Hamlin cultivar. Valencia data is now being collected.
As previously reported, trees were steam-treated as described in our research proposal during July 2015. Adults and nymphs were enclosed in mesh sleeves on trees for acquisition feeding approximately 5 weeks following treatments. Following acquisition feeding, insect and leaf samples were collected (45 d post-treatment) from trees and taken to the lab for subsequent nucleic acid extraction and analysis. Acquisition feeding assays were repeated approximately two months later, with samples collections beginning 114 d post-treatment. Nymphs were collected from plants after adult emergence, until no psyllids remained in the mesh sleeves. The titer of Candidatus Liberibacter asiaticus (CLas) in trees receiving steam treatment did not significantly differ from untreated trees on days 0, 45, or 114 post-treatment (p = 0.99, 0.11, and 0.81, respectively; Tukey’s Honestly Significant Difference (HSD) test); however CLas titers in treated and untreated trees were lower at 45 d post treatment as compared to days 0 and 114 post-treatment. This is likely due to naturally-occurring seasonal decreases in CLas titers. CLas titers were significantly higher in steam-treated trees than untreated trees on day 0 as compared to day 45. CLas acquisition by adult psyllids enclosed on trees receiving thermal treatments did not differ significantly from acquisition by adult psyllids on untreated trees. Samples from CLas acquisition feeding assays with psyllid nymphs are still being processed. Based on these results, which indicated the thermal treatments applied during July 2015 did not reduce plant CLas titer or psyllid acquisition, a second thermal treatment was applied during late November 2015. In early January 2016, adults and nymphs were enclosed in mesh sleeves on trees for acquisition feeding approximately 5 weeks following treatments. Insect and leaf samples were collected after 10d of acquisition feeding or upon adult emergence to assess adult and nymph acquisition, respectively. In addition, we have initiated a complementary laboratory study to evaluate the effect of thermal therapy on acquisition of CLas under controlled conditions. Two year old Valencia trees were inoculated with CLas by enclosing plants with CLas-infected psyllids for two weeks. Currently, plants are being held in a secure, insect-free greenhouse until they are determined to be positive for CLas. At that time, a controlled environmental chamber will be used to apply heat treatments to trees for use in subsequent acquisition experiments
The goals of this project are to: 1. Continue monitoring ACP field populations for insecticide susceptibility in Florida. 2. Develop a useful tool to improve monitoring for resistance and to make such monitoring quick and easy. 3. Refine and implement effective rotation schedules based on understanding what is taking place in the field and our understanding of the fundamental resistance mechanisms in ACP. We are looking for development of an inexpensive, standardized diagnostic method to monitor insecticide resistance for all citrus grower against Asian citrus psyllid (ACP), Diaphorina citri, in Florida. This experiment used susceptible laboratory population of ACP was reared in a greenhouse at University of Florida, the Citrus Research and Education Center, Lake Alfred, FL. Tested insecticides were analytical grade and included bifenthrin (98%), dimethoate (99.5%), fenpropathrin (99.1%), imidacloprid (99.9%), cyantraniliprole (98.%), sulfoxaflor (99.5%), flupyradifurone (99.5%) and spinetoram (76.2% J-21.0% L) representing several insecticide classes. According to the manufacture recommendation, the insecticides were stored in freezer, room temperature and a cool dry place, respectively. About 0.01 or 0.1g of each chemical was weight and mixed with acetone (assay 99.5%). Various concentrations of the chemicals were prepared using the serial dilution procedure. Dilutions of technical insecticide in acetone were made to generate test concentrations ranging from 0.0001 to 10000 ng/ l. The bottle bioassay method was used to evaluate direct toxicity of insecticides against ACP adults to determine the LC50 and time to 100% knockdown rate. Each experiment was repeated three times and replicated five times. Toxicity of ACP was assessed 24 hours after treatment. Time to 100% knockdown rate was observed after 15, 30, 45, 60, 75, 90, 105 and 120 min. Results showed LC50 values of test insecticides ranged from 0.05 to 0.84 ng/ l. The 100% knockdown rate within the 1 hour period time was between 1000 to 10000 ng/ l with the exception of sulfoxaflor, cyantraniliprole and spinetoram. This study showed that a bottle bioassay is suitable for assaying insecticide resistance against adult ACP in the laboratory and should be useful to monitor insecticide resistance in the field. It should be a feasible and flexible tool for rapid testing of insecticide resistance.
The goals of this project are to: 1: Determine the fight initiation thresholds of ACP depending on temperature and humidity. 2: Determine the effect of wind speed on flight and the direction of psyllid flight with respect to wind. 3: Determine the effects of barometric pressure changes on psyllid dispersal. 4: Measure how psyllid dispersal is affected by abiotic factors in the field. 5: Establish a model to predict the risk of ACP dispersal/invasion based on prevailing abiotic conditions. Deliver this model as an online tool for growers. We are continuing experiments to study the effect of ambient temperature and relative humidity on the dispersal behavior of the Asian citrus psyllid (ACP). The experiments are set up in a climatic chamber where temperature and relative humidity are controlled precisely. Humidity and temperature are varied in a range that is in accordance with the conditions observed in Florida during spring and summer. The temperature treatments tested in the chamber were 15, 18, 21, 25, 30, 35 C and the humidity treatments 35%, and 75% and 95% RH. We obtained the highest percentage of dispersing individuals (67.80% after 3 days) at 30 C and observed slightly decreased dispersal between 33 C (63.00% after 3 days) indicating that the optimal temperature for short range movement in ACP is comprised between 30 and 33 C. Humidity did not affect the dispersal behavior of ACP except at low temperatures. Whereas 5.8% of ACP dispersed after 3 days at 18 C with 70% RH, 17.00% dispersed after 3 days at 18 C with 35% RH. The dispersion of ACP was the same at any humidity level after 21 C. In summary, our experiments indicate that the minimal threshold for psyllid movement is comprise between 15 and 18 C at 35% RH and is close to 18 C at 70% RH. We finished our automatized flight mill and are now capable to record the flight of four psyllids at the same time and for a longer time than previously. We will use this flight mill to investigate the effect of temperature and relative humidity on long-range flights. We also built a new wind tunnel adapted for Asian citrus psyllid; this wind tunnel has been controlled for regular air flow and absence of air disturbance. We will use this wind tunnel in the next weeks to determine how psyllids move depending of wind direction, and the maximum and minimal thresholds for wind speed regarding flight initiation. Finally we have developed a pressure chamber for psyllid behavior where we will be able to measure psyllid dispersion depending of controlled barometric pressure changes.
Currently, objective 1, investigating the specificity and efficacy of immune priming response in ACP, is underway. The objective of this experiment is to identify if ACP will survive a lethal dose of pathogenic bacteria, Serratia marcescens, if they have had prior immune challenge by a sublethal dose of the same or other species of bacteria. We are currently investigating the effect of: a) pathogen and b) non-pathogen to determine whether immune priming regulates the response of ACP to subsequent infections. Fifty experimental 3 day old ACP adults were collected and starved for 3 h. Half of the insects were orally primed by feeing on an artificial diet solution containing a sublethal dose priming treatment, corresponding to a lethal dose response of 1% (LD1) of the insect pathogen S. marcescens, while they other half were fed a control artificial diet solution containing bacterial media only (Tidbury et al. 2011) (Table I, Objective 1a; Fig 2A). The diet solution consist edof 15% (w:v) sucrose and 1/10 (v:v) green food coloring (McCormick & Co.) sandwiched between two parafilm layers, as described in Hall et al. (2010). After 24 hours, each treatment group was enclosed on separate branches of a potted citrus plant (var. ‘Swingle’). Seven days after priming, insects were removed from plants, starved for 3 h, and either injected (experiment 1) or orally inoculated (experiment 2) on a diet solution containing a lethal dose pathogen challenge or control treatment. After feeding or injection, the ACP were returned to the ‘Swingle’ plants for 14 days or 100% mortality. Mortality throughout the experiment was recorded every 24 hours. After 14 d, ACP were examined for the presence of S. marcescens by plating crushed insects on Petri dishes containing LB nutrient agar. Samples of colonies formed after 3 d were collected for DNA extraction and confirmation of bacterial identity using conventional PCR. Each experiment was replicated six times. We are currently analyzing the results of these experiments. Subsequent experiments to address the effects of non-pathogen immune priming to pathogenic bacteria (objective 1b) are still underway. Additionally, we have initiated the second project objective, to determine the specificity of RNAi immune priming in D. citri. We have begun construct dsRNA using a GFP sequence. GFP is not naturally present in ACP; therefore, introduction of the dsRNA for this target should activate the RNAi response without inducting psyllid mortality. Additional targets will be constructed during the current project quarter.
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. Seven Bt strains showed toxicity against ACP with significant ACP mortality at 500ug/ml relative to control treatments. One Bt strain was selected for further identification of individual toxins by LC-MS/MS analysis. Based on the results, three specific Cry toxins were identified. The identity of these toxins was confirmed by comparison with the Bt Cry toxin holotype list, using ClustalW2-nucleotide. Further ACP bioassays with two of the toxins from the selected strain indicated that both toxins show toxicity against ACP at 500ug/ml. One of these toxins was selected for modification with the gut binding peptide. A phage disulfide-constrained heptapeptide library was screened and four ACP gut binding peptides were isolated. These peptides were expressed as peptide-mCherry fusion proteins as described in the previous report. Pull down assays were used to assess the binding of peptide-mCherry fusion proteins to ACP brush border membrane vesicles (BBMV) to confirm binding and to assess the relative strength of binding of the different peptides. mCherry alone and a random sequence peptide-mCherry fusion protein were used as negative controls for these assays. Specific binding of peptides to BBMV proteins was supported by the results of binding competition assays with mCherry or with a competitive synthetic peptide. Two dimensional gel electrophoresis coupled with ligand blot analysis showed that four peptide-mCherry fusion proteins bind to 50kDa, 37kDa and 25kDa BBMV proteins all with the same pI (~9). The negative control, mCherry showed non specific binding to an abundant 50kDa protein with a pI ~5. Analysis of peptide binding to the psyllid gut in vivo using fluorescence microscopy indicated that peptide 15-mCherry and peptide 18-mCherry bind the ACP gut, with minimal background fluorescence from the mCherry negative control.
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 fifteen months. Irrigation water was acidified at one of four target water pH (7.5, 6.0, 5.0, and 4.0). A controlled release form of elemental sulfur was applied to half of the trees in each pH treatment (main effect) including the non-acidified control (pH~7.5). 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 June. Soil samples collected in August indicate that soil pH in the plots recieving sulfur applications remained near target pH level at both sites. However, plots not receiving sulfur applications increased in soil pH levels by an average of 0.75 pH units with plots to have pH between 6.0 and 7.0 returning near the pH of the irrigation water . This result is similar to data collected in 2014. The cause of soil pH is the low amount of irrigation required during the summer months. At both the mature and young tree site, no significant difference in root density was found in samples collected in August 2015. Significant increases in nutrient concentrations of leaves collected in June 2015 were been found in plots at both sites with reduced water and/or soil pH. The greatest increase in leaf nutrient concentrations were found for Mg, Mn, Zn, and B. These results may indicate increased nutrient uptake from soils with soil solutions below 6.5. Nutrient deficient symptoms consistent with HLB positive trees have lower nutrient concentration in their woody tissues and thus can not provide nutrients to leaves until these reserves are replenished by higher nutrient availability presumed at lower soil pH levels (5.5 to 6.5). Average Hamlin and Valencia tree water uptake under greenhouse conditions were not significantly different from one another. However, water uptake by trees affected with HLB were 20%-25% lower than healthy trees. These data have been consistent for the past year. There is increasing evidence of reduced water uptake for trees receiving water supplemented with calcium bicarbonate. The cause of reduced water uptake appears to be lower but non-significant reductions in root density and soil pH increases in soil irrigated with higher concentrations of calcium carbonate. Reduced water uptake by trees receiving calcium carbonate in irrigation water would account for reduced leaf area and trunk diameter.
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