To investigate the effects of double-stranded RNA (dsRNA) treatment on D. citri s biology, we conducted dsRNA feeding assays on adult D. citri sampled from a laboratory colony. Briefly, D. citri (separated by gender) were fed with 20% sucrose solutions supplied with 0.5% green food coloring dye and 50 ng/ l of dsRNA. The dsRNA solutions was prepared using a V-type proton ATPase [v-ATPase]) gene or a bacterial green fluorescent protein (GFP) gene fragment. For each gender, additional groups of individuals were kept either on a blank sucrose + buffer diet or in a container without food. After monitoring the survivorship of these insects overtime, we found that D. citri under the starvation treatment had the lowest survivorships, indicating that those in the other treatment groups do feed on the dsRNA-containing diets. In females, the survivorships of both the GFP-dsRNA and the v-ATPase-dsRNA treatment groups were lower than the buffer-only group. No significant difference was detected between the survivorship of the two dsRNA treatment groups. The patterns were different in males; treatments other than the starvation group all exhibited similar survivorship over time. We are currently testing whether RNAi treatments could influence D. citri s ability to acquire and transmit Candidatus Liberibacter asiaticus (CLas). D. citri adults collected from a laboratory colony free of CLas infection were subjected to dsRNA treatments. For such treatments, the insects were fed on a diet consisting of 100 ng/ul dsRNA, 0.5% green food coloring dye, and 20% sucrose. The diets were enclosed within stretched Parafilm membranes serving as the bottoms of plastic petri dish arenas (Fig. 1A). As a negative control, dsRNA was replaced with products of blank dsRNA synthesis reactions (i.e., no-template reaction product) of the MEGAscript T7 Transcription Kit (Ambion, Inc.). The feeding assays were conducted in a growth chamber under 28 C. After 72 h of treatment, the insects will be moved onto flushing, CLas-infected citrus plants (Citrus macrophylla; Fig. 1B). The infection statuses of these plants were determined using quantitative PCR (Li, Hartung, and Levy 2006). Approximately 8-10 adults (male and female ratio ~1:1) will be moved to an individual plant. Four to five replicates (plants) will be tested for each treatment group. The insects will be allowed to reproduce and oviposit on the plants. If the insects successively reproduce on the plants, the original adults will be removed after the nymphs emerge. The second-generation adults developed from the nymphs will be relocated onto leaf discs of healthy citrus plants. After a week, the individuals will be sampled and stored at -80 C. The collected samples will be tested for the presence and abundance of CLas using quantitative PCR (Li, Hartung, and Levy 2006) and the measurements will be compared.
The objective of this research project is to investigate and develop a potential non-phytotoxic, environmentally-friendly film-forming ACP repellent solution for preventing HLB infection. In the last reporting period OS-SG 15 was optimized for potential use in future ACP infection trials. In this report period, a new formulation called OS-SG-16 was developed to further build on the concept of OS-SG 15. OS-SG 16 was synthesized using an all natural silica substrate, along with both an EPA approved “For Food Use” polymer and “For Food Use” surfactant. Additionally an industrially approved dispersant was introduced to further stabilize and increase the film forming capabilities of the material. This new version was expected to display high colloidal stability in aqueous solution, high surface coverage and moderate rain-fastness properties. The colloidal stability of the formulation was checked via measuring %Transmittance (%T) of the supernatant collected from the solution left undisturbed. The formulation revealed less than %50 transmittance up to 6+ hours which was found to be better compare to commercial control -Surround WP- which is currently commercially available for growers. OS-SG 16 was characterized using UV-Vis and FTIR spectroscopy which revealed the presence and interaction between the individual components such as silica, surfactant and polymer. Peaks in the FTIR were found for Si-C stretch (788 cm-1) for the silica source, C-N stretch (1466 cm-1) for the surfactant and C=O stretch (1220 cm-1) for the polymer. Safety analysis and plant leaf surface coverage of OS-SG 16 formulations were conducted using Cleopatra orange sp (common citrus variety) as a model plant. The formulations were sprayed in triplicate at the application rate of 0.5 lbs/gallon (recommended rate for the commercial control) based on active content. The formulations revealed high plant leaf surface coverage at the application rate which was comparable to the commercial control. Compared to the previous OS-SG 15 version, the inclusion of the industrial dispersant increased the overall leaf coverage of the material and improved the film formed. 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, 32 Celsius). OS-SG 16 formulation did not cause any plant tissue damage at the applied rates, matching the commercial control. To fully ascertain the potential of recent OS-SG versions, the possibility of new ACP trials are currently being discussed.
The objective of this research project is to investigate and develop a potential non-phytotoxic, environmentally-friendly film-forming ACP repellent solution for preventing HLB infection. In the last reporting period OS-SG 15 was optimized for potential use in future ACP infection trials. In this report period, a new formulation called OS-SG-16 was developed to further build on the concept of OS-SG 15. OS-SG 16 was synthesized using an all natural silica substrate, along with both an EPA approved “For Food Use” polymer and “For Food Use” surfactant. Additionally an industrially approved dispersant was introduced to further stabilize and increase the film forming capabilities of the material. This new version was expected to display high colloidal stability in aqueous solution, high surface coverage and moderate rain-fastness properties. The colloidal stability of the formulation was checked via measuring %Transmittance (%T) of the supernatant collected from the solution left undisturbed. The formulation revealed less than %50 transmittance up to 6+ hours which was found to be better compare to commercial control -Surround WP- which is currently commercially available for growers. OS-SG 16 was characterized using UV-Vis and FTIR spectroscopy which revealed the presence and interaction between the individual components such as silica, surfactant and polymer. Peaks in the FTIR were found for Si-C stretch (788 cm-1) for the silica source, C-N stretch (1466 cm-1) for the surfactant and C=O stretch (1220 cm-1) for the polymer. Safety analysis and plant leaf surface coverage of OS-SG 16 formulations were conducted using Cleopatra orange sp (common citrus variety) as a model plant. The formulations were sprayed in triplicate at the application rate of 0.5 lbs/gallon (recommended rate for the commercial control) based on active content. The formulations revealed high plant leaf surface coverage at the application rate which was comparable to the commercial control. Compared to the previous OS-SG 15 version, the inclusion of the industrial dispersant increased the overall leaf coverage of the material and improved the film formed. 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, 32 Celsius). OS-SG 16 formulation did not cause any plant tissue damage at the applied rates, matching the commercial control. To fully ascertain the potential of recent OS-SG versions, the possibility of new ACP trials are currently being discussed.
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. Grove 1 has ~25 years old of Flame grapefruit on Swingle rootstock. Grove 2 utilizes ~7-year-old Ruby Red on Sour orange trees. Combinations of macro and micronutrient treatments initiated on all three trials in February 2014 and applications have been made quarterly since. Both research locations were maintained during this quarter, with foliar sprays continuing on schedule. Tree growth assessments of canopy volume, tree height, and leaf area index were made, along with assessments of insect counts. Presentations of research results were made to Florida State Horticultural Society and discussion of preliminary results was made to citrus growers and other scientists. The enhanced nutritional programs generally did not significantly influence tree growth parameters during this time period, as both leaf area index and canopy volume were generally unaffected by the foliar treatments. Although analysis indicated that there were differences in these parameters between years caused by pruning and topping, none of these differences were attributed to the enhanced nutritional programs. Leaf nutritional sampling and continuation of the spray programs will progress in late summer 2016 and extend to the final harvest in Winter 2016.
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. Grove 1 has ~25 years old of Flame grapefruit on Swingle rootstock. Grove 2 utilizes ~7-year-old Ruby Red on Sour orange trees. Combinations of macro and micronutrient treatments initiated on all three trials in February 2014 and applications have been made quarterly since. Although no microelement deficiencies of leaves were detected, the results indicated significant differences in the concentration of Mn, Zn and B within the treatments evaluated for grove 2. Soil tests reflected similarities in the concentrations of nutrients in the soil between treatments, where the application of enhanced nutritional programs generally had a low influence on nutrient concentrations in the soil. Few effects of nutrient application on canopy growth and tree density in terms of LAI was detected. The addition of macroelements such as N and K did not affect the volume of the tree in ENP treatments compared to the control. However, for grove 1 the LAI results suggest a decrease in the LAI through time but the trees in grove 2 showed increases, suggesting differences between the groves in response to nutrient application. For grove 1, the control had the lowest yield with an average of 4.0 boxes per tree, whilst the maximum yield occurred for the treatment DKP+KP with 4.8 boxes, showing an increase of 20% compared to control. For grove 2, the control treatment tended to show the lowest yield, while the DKP+KP+M and KN+KP treatments showed increases of 50% and 53% more boxes per tree, respectively. The treatment with the highest percentage of large fruit was DKP+KP+M, showing a significant increase of 42% relative to the control. The variation in the production of small, medium and large fruit affected the GPV calculated for each treatment, resulting in low values for the control treatment with $77 per tree and high values for the treatments DKP+KP+M and KN+KP with $127 and $125 per tree, respectively.
We have continued to investigate movement of Asian citrus psyllid (ACP) as it relates to biotic and abiotic factors. Climate change increases the duration and intensity of heat waves, causes earlier spring arrival, and more frequent drought stress events. We investigated the response of ACP and its parasitoid to pathogen and herbivore-induced plant volatiles released from plants with and without drought stress. ACP vectors were attracted to headspace volatiles of CLas-infected citrus plants at 95% of their water holding capacity (WHC); such attraction to infected plants was much lower under drought stress. Attraction of the vector to infected and non-stressed plants was correlated with greater release of methyl salicylate (MeSA) as compared with uninfected and non-stressed control citrus plants. Drought stress abolished MeSA release from CLas-infected plants as compared with non-stressed and infected plants. Similarly, the parasitoid wasp, Tamarixia radiata, was attracted to headspace volatiles released from ACP-infested citrus plants at 95% of their water holding capacity (WHC). However, wasps did not show preference between headspace volatiles of psyllid-infested and uninfested plants, when they were at 35% WHC, suggesting that herbivore-induced defenses did not activate to recruit this natural enemy under drought stress. We also investigated learning in ACP as it relates to movement. In ACP, which mate throughout their lives, learning in the context of sexual behaviors may increase the likelihood of successful mating. In females, two aspects of behavior were investigated: 1) the influence of natal host species on adult settling and oviposition host preference, and 2) the effect of the initial mating experience on future mate selection. In males, we investigated whether female odor is learned after mating experience. Our results indicate that females prefer to oviposit onto host plants similar to their own natal environment. However, this only occurs when the natal host plant may be associated with fewer plant defence compounds than the alternative host species. Females also demonstrate the ability to discriminate between males based on abdominal color and later avoid males bearing certain traits, perhaps associated with reproductive immaturity. Additionally, male psyllids appear to learn odors associated with receptive females. We conclude that in ACP, learning may modulate oviposition preference and mate choice in females, and male recognition of female conspecifics. These data suggest an adaptive significance of learning in the context of reproduction and that learning may increase the likelihood of successful reproduction in ACP.
The overall goal of this project is to improve insecticide resistance management for Florida populations of Asian citrus psyllid (ACP). We are achieving this through investigations of the mechanisms of resistance, monitoring resistance in the field, development of optimized rotation skills, and evaluations of new tools for implementation into these rotation schedules. One of the major obstacles facing Florida citrus growers is a lack of a sufficient number of modes of action for management of to achieve efficacious and cost-effective rotations season-long. Currently, and unfortunately, this sometimes requires application of the same mode of action more than one time per year. We therefore continue to investigate the psysionlogical consequences and effectiness of alternative modes of action against ACP. The objective of the previous quarter’s research was to determine whether the next generation insecticide, flupyradifurone, induced stress to behavior and hormesis of ACP. Therefore, we investigated thew dose-mortality response of ACP to flupyradifuron and the lethal and sublethal effects of flupyradifurone on flight behavior and hormesis of ACP. Experiments were conducted with both ACP immature stages and adults. Flight behavior of adult ACP was investigated five days after treatment with flupyradifurone. All flight data were collected using a laboratory flight mill apparatus that was custom-made by us for measurement of flight capacity by ACP. Data were automatically recorded onto a computer DATAQ data logger. After recording, the flight number (n), total flight duration (sec.), average and maximum flight speed (m/s), total flight distance (m), flight trial number (n), and time to elapsed to the 1st flight were auantified. We then determined whether there were differences in flight behavior of psyllids as effected by dose of flupyradifurone treatment. Our results indicated that percentage of flyers treated with the LC50 concentration of insecticide was 88.9%; which was 60.0% higher than the control. The number of ACP flying following treatment with LC50 concentration was over 5 times greater than the control. There was a significant difference between the LC50 and control treatments (F = 29.036; t = -2.288; p=0.035) with respect to the number of ACP initiating flight. As compared with control, both average and maximum flight speed of psyllids were increased with treatment at the LC50 concentration of insecticide. However, there was a significant difference in only maximum flight speed statistically (F = 12.592; t = -2.417; p = 0.042). The result indicated that the sulthletal concentration affects ACP flight behavior. For the hormesis research, the experiment was set up in a randomized complete block design comprising five lethal and sublthal concentrations and each concentration was replicated 4 times. Each plants was sprayed with one of five concentrations of flupyradifurone or water until runoff using a handheld atomizer. Plants were allowed to air dry and then exposed to four pairs of adult ACP for mating and oviposition. Thereafter, adults were removed from each plant and the number of eggs laid per plant was recorded under a stereomicroscope. The result indicated that the number of eggs produced per plant significantly affected by concentration of applied flupyradifurone (p <0.001). After exposed to the LC10 and LC25, the number of egg was higher than for the control. The result overall indicated that ACP exposed to sublethal dconcentrations of flupyradifurone show increased hormesis as compared with the control. This insecticide should be an effective additional tool for management of ACP. Further field testing is needed and will be conducted in future testing.
December 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 2016 leaf litter and the presence was more consistent than 2015. From 2014 to 2016 the number of Phyllositca spp. structures consistently increased significantly (P < 0.05). 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 fruit from the 2016 application has not yet been evaluated. 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 final publication was the citation: van Bruggen, A.H.C., Sharma, K., and Shin, K. 2017. Sugar cane processing residue, bagasse, enhances decomposition of citrus leaves and could contribute to citrus black spot management. Crop Protection 93: 89-97. The detached leaf assays for assessment of leaf susceptibility continue. There was an out break of mites in the greenhouse that slowed the leaf production process for the leaf assay. We have treated for mites and the experiment continues. 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 was an increase in pathogen presence in 2015, we have decided to continue sampling since levels were very low in 2014. There appears to be more asexual structure formation in the spring of 2016 than 2015 and greater conidia production. In 2016, fewer ascospores were observed in the spring than in 2015 but in general the overall trends were similar. The summer data had similar trends from previous years but the level of P. citricarpa DNA increased overall and was more consistently found among samples. In Australia, confirmation of the ascospore and conidia production results continues. Sampling of leaf litter in two groves in the Queensland mandarin growing region was completed in April and samples are being processed. In the three years of sampling, the fruitifcation patterns have been different but we are not sure why. Pycnidia are found most of the sampling season but pseudothecia tend to be clustered towards the end of the season. qPCR data are being generated but are not yet complete. Inoculations of fruit are complete and preliminary symptoms have been confirmed on susceptible fruit. They have identified several potential candidates for resistance after 2 seasons of inoculations. They repeated the fungicide work to confirm previous results. In 2015, mulch was the best treatment to reduce the amount of leaf litter under trees and in 2016 the added bagasse treatment was even better. The high volume fungicide applications did slightly reduce decomposition of the leaf litter but may not be significant. These results were confirmed in the 2016 trials. The work was submitted to Citrus R&T: Miles, A.K., Tran, N. T., Shuey, T.A., Drenth, A., and Dewdney, M.M. 2015. Does fungicide run-off from citrus delay leaf litter decomposition? Citrus Research and Technology 36:(submitted)
Low concentrations (1/4 rate) of 2, 4-D and Max-Cel were applied every 45 days to Hamlin and Valencia tree canopies at two locations in central Florida starting in Spring 2014. Concentrations were 12.6 ml Citrus Fix and 480 ml Max-Cel/acre. Every other 45 day period, GA3 (0.04 g ai/tree) was applied in 3 gal of water per each microjet irrigation zone. Treatments were applied from Spring through October. Trees were sampled in late spring for phloem development at four locations in the scaffold system (root flare, trunk, small scoffolds and leaf main veins). Trees were again sampled in late fall for phloem development at the same four locations in the scaffold system. Comprehensive PCR profiles on the treatments were run by Dr. Killiny and blight analyses were run in Albrigo s lab. Almost all of the treatment trees were positive for HLB and about half were positive for citrus blight. Fruit drop and fruit per tree data were collected for Hamlin plots and similar Valencia data was collected also in year one. In the Hamlin test 2, 4-D + Cytokinin with or without GA had less fruit drop than the Control trees, 11.5 and 13.1, respectively, versus 17 % drop for the Control, significant at the 1 % level. The Valencia trial had drop rates of 8.0 and 9.8 % for 2, 4-D + Cytokinin with and without GA compared to 10.5 % drop for the Control with no significant differences. No differences in leaf drop were detected. In year two, root densities and spring flush/leaf counts and 2015-16 crop fruit counts were collected using 8 limb counts per 2 tree conditions per plot. Root densities averaged about 10 gm fresh weight per tree with no difference between treatments. Total leaf drop per tree averaged 249 leaves for the Control trees and the treated trees had 3 % more dropped leaves for the MaxCel + 2, 4-D and the GA treatments and the combined treatment of all three PGRs had 4 % less leaf loss. Percentage fruit drop was equal to the Control (22.5 %) or higher by one or four %. The low concentrations (1/4 rate) of PGRs every 45 days to Valencia orange trees in the 2015-16 growing season did not reduce fall leaf drop nor preharvest fruit drop of HLB infected trees in the second year. Total leaf drop per tree averaged 400 leaves for the Control trees and the treated trees had 7 to 17 % more for the 3 treatments (MaxCel + 2, 4-D, GA treatments and combined). Most of the difference was in the accumulated summer leaf drop, August count. A similar result occurred with the Hamlin trial. No difference in phloem development was detected. Percentage fruit drop was near 15 % for the Control and all the treatments. The fruit per tree in the second year of treatments was plus or minus 4 % of the Control for all the treatments and not significant. Further no improvement in tree condition occurred so the experiment was terminated. Two greenhouse experiments were conducted to see if GA soil applications improved root growth. GA rates were 0, .00125, .0025, .005, and .0063 g ai/plant. The number of new roots was increased 38 and 31% over the Control by the two higher GA concentrations after 3 weeks. Total and average root length increased by 27 and 28%, respectively, 6 weeks after application. The test was rerun on HLB affected nursery trees. Some increase in root growth occurred, for the bottom third of the pots averaged root length was 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 lowest GA concentration (0.00125) but only at 8 weeks, 18/27 versus 23/23 for the control. Little difference was found in the top third of the pot. GA did not cause as much response in HLB affected trees and tree to tree variability was much greater.
The evaluation of the up-graded on-line ‘Citrus Flowering Monitor System’ after the first year of data collection was completed. In spite of the totally abnormal weather pattern this past fall and early winter, essentially no cold induction before January 1st, the flowering cohorts and timing were very close to the actual time of flowering experienced by many growers. No growers reported problems with using the new on-line format. With one year of data, 5 to 10 % open flowers was 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. 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. This short window would only allow for an aerial spray. Flowering for the second spring bloom period under this project had major blooms in March and April. The second major wave was in early April. Preliminary evaluation of flower and flush development indicated that a longer period of time occurred between the feather flush of leaves and 5 to 10 % open flowers. These major cohorts of normal flowers plus earlier off-season flowering as a continuous winter-spring flowering event probably contributed to very heavy incidence of PFD this past spring.
Three growers agreed to apply 1/4th the regular rate of Citrus Fix and MaxCel every 45 days on approximately 1 acre, about 2 rows, each of Hamlin and Valencia orange trees. Grove locations are Sebring, Babson Park and Ft. Meade. The last application for the first growing season occurred in October, 2014. Growers were diligent in making timely applications. Comparable control rows are being monitored two rows from the sprayed trees. Approximately one acre was treated in each cultivar and these trees plus controls were categorized as to tree health. No apparent difference in flush growth or any phytotoxicity was detected. Tree condition (decline status) was monitored and evaluated at the end of the fall period but no difference was detected. Fruit drop was measured and yields obtained. Flowering will be checked for any difference in timing or intensity. Fruit drop was measured and yields obtained during the harvest period. Decline rated trees (3) had less fruit than 1 rated trees and 3 % more drop. Yields were not different for year one between Control and treated trees, but fruit drop was numerically less for the treated trees by 6 % for Hamlins and 3 % for Valencias compared to the Control plots for all sites combined. Fruit dropped Total fruit Total fruit dropped n per tree (no.) per tree (no.) per tree (%) Tree condition Trees rated 1 145 97 a 429 A 24 B Trees rated 3 147 99 a 396 B 27 A Hamlins 4 sites evaluating 2,4-D+ Maxcel Controls 16 100 a 354 a 32 A 2,4-D+ Maxcel 16 92 a 399 a 26 B Valencias Controls 18 51 a 399 a 14 A 2,4-D + Maxcel 18 46 a 429 a 11 B Each spray application included 5 gm ai ProGibb per acre with the Citrus Fix and Maxcel in the second year. Flowering was evaluated in March of year 2, but no differences were detected in time nor intensity of flowering. Treated and Control trees were categorized by three levels of tree health and were monitored for fruit drop, leaf drop, flushing, yield, etc. We monitored leaf drop to see if these frequent applications may affect leaf drop as well as fruit drop. If they do, it would more than compensate for keeping some additional fruit on the tree longer than normal for HLB affected trees. 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 for Hamlins, and leaf drop was heavier for Valencias in December than later in the spring. In one case almost 70 % of the leaf loss occurred by October 8th. No differences were found between PGR-treated and Control trees in the amount of leaf drop. 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 Hamlin trees were 19 vs 15.5, 18.8 vs 19.4 and 24.5 vs 25.1, respectively. There was usually only 1 or 2 % difference between 1 and 3 rated trees. Overall fruit drop rates in year two were 22 % and 19 %, respectively, for Hamlin and Valencia trials at Babson Park, 22 % and 20 % at Sebring and 18 % and 15 % at Ft. Meade, respectively. No differences were found between the low concentration PGR treatments and Controls at any of the sites for either healthier or more declined trees in the Valencia trials. The reduced fruit drop in treated trees after the first year did not occur in the second year. No differences in juice content, brix or acidity were found between Treated and Control tree fruit. At each site there was no difference in the fruit per tree, fruit or leaf drop or tree condition between the Control and PGR treatments in the second year after two years of treatments. Therefore the experiment was terminated.
To examine the general effects of double strand RNA (dsRNA) uptake on D. citri fitness and biology, we have conducting dsRNA feeding experiments on D. citri adults of a CLas-free laboratory colony. dsRNA of genes that exist (e.g., V-type proton ATPase [v-ATPase]) or do not exist in D. citri (a bacterial GFP gene) were used for this study. Briefly, primers targeting each DNA fragment were designed and synthesized with T7 promoters tagged (5 ends). PCR were carried out and purified amplicons were used for dsRNA synthesis using the MEGAscript RNAi kit (Ambion, Austin, TX). dsRNA treatments were conducted by feeding D. citri with 20% sucrose solutions supplemented with 50 ng/ l of dsRNA and 0.5% of green food coloring dye, using previously described methods (Wuriyanghan, Rosa, and Falk 2011). The insects were separated by gender and 25 to 30 insects were used for each Gender x Treatment. For the negative control, blank elution buffers were added to the 20% sucrose solution. To determine whether the insects were feeding, we included a treatment group in which the insects did not have access to any diet. All insects were kept at a growth chamber and their mortality was recorded daily. Our preliminary data showed that insects that did not receive any diet had the lowest survivorship, indicating that D. citri in the other treatments were feeding on the sucrose diets. In females, the survivorship of both the GFP-dsRNA and v-ATPase-dsRNA treatment was lower than the buffer-only group. No significant difference was detected between the survivorship of the two dsRNA treatments. In males, all treatments other than the no-diet treatment had similar survivorship. Overall, these findings suggest that male and female D. citri may respond differently to the same dsRNA treatments; whether such difference is related with their physiology or behavior deserves further investigation. The reason why GFP-dsRNA and v-ATPase-dsRNA treatment resulted in similar effect on D. citri survivorship (particularly in females) will also be studied further. In the future, we will also be testing how similar RNAi treatments may influence D. citri s immune system and ability to acquire or transmit CLas.
July 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. We have been working on a complete analysis of the first two years of data but this is not yet completed. 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 but the Co-PI had no further updates. 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 was an increase in pathogen presence in 2015, we have decided to continue sampling since levels were very low in 2014. There appears to be more asexual structure formation in the spring of 2016 than 2015 and greater conidia production. In 2016, fewer ascospores were observed in the spring than in 2015 but in general the overall trends were similar. In Australia, confirmation of the ascospore and conidia production results continues. Sampling of leaf litter in two groves in the Queensland mandarin growing region was completed in April and samples are being processed. Inoculations of fruit are complete and preliminary symptoms have been confirmed on susceptible fruit but the less susceptible fruit are still incubating, including the promising accections. They repeated the fungicide work to confirm previous results. 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. These results were confirmed in the 2016 trials.
Major accomplishments 1. We identified an optimal 2-component blend (1.9:1 formic:acetic acid) and an optimal 3-component blend (3.5:1.6:1 blend of formic acid:acetic acid:p-cymene) for maximal probing by ACP of a wax substrate. This could be used as a phagostimulant for attract-and-kill strategies for trapping psyllids. 2. We found that CLas-infected ACP probed significantly more often compared with healthy ACP on our phagostimulant blend. 3. This is the first report of a behaviorally active semiochemical blend that could be used for psyllid trapping strategies. 4. We determined that the presence of a thick, well-developed fibrous ring around phloem tissues of mature leaves acts as a barrier to frequent or prolonged phloem ingestion by D. citri from citrus leaves. This may have an important role in limiting or preventing CLas acquisition and/or transmission by D. citri, and could be used for identification and development of resistant citrus cultivars. 5. Our findings were reported in four peer-reviewed articles in prestigious journals including Chemical Senses, the Journal of Chemical Ecology and PLOS Computational Biology. Publications related to this project: Hall, DG, George J, Lapointe SL 2015. Further investigations on colonization of Poncirus trifoliata by the Asian citrus psyllid. Crop Protection 72:112-118. George J, Robbins PS, Alessandro RT, Stelinski LL, Lapointe SL 2016. Formic and acetic acids in degradation products of plant volatiles elicit olfactory and behavioral responses from an insect vector. Chemical Senses 41(4). doi: 10.1093/chemse/bjw005 Lapointe, S.L., J. George, and D.G. Hall. 2016. A phagostimulant blend for the Asian citrus psyllid. J. Chem. Ecol. 42(9): 941-951. DOI 10.1007/s10886-016-0745-4. Willett DS, George J, Willett NS, Stelinski LL, Lapointe SL 2016. Machine learning for characterization of insect vector feeding. PLOS Comp Biology 12(11): e1005158. Doi:10.1371/journal.pcbi.1005158 Manuscript submitted: George, G., E. Ammar, D. G. Hall, S. L. Lapointe. 2017. Sclerenchymatous ring as barrier to phloem feeding by Asian citrus psyllid: evidence from electrical penetration graph and visualization of stylet pathways. PLOS ONE (submitted 10/26/2016)
CRDF quarterly report July 2016 Electrical Penetration Graph (EPG) is a powerful tool for monitoring the cryptic feeding behaviors of piercing-sucking insects that feed on plant xylem and phloem tissues. While the insect probes, ingests, and salivates within the plant, characteristic voltage waveforms are produced that, in conjunction with histological studies, allow researchers to determine feeding patterns associated with pathogen acquisition and inoculation. We have shown that EPG can be used for detecting resistance in trifoliate citrus genotypes. Waveform annotations of feeding behavior of Asian citrus psyllids on trifoliate and citrus cultivars showed reduced phloem feeding on trifoliate accessions compared to trifoliate/citrus hybrids and citrus cultivars. Phloem feeding is required for acquisition and transmission of CLas. The observed low incidence of phloem feeding on P. trifoliata and trifoliate hybrids suggests a mechanism to explain the observed tolerance of citrus genotypes in the field, despite demonstrated susceptibility to the pathogen by graft inoculation. Poncirus trifoliata may possess physical traits that confer resistance to transmission by interfering with the vector’s ability to attain the phloem. Psyllid feeding may be hindered by physical barriers to stylet passage conferred by fibrous rings of sclerenchyma cells associated with vascular tissue in P. trifoliata. The presence of sclerenchymatous cells associated with the phloem may be a valuable trait for citrus breeding to produce scions that reduce or prevent ACP feeding and reduce CLas transmission. To study the role of the fibrous rings on ACP feeding behavior on emerging and fully expanded leaves of susceptible citrus, we recorded xylem and phloem feeding by EPG on adaxial (upper) and abaxial (lower) surface of young and old leaves. ACPs were allowed to feed on upper or lower surfaces of young and old leaves of Valencia (Citrus sinensis) for 21 hours. We monitored ACP feeding behaviors (np-non probing, c-intercellular stylet penetration, d-phloem penetration, e1-phloem salivation, e2-phloem ingestion, g-xylem ingestion). The waveforms produced from leaf tissues were manually annotated based on prior studies. Leaves fed upon during the EPG runs were sectioned and examined by flourescence microscopy to visualize the stylet paths and to correlate stylet position with EPG wavefoms. EPG waveform annotations showed that ACP feeding behavior on younger and older leaves was modified by the presence of sclerenchymatous cells associated with the phloem. Significantly longer phloem feeding was observed on upper surface of young leaves compared with lower and upper surfaces of older leaves. Although fewer phloem penetrations (d-e1-e2) were observed on younger leaf surfaces, they resulted in longer, sustained bouts of phloem feeding. This could be attributed to the reduced presence of sclerenchymatous rings in younger leaves that facilitates access to phloem compared with older leaves. Histological correlations showed that the fibrous rings are better developed in lower surface of older leaves, which may have resulted in decreased phloem feeding by ACPs. These findings have a significant role in understanding the feeding behavior of ACPs on young and older leaves of susceptible cultivars, and how it affects the acquisitions and transmission of HLB pathogen. Publications related to this project: George, J, PS Robbins, RT Alessandro, LL Stelinski, SL Lapointe. 2016. Formic and acetic acids in degradation products of plant volatiles elicit olfactory and behavioral responses from an insect vector. Chem. Senses 41 (4): 325-338. Lapointe, SL, J George, DG Hall. 2016. Phagostimulants for the Asian citrus psyllid (Hemiptera: Liviidae). J. Chem. Ecol. (accepted 6/29/16).
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