This project looked at the in vitro sensitivity of Guignardia citricarpa, causal agent of citrus black spot (CBS) for demethylation inhibitor fungicides (DMI; FRAC 3), succinate dehydrogenase inhibitor fungicides (SDHI; FRAC 7), an aniline-pyrimidine (FRAC 9), and a phenylpyrrole (FRAC 12). This allowed for the establishment of baseline sensitivities for those fungicides registered for citrus along with estimating whether it was worth placing them in a year-long field trial. Three DMI fungicides showed promise: difenoconazole, fenbuconazole, and propiconazole. The difenoconazole and fenbuconazole were found to be efficacious in the field trials and were recommended for CBS management. Imazalil is used post-harvest but did not have great in vitro efficacy or an ability to suppress lesion development post-harvest. The three SDHI fungicides were all efficacious in vitro and boscalid was recommended for CBS management based on field trial results. The two newer SDHI fungicides have not been field tested as of this date. The aniline-pyrimidine and the phenylpyrrole were not pursued further based on in vitro results. There was a spatial distribution study of G. citricarpa in the field and it was found that there was a clustered distribution of the disease in groves. There were several post-harvest studies undertaken. One looked at the effect of heated fungicides. It was found that heating the fungicide solution did not improve the efficacy nor did hot water treatments by themselves. Chitosan was also tested as a post-harvest control but did not have any particular effect on lesion development.
The objective of this project was 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 crop loss. The experiment was set up in a 10 ha plot planted with Valencia sweet orange grafted on Rangpur lime located in the municipality of Xambre, Paran , Brazil. The different treatments were the combination of up to three control measures (Cu, Wb, Lc) or none. The presence or absence of windbreak represented a plot. The presence or absence of copper sprays and leafminer control represented a subplot. Each subplot was composed of 112 trees. Each of the eight treatments had three replicates. Cu treated plots were sprayed with Kocide (35% metallic copper) at 1 kg metallic copper/ha every 21 days. Lc was performed with application of abamectin at 150 ml/ha every 21 days. Casuarina was used as a natural Wb around the plots. Disease evaluations started in December 2013 and included 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 covered the period of November 2015 to October 2016, which corresponds to the second entire season assessed in the trial. The results observed during this season (2015-2016) followed the same trend of the previous one. Although the incidence of trees with citrus canker reached 100% for all treatments, the progression rate was lower in plots under complete management than trees with no management. The incidence of diseased trees in non-managed plots reached 100% in March 2015, 16 months after epidemics started, whereas, in managed plots this incidence was observed only a year later. Peaks of leaves with ACC symptoms in the last season for plots with complete management and no management were 2 and 42%, respectively. Fruit drop per tree due to canker was 109 and 18 for unmanaged and managed trees, respectively. These drops represented 37 and 5% of the crop loads, respectively. At harvest, incidence of fruit with ACC was 8 and 70% for managed trees and non-managed trees, respectively. Finally, average yield of trees treated with Cu+Wb+Lc was 84 kg, 83% higher than in trees under no management, which produced 46 kg. All measures reduced disease losses, but the combination of Cu and Wb provided the greatest disease control. Lc did not significantly contribute to reduce ACC incidence and disease impact on fruit loss.
June 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. The results were gathered at 5 weeks post-inoculation but again the results were inconclusive because a mistake was made in the incubation conditions by a new employee. A site has been found to conduct field experiments of inoculum potential and preliminary work is continuing. We have improved our method of collection of conidia from twigs. Collection continues at two week intervals. Methods to improve sampling for DNA extraction with the quantity of twigs are being examined. Experiments were started to look at the effect of temperature on the level of sporulation of P. citricarpa. It can be quite difficult to get consistent sporulation even under controlled conditions. The temperatures that are being tested 15, 20, 24, 28, 32, and 36C. Data has not been collected yet. Work on the effect of FDACS recommended disinfectants (200 ppm bleach or 2000 ppm quaternary ammonium) on conidia germination was conducted. Effective concentrations to inhibit either 50% or 90% of conidia germination for 2 quat products, Canker Solve and C-Quat, and bleach. were found to be well below 5 ppm for all products. Bleach was about ten times more effective but is not as stable as quat. New experiments are being planned to look at the effect of plant debris on the efficacy of these products to have a more ‘real’ world test of these products.
September 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. The results were gathered at 5 weeks post-inoculation but again the results were inconclusive because a mistake was made in the incubation conditions by a new employee. We have not repeated this experiment again at this point but plan to. In the mean time, we have been working on the experimental design for the larger experiment. It will be an unbalanced complete block design because we do not have the number of incubators to run all temperatures at once. A site has been found to conduct field experiments of inoculum potential and preliminary work is continuing. We have improved our method of collection of conidia from twigs. Collection continues at two week intervals. We have settled on a way to sample the twigs in an unbiased, yet manageable method, for DNA extraction and have begun processing the samples. Experiments were started to look at the effect of temperature on the level of sporulation of P. citricarpa. It can be quite difficult to get consistent sporulation even under controlled conditions. The temperatures that are being tested 15, 20, 24, 28, 32, and 36C. After incubation in complete darkness to avoid the confounding effects of light, it was found for 5 isolates that 24C was the best temperature for sporulation (P < 0.05) followed by 28C. The experiment is being repeated. Work on the effect of FDACS recommended disinfectants (200 ppm bleach or 2000 ppm quaternary ammonium) on conidia germination was conducted. Effective concentrations to inhibit either 50% or 90% of conidia germination for 2 quat products, Canker Solve and C-Quat, and bleach. were found to be well below 5 ppm for all products. Bleach was about ten times more effective but is not as stable as quat. The disinfectants have been preliminarily evaluated in the presence of finely ground plant debris (twigs and leaves as would be found on mowers or hedgers). The low concentrations of 20 ppm of quat have not been found to be effective in the presence of debris and an expanded concentration range is being explored to further evaluate the effect of debris.
September 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 analysis has been more complicated than anticipated so not yet completed. The work was presented in as a poster at the annual conference for APS in 2016. 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 was accepted with the citation: van Bruggen, A.H.C., Sharma, K., and Shin, K. 2016. Sugar cane processing residue, bagasse, enhances decomposition of citrus leaves and could contribute to citrus black spot management. Crop Protection (accepted). The detached leaf assays for assessment of leaf susceptibility continue. ince it was difficult to coordinate leaf ages between CREC and the USDA, new trees were grafted at the CREC and have just become large enough to harvest leaves for a second repeat of the experiment. 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. Summer data has not been fully processed yet. We have been refining the spore DNA extraction and qPCR technique and anticipate getting that data generated in the next quarter. Some of this work was presented as a poster at the annual meeting for APS this year. 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. 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. 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 fungicide work was presented at the ICC meeting and will be written up for the proceedings.
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. We identified two Bt toxins that have toxicity to ACP in bioassays. The most toxic of these ACP-active toxins has been modified with gut binding peptide 18. The sequence encoding ACP gut binding peptide 18 was introduced into the toxin at four different sites. The specific sites for insertion of the sequence was delineated based on PyMol modeling to increase the likelihood that the peptide would be exposed on the surface of the toxin. As some of the modified toxins did not express well in E. coli, additional approaches for toxin expression are being screened.
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 July indicate that soil pH in the plots receiving sulfur applications increased to about 0.5 pH unit above the target pH level at both sites. The increase in soil pH is the low amount of irrigation required during the spring and summer months because of higher than normal rainfall. At both the mature and young tree site, no significant difference in root density was found in samples collected in July. Likewise, no significant increases in nutrient concentrations were found in leaves collected in August were found among treatments because of lower soil pH. Average water uptake by trees affected with HLB were 15% lower than healthy trees. These data have been consistent for the past year. Therefore increasing evidence of reduced water uptake for trees receiving water supplemented with calcium bicarbonate have been documented. 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.
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)
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