Our objective for this project has been to evaluate botanical compounds as repellents for Asian citrus psyllid (ACP) with the purpose of developing possible repellent formulations for use in the field. Over the course of this research, we have identified several botanical insecticides that may be useful for management of ACP. Initially, we found that a sesquiterpenoid, citronellol, from lemon grass oil repelled psyllids. However, it was not toxic to ACP with topical applications. Beta-damascone, 9-decen-ol, and geraniol did not influence psyllid behavior. We found that clove oil was the most toxic botanical oil tested and killed nearly 100% of psyllids at a10ng/mL concentration. While both citronella and litsea oils caused 100% mortality at a 100ng/mL dosage, the lethal concentration was lower. Fir and camphor oils exhibited minimal mortality at 100ng/mL suggesting that these oils are not very toxic to psyllids. We selected five botanical oils with known repellency to other insects, to evaluate behavioral activity on ACP. In olfactometer assays, fir oil was repellent to female ACP. However, clove and camphor oils were attractive, while litsea and citronella oils elicited no response from ACP females. In no-choice settling experiments, neither the low (5mg/day) nor high (9.5 mg/day) fir oil dosages tested deterred ACP from settling on citrus. Subsequently, ACP were presented with a choice test between control plants and plants treated with the high dose of fir oil. ACP disproportionately settled on control plants, avoiding fir oil-treated plants completely. We conducted two field trials to determine if these oils affected ACP behavior in citrus groves. We found no attraction of ACP to yellow sticky traps baited with clove or camphor oil deployed from polyethylene vials as compared to unbaited controls. We found no repellency of ACP from sweet orange resets when treated with high release fir oil devices (10 g/day/tree). Our results suggest that ACP behavior may not be easily modified under field conditions by olfactory cues from botanical oil treatments. Volatiles from essential oils of coriander, lavender, rose, thyme, tea tree oil and 2-undecanone, a major constituent of rue oil repelled ACP adults compared with clean air. Also, coriander, lavender, rose and thyme oil inhibited the response of ACP when co-presented with citrus leaves. Volatiles from eugenol, eucalyptol, carvacrol, b-caryophyllene, a-pinene, a-gurjunene and linalool did not repel ACP adults compared with clean air. In an effort to isolate the repellents and toxicants from effective essential oils, the headspace components of coriander and lavender oil were analysed by gas chromatography-mass spectrometry and revealed that a-pinene and linalool were the primary volatiles present in coriander oil while linalool and linalyl acetate were the primary volatiles present in lavender oil. Coriander, lavender and garlic chive oils were also highly toxic to ACP when evaluated as contact action insecticides using a topical application technique. The LC50 values for these three oils ranged between 0.16 and 0.25 lg/ACP adult while LC50 values for rose and thyme oil ranged between 2.45 and 17.26 lg/insect. The repellent essential oil volatiles have potential for development into filed-based tools. In the field experiments conducted throughout the study, results were mixed and often negative. We cooperated with multiple companies in an effort to develop dispensers for field deployment of botanical insecticides. However, in most cases, the devices investigated to date were not comparable to standard toxic insecticides with respect to efficacy and population control of ACP in Florida.
In this enhancement phase of the Asian citrus psyllid vibration trap study, heavy-duty speakers have been operated with amplifier systems that produce airborne signals of 75-97-dB amplitudes with the goal of inducing vibrations in citrus trees that interfere with the mating duets of male and female Asian citrus psyllids. Tests have been conducted with signals produced at multiple harmonics of 200 Hz, i. e., at 200, 400, 600, . . ., up to 4000 Hz, to mimic the harmonics of the signals produced by the psyllids themselves. As expected, we found that additional harmonics are generated by the tree structure itself and the relative amplitudes of the harmonics are affected by the size and shape of the tree. Surprisingly, however the frequencies between 200 and 2000 Hz elicit high rates of calls by males on the tree, but they did not elicit movement. In eight tests completed with male and female psyllids placed on a citrus tree in the laboratory, no mating occurred while the speakers were producing high-amplitude sounds at any of the tested frequencies, but males often produced calls at when the speaker signals were produced between 200 and 2000 Hz. In one case, mating occurred after the speakers were turned off. These studies on the feasibility of interference with psyllid mating are continuing.
In this phase of the vibration trap research project, four different designs of trapping surfaces were tested in the laboratory with a “Bugphone” microcontroller device that detects Asian citrus psyllid male vibrational calls, plays back female replies, and attracts the males to the trap location. Three of the traps employed sticky surfaces to retain the males that were attracted and one used a trap door that shut when the male entered the trap. None of the new designs were considered effective enough to develop further for field usage. Consequently, we have begun consideration of a second option to count the numbers of male calls detected over time and save this information on a secure digital (SD) non-volatile memory card so that it can be retrieved later when the trap is serviced and the microcontroller system battery is replaced. A count of the rate of calls then could be used to estimate the number of males within the detection range of the trap. SD cards have been added to multiple Bugphones and testing will be done to determine their effectiveness in providing estimates of the psyllid population. In addition, electrical engineering colleagues at the University of Florida have begun studies to improve operation of the Bugphone by reducing its energy usage. We have constructed and are beginning tests of a version of the Bugphone that is expected to operate on three AA batteries for a week instead of the previous version that worked for four days on eight AA batteries. Ultimately the goal is to enable operation for a month or more without battery replacement.
The large-scale validation of citrus leafminer (CLM) disruption with the ISCA Dcept CLM technology is underway. Since the project began in the spring (2014) we assisted in applying the Dcept CLM product by hand to approximately 3,000 acres at three locations in southeast and southwest Florida. Our objective has been to determine how scale of application and proximity of pheromone-treated groves to untreated sources of mated CLM females will affect the efficacy of mating disruption with this product. Therefore, the tests have been established at three contrasting locations. For example, Emerald grove (The Packers of Indian River) in NW St. Lucie County is surrounded by extensive citrus plantations. VPI% (Golden River Fruit Co.) in SW St. Lucie County is surrounded by natural areas or pastures and receives relatively few immigrating females representing what we may expect from area-wide application of pheromone. To date, we have been collecting efficacy data by trapping CLM males with pheromone traps as a surrogate measure of mating disruption. Thus far it appears that efficacy is lowest on border areas and areas where there are a significant number of missing trees, as we would predict. However, thus far we have found the treatments to be efficacious overall. In addition to continuing monitoring of CLM populations with trapping in the field, we will conduct flush damage assessments in the next quarter.
We have almost completed the annual field survey of Asian citrus psyllid (ACP) susceptibility to insecticides for 2014. Our findings have been consistent over the past two years in that the levels of resistance have been declining and have remained consistently lower than what we observed between 2009-2012. However, during both 2013 and 2014, the rates of response to insecticides of field populations were significantly different from that of the laboratory strain. In most cases, the rates were much slower with respect to resultant mortality of ACP in field-collected, as compared to, the lab strain ACP. This means the ACP remain alive over a wider range of insecticidal doses. This suggests that there are underlying differences in metabolic processes, such as the activity of detoxifying enzymes, in the field-collected psyllids as compared with the laboratory strain. Once we have collected the remaining data, we will be analyzing these rates between the two years collected for each independent population to determine if differences have occurred within the same site. This should indicate how much gene flow occurs within a localized site among ACP populations. In addition to LD50 data, we also determined the infection rate of the psyllids collected at each site, which varied significantly: from 38.5 – 100% infection. Due to the fact that the ACP were collected within a short period of time (several weeks), we believe this has to do with spatial rather than temporal factors. While most of the LD50 estimates for the field populations were not statistically different from the laboratory strain, the rates of response from field populations were different, meaning that the field insects remained alive over a wider dose-range than the susceptible laboratory strain. This suggests that insecticidal treatment has resulted in changes within the genetic background of some field populations. We are monitoring this parameter carefully because it could mean that the genetics of these insects within these field populations is ‘primed’ so that with further selection pressure from insecticides, a resistant phenotype could develop quickly in these populations. It also tells us that we need to continue our efforts to understand the behavior of the insect, including how far they migrate and how they survive the winter. It also suggests underlying differences in metabolic processes among ACP in populations that have been heavily sprayed with insecticides in the past.
Abandoned citrus groves in US citrus-producing regions are potential sources for both Asian citrus psyllid (ACP), and Candidatus Liberibacter asiaticus (CLas), the bacterium that causes Huanglongbing (HLB). Since ACP adults are highly mobile, they can disperse from abandoned to productive citrus groves. If not controlled, these psyllids will stymie the effectiveness of area-wide management programs aimed at containing the spread of HLB in commercial citrus. ACP is susceptible to a native entomopathogenic fungus, Isaria fumosorosea (Ifr). For our project, we are developing a novel autodissemination system that will inoculate ACP with Ifr and use these infected psyllids to instigate epizootics and rapidly reduce ACP populations in abandoned groves. To accomplish our project goals, we have been collaborating with Paramount Citrus to conduct field trials in Hidalgo County with three of their managed groves and three adjacent abandoned groves. The first site is located in Edinburg, TX, and consists of an abandoned Rio Red Grapefruit grove with around 4500 trees that is adjacent to a managed Rio Red Grapefruit grove with around 4800 trees. The second site is located in McCook, TX, and consists of an abandoned Rio Red Grapefruit grove with around 400 trees that is adjacent to a managed Valencia Orange tree with around 1500 trees. The third site is also located in McCook, TX, and consists of an abandoned Valencia Orange grove with around 1000 trees that is adjacent to a managed Rio Red Grapefruit grove with around 1700 trees. We also had a fourth field site located near Doolittle, TX, that consisted of an abandoned Valencia Orange grove with around 2400 trees that was adjacent to a managed Rio Red Grapefruit grove with around 2200 trees but the abandoned grove was pushed out during the first week of our trials. During the first week of June 2014, we received our first shipment of prefabricated autodisseminator components from AlphaScent Inc. The field trials were initiated during the second week of June 2014 approximately one to two weeks after the managed groves at the four sites were flood irrigated. With these trials, we are evaluating impact of Ifr autodisseminators on ACP movement, infestation, and Ifr infection in plots of abandoned and managed citrus trees. At each of the three field sites, we have established both treatment plots and control plots. For treatment plots, we have hung pairs of autodisseminators with Ifr formulation and citrus-blend lures in trees on the edge rows of the abandoned groves. For control plots, we hung autodisseminators with only citrus-blend lures. To monitor ACP populations and movement, we are releasing every week up to 1200 ACP adults marked with fluorescent powder on abandoned trees at each field site and placing ACP sticky traps in the managed trees that directly face the abandoned trees. During the four consecutive weeks of each trial, we are replacing dispensers and inspecting ACP traps every 7 days. To evaluate the effect of field exposure on the efficacy of our Ifr formulation, we are comparing infection of ACP adults dusted with fresh formulation or formulation extracted from recovered autodisseminators. These trials will continue throughout the summer (June-July), fall (September-October), and winter (November-December) of 2014 and also the spring (March-May) of 2015. We are continuing to scout the Rio Grande Valley for other abandoned citrus groves that are located near managed citrus groves. If additional sites are found, they will be used for trials during the fall and winter of 2014 or the spring of 2015.
In the 6 year old block of Valencia on Swingle in Lake Placid, PCR positive trees with symptoms or pre-symptomatic were were treated with soil drenches of Magna-Bon (MB), Cop-R-Quik (CQ) and an experimental compound (EXP) with well demonstrated systemic activity against citrus canker (caused by Xanthomanas citri subsp. citri) as a soil drench. After two seasons of spring and fall soil drench applications with high and low rates of each of these compounds in 3 replicated blocks, visual tree health ratings on a scale of 1-5 (1=Vigorous, asymptomatic, 2=Slight decline; 3=Moderate decline; 4=Severe decline; 5=Non viable, won’t recover) were higher for treated trees than the untreated check. Tree responses indicate that these treatment were having phytotoxic effects on HLB infected trees rather than achieving reduction in bacterial infection and or reduction in HLB symptom expression.
The winter season is the ‘weak link’ in the seasonal survival of ACP and thus the time when populations of psyllids can be targeted to efficiently reduce their numbers. The original scope of work for this project was limited to identifying within-tree overwintering preferences for ACP populations in commercial groves and surrounding alternate hosts. During the course of this study, we have identified potential overwintering sites based on management practices and surrounding host vegetation. An unexpected result of our work so far indicates that significantly more ACPs are found in poorly managed groves than in groves under any other management regime. The objective of this supplemental project was to expand sampling from the five sites identified in the original proposal and to track psyllid movement between these sites (classified as well-managed, poorly managed, and abandoned) and surrounding alternate hosts throughout the winter. During the course of this study, we have identified potential overwintering sites based on management practices and surrounding landscape characteristics. We found significantly more ACP in intermittently managed groves in winter of 2012/2013 and groves under intermittent and organic management in winter of 2013/2014, rather than in groves under any other management regime. We have completed testing our hypotheses as to whether ACP, a) move to and ‘shelter’ in these groves during winter months, or b) whether they ‘hunker down’ and remain in place before dispersal to other groves in the spring. We have conducted movement studies using capture sticky traps set up at canopy height in concentric buffers surrounding groves to understand movement and directionality of ACP populations within citrus growing areas. During the winter season of 2013/2014 we have studied three local citrus area landscapes, under three different management regimes: organic, conventional and intermittent management. We found expected abundance relative to grove management of ACP within each grove throughout the winter using tap sampling, sticky trap sampling, and vacuum insect collection. However, there was no evidence of ACP movement between groves within our experimental areas during the winter and early spring months. We found that while movement increases in late spring, ACP abundance within groves is significantly higher in spring. These data indicate that within grove survival, rather than between grove movements, is the contributing factor to population densities found in groves under differential management. These data support the use of targeted dormant sprays (such as those used in groves under conventional management) and suggest that organic and intermittent managed groves may benefit from a targeted approach in ACP management in order to stem population resurgence in late spring.
Citrus greening is a devastating disease of citrus that has cost the Florida citrus industry over $3.64 billion. ‘Push-pull’ strategies because of their multi-component approach are often synergistic and have been successful in controlling insect vectors. The overall long-term objective of this research was to develop a push-pull system for the Asian citrus psyllid (ACP) that can complement integrated management systems in young citrus plantings. Towards that goal, in this study, visual factors that affect psyllid takeoff into flight and landing were examined to guide the development of an optimal pull component. Optimal visual responsiveness of ACP relating to flight or walking occurred in during the afternoon with less activity during the evening and night hours when phloem may be more nutritious to psyllids. A vertical bioassay demonstrated a strong attraction to green and UV light which related to flight orientation towards vegetation or the sky. Studies of horizontal movement of ACP revealed strong orientation to both UV and green light. Young ACP responded strongly to green visual targets with a decrease in attraction to green with age. Older females demonstrated a strong preference for UV possibly related to the need to find new host plants for oviposition or better food quality for egg development. A flight mill system based on Arduino microprocessors and programming was developed to simultaneously evaluate 6-12 individual ACP on flight mills. Effects of different rearing conditions of ACP on flight characteristics are being evaluated under different qualities of lighting. Effects of different rearing conditions of ACP on flight characteristics are being evaluated under different qualities of lighting. Results from these studies providing a better understanding of the specific behaviors of ACP in response to visual stimuli form the basis for development and optimization of push-pull elements for management of dispersing ACP onto citrus trees.
Characterization of antennal responses of male and female Asian citrus psyllid to host and nonhost plant volatiles using gas chromatograph-coupled electroantennographic detection (GC-EAD) and simple electroantennography (EAG) was largely completed. Dose-response curves for active compounds were constructed and a manuscript is in preparation. Certain common plant volatiles present in citrus and other ACP hosts were determined not to elicit ACP antennal response. However, degradation products of those same chemicals were discovered to be highly active in eliciting depolarization of antennal chemoreceptors. These compounds are being further studied for their practical application. We continue to develop new bioassays to discover the basis for resistance in certain accessions of Poncirus trifoliata to the Asian citrus psyllid. Quantitative probing assays suggest the presence of plant compounds that deter probing in some trifoliate accessions. This is now being examined through the use of an electronic penetration graph that allows determination of stylet location and feeding behavior over 24 hours or longer. ACP feeding behavior varies greatly between accessions. Stylet position will be confirmed histologically by thick sectioning, staining and visualization including scanning electron microscopy. Results of comparative SEM of resistant and susceptible trifoliate accessions suggest that physical barriers may play a role in blocking stylet access to phloem elements in resistant accessions.
June 2014 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. The small plot work of the leaf litter was completed and the data processing has begun. The field site for the large trial with 3 treatments was mapped for disease incidence and laid out. To look at the effect of bagasse, a controlled experiment portion was conducted twice. It involved an in vitro study of the decomposition of citrus leaves and inactivation of G. citricarpa by various amendments including bagasse including the microbial consortium of fungal and bacterial strains to aid in the decomposition of bagasse and of citrus leaves. Leaves and other components were collected at time zero and at 10 day intervals. At each collection time, leaves and other components were oven dried and weighed. Soil weight, leaf weight, and then the total weight of each box were recorded. Leaf infection by G. citricarpa was verified by plating and qPCR will also be used. Data collection and analysis is ongoing. Collection of leaf litter material has begun in Florida and is being collected every two weeks from a grove with moderate black spot incidence in the previous season. We also have done many isolations from fruit collected as part of the CHRP surveys to get as wide a population of G. citricarpa isolates as possible. The subcontracting process with the University of Queensland has been completed and the UQ and HAL contact was signed so work will begin in earnest. Collection of the different spore stages was completed in March and preliminary analysis indicated the need for cardinal sampling. The PI visited our Australian sites and we were able to get the DNA extraction to work as hoped. The DNA has arrived in FL for qPCR detection of the two Guignardia spp. Some symptoms were appearing on the inoculated fruit in Bundaberg but not enough to make any conclusions yet. This round of inoculations allowed us to find out how many fruit will need to be inoculated in the future for reliable results. Many abscised over the year.
June 2014 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: All preliminary experiments with the DMIs are completed and tests are underway. Our main limitation is suitable incubator space at the moment. A student has been found to work with the SDHI compounds. Objective 2: Data from spray trial have been taken and are still being analyzed. Sites have been selected for then next trial and planning has begun for the 2014-2015 season. Objective 3: Suitable sites to conduct the spatial studies have been located. Blocks were scouted and initial disease incidence and severity were collected. Objective 4: Asymptomatic and symptomatic fruit from a known symptomatic were harvested and a set of experiments were initiated to look at the effect of postharvest fungicides and heat treatments. In vitro heat treatments were also conducted and heat did have an effect on G. citricarpa growth on petri dishes. Some experiments were also started on fruit with heat and carvacol. Experiments are on-going and results are expected soon.
Sept 18, 2014 The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. Guignardia mangiferae sequencing is complete and the assembly is continuing. To get better resolution with the G. citricarpa genome, the organism was resequenced, giving far better resolution from the genome data. Analysis of the gene structure is being continued. A manuscript on mating type genes is in preparation with all of the data for figures collected. Materials and methods were drafted and are under revision. We decided to run another set of confirmatory experiments after the results of the last experiments were somewhat inconclusive. Sample collection for the greenhouse inoculation experiment continues and the samples are being fixed and embedded. We moved from weekly sampling to biweekly sampling. Some fungal penetration structures have been observed in the samples. Irradiated and autoclaved citrus twigs were inoculated with a replacement series of G. citricarpa and D. citri inoculum. In initial results of the first trial of the experiment, there was greater initial colonization by D. citri than G. citricarpa.
June 4, 2014 The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. Guignardia mangiferae sequencing is complete and the assembly is continuing. To get better resolution with the G. citricarpa genome, the organism was resequenced, giving far better resolution from the genome data. A manuscript on mating type genes is in preparation with all of the data for figures collected. Materials and methods are being written. We continuing the confirmation of some of the results in one section. An inoculation experiment with conidia on leaves was preformed and samples continue to be collected at regular intervals for microscopic observation for when infection structures are formed and when the fungus starts to spread through the leaf. The paraffin embedding technique was fine tuned. Irradiated and autoclaved citrus twigs were inoculated with a replacement series of G. citricarpa and D. citri inoculum. The fungal structures and qPCR are being counted bi-weekly
Reduction/suppression of inoculum and fruit infection in grapefruit using trunk applications of non-insecticidal SAR inducers:SAR induced by soil-applied insecticides provides substantial benefits for canker disease management on young citrus trees that may be augmented with ASM. Currently, use of neo-nicotinoids insecticides for control of the psyllids and leafminer is limited to trees less than 2.75 m tall. Soil drench or trunk applications of IMID and THIA and ASM are bing compared to untreated controls and 9 or 10 standard 21-day interval copper sprays for protection of foliage and fruit on 5 to 7-yr old ‘Ray Ruby’ grapefruit trees. Actigard is commercial SAR inducer which is non-insecticidal and does not have a tree size restriction for use. Collaboration with Syngenta in an EPA-approved Experimental Use Program (EUP) in two east coast grapefruit groves is being repeated again in 2014 to support the labeling of soil applied Actigard integrated with copper sprays for control of canker on bearing grapefruit. Reduction/suppression of fruit infection in citrus trees using foliar applications of soluble copper novel bactericidal formulations in comparison with film-forming copper formulations: In 2014 trials, fixed copper formulations (e.g. copper hydroxide and copper oxide) sprayed at 2 to 4 lb of product per acre (0.75 to 1.4 lb per acre of metallic copper) are being compared with commercial and experimental soluble copper formulations at much lower metallic rates in grapefruit and Hamlin blocks. Improving performance of copper formulations: Reducing wind speed below 18 mph greatly increases the efficacy of the copper film for protection of the fruit surface from infection through natural openings and rind wounds. 6 and 7 yr-old red grapefruit blocks surrounded by a 20-30 ft tall Corymbia torelliana windbreaks are under study. In each block, weather stations are deployed to measure wind gusts in proximity to the windbreak. Cu bioavailability and residual activity of copper formulations: Copper residues of fixed and soluble copper formulations continue to be evaluated at different periods of season as rainfall and fruit expansion rates vary.
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