July, 2010 ‘ Met with Dr. Steve Rogers in Winter Haven to discuss and submit raw data and reports developed from the project. August, 2010 – No Activity September 2, 2010 ‘ Submitted a no-cost extension to FCPRAC due to the volume and complexity of the data resulting from the project.
No progress in the field trials has been made since the October update. Currently we are planning multiple field trials to begin in spring 2011 that will validate the greenhouse studies on the effects of altering host plant nutrition on psyllid population dynamics. These trials will evaluate use of differing nutrient strategies to help manage psyllid populations using certain nutrients applied at rates that would be feasible for commercial citrus production without undue harm to the overall grove production or tree health. We are also in the process of propagating more ACP resistant and susceptible Cleo hybrids for more in depth studies on the mechanism of resistance to ACP observed in some Cleo plants in the greenhouse and field trials.
The results so far indicate that with regional management of HLB: – The disease epidemics starts later (299 days) and is slower (disease progress rate reduced in 75%); – Increases the efficiency of local control measures because reduces the migrant psyllid population (ACP population is decreased year after year even in plots without insecticide application), reduces the regional inoculum sources, and finally reduces the migrant infective psyllid population that reach the local plots. – Reduces the control costs because with less intensive vector control program (insecticide sprays every 28 days, or even no insecticide sprays) it is possible to be more efficient than a high intensive vector control program (insecticide sprays every 14 days) applied locally within a region without regional management. – Allows resets and solid block replanting with the guarantee of yield. In plots of E2 without and with vector control the yield still increasing (2 ton/ha in the first, 16 ton/ha in the second and 38 ton/ha in the third harvest) while in plots of E1 the yield decreased year after year (2 ton/ha in the first, 9 ton/ha in the second and 10 ton/ha in the third harvest for plots with insecticide application, and 2 ton/ha in the first, 4 ton/ha in the second and 3 ton/ha in the third harvest for plots without insecticide application). Now we are interpreting the outcome results from spatial analysis of annual maps of HLB-symptomatic trees to verify the effects of each treatment on primary and secondary spread of HLB. Experiment 1 (E1) under high external inoculum pressure was finished at the end of November 2010 with 76.7% of infected trees in plots without vector control and 53.0% in plots with vector control. Experiment 2 (E2) under low external inoculum pressure is with 4-6% of eradicated trees and will be assessed for more time for better understand about the temporal longevity of a grove under regional management of HLB. These preliminary results will be presented in the 2nd International Research Conference on Huanglongbing in Orlando ‘ January/11. A paper with the temporal analysis of both experiments is on preparation and would be submitted for publication until April/11. The results of this project are also being presented to growers to help the establishment of growers’ groups for regional management of HLB in other countries.
Research on low volume spray application technology has been continued with complementing field and laboratory investigations. Field investigations were conducted to gain a better understanding of how efficacy of various chemistries may differ when applied at different times of the year. In addition, the effect of various adjuvants on droplet size and efficacy of low volume sprays has been investigated. Laboratory investigations have focused on the impact of spray droplet size on mortality of Asian citrus psyllid (ACP) as well as how adjuvants impact droplet size range. Results from the field investigations suggest that applications of the pyrethroid, Danitol, are not equally effective against ACP at all times of the year and under all environmental conditions. The results suggest that effectiveness of this treatment applied by low volume may be lower later in the summer as compared with winter and early spring applications. This may be due to increased humidity and wetness of foliage in the summer as compared with the fall and early spring. Our laboratory evaluations with several pesticides to date (Danitol, Lorsban, Dibrom) consistently show that mortality of psyllid eggs, nymphs, and adults increases as droplet size is decreases. With all products tested in the laboratory to date optimal psyllid kill appears to be with about 100 micron droplets. However, droplet size measurement of Danitol, when sprayed with a commonly used low volume applicator, revealed larger than the optimum and more variable droplet size range. To remedy this problem, we have investigated whether addition of various adjuvants to Danitol could reduce average droplet size. We found that addition of the organosilicone adjuvant (Sylgard) at 5 % by volume could reduce average droplet size of the low volume Danitol spray for optimal psyllid kill and compliance with the label requirement. Addition of this adjuvant also makes droplets much more uniform in size. The effect of additional organosilicone adjuvants on droplet size of low volume application is currently being investigated. We are currently investigating whether those adjuvants could improve efficacy of low volume sprays with insecticides such as Danitol and whether their addition could improve efficacy when applied to wet foliage.
This project started in May and the previous report was submitted in August. The main objective of the project is to evaluate impact of psyllid control programs on non target pests and beneficial insects in citrus groves. Increased use of insecticides to control psyllid continues to show negative impact on non target pests of citrus. In a recent survey of grove managers in southwest FL, 30% reported 6-11 sprays of insecticides to control psyllids during the past 12 months and 100%, 67%, and 33% of them reported seeing an increase in the populations of citrus leafminer, scales and mites, respectively. In contrast, 70% reported 5 or less sprays and only 14% and 29% of those reported increased citrus leafminer and mites, respectively. Studies conducted before the advent of HLB in Florida in 2005 showed huge impact of predation, particularly by ants, on citrus leafminer populations. It appears that increased use of insecticides in conventional groves is significantly impacting populations of ants and/or other natural enemies. A recent comparison of organic and conventional groves in Charlotte county showed huge difference in the populations of ants and spiders between the two practices. An average of 22 ants and 11 spiders were observed per 140 tap samples conducted in the organic grove compared to 0.3 ants and 4 spiders observed in the conventional grove. Trees in both groves were not flushing at the time of sampling, but old damage indicated a very serious infestation of citrus leafminer in the conventional grove compared to the organic grove. Ants and spiders were also the only common predators in the citrus block in Glades county which was also sampled in September and is being treated with nutritional mix and 435 Oil @ 2%. Ants and spiders averaged 0.5 and 0.3 per 10 tap samples in the treated blocks respectively, compared to 1.4 and 0.6, respectively, in the untreated plots. Only a few young shoots were available and 38% and 75% were infested in treated and untreated plots, respectively. A trial comparing low vs. high volume applications of Movento along with Mustang Max, Baythroid, Provado, and Agrimek was initiated to look at impact on psyllids and non target pests and beneficial insects. Data is being collected and will be presented in the next report. Two more replicated trials are being initiated on oranges in Hendry county, one in a 35 acre block of ‘Early Gold’ and the other in a 16 acre block of ‘Valencia’. There are 4 treatments: three spray programs one receiving monthly calendar sprays, two intended to maintain ACP populations below thresholds of 0.2 or 0.7 psyllids per tap sample and an untreated check. The plan is to use exclusion cages to evaluate mortality from predaceous and parasitic arthropods in each plot and evaluate the relationship with intensity of insecticide use. Additionally, we are monitoring infestations of Florida red scale, lesser snow scale and black fly in commercial groves.
We are attempting to develop an RNAi-based approach for controlling psyllids. Our goal is to identify and then use specific psyllid RNA sequences to induce RNA interference activity in recipient psyllids. To make rapid progress we are using the tomato psyllid (Bactericerca cockerelli), which colonizes herbaceous plants and is the vector of Liberibacter spp. (Ca. L. psyllaurous), which is believed to cause psyllid yellows in tomato. Currently, sequence information of B. cockerelli is very limited in available databases. However, we were able to clone 28 gene fragments from B. cockerelli by using D. citri homologous primers from the D. citri midgut cDNA library database. We also generated a B. cockerelli normalized cDNA library and so far have sequenced less than 100 targets. We have developed three approaches for delivering candidate RNAs to psyllids including a Tobacco mosaic virus (TMV)-based expression system and tomato plants; micro-injection of dsRNAs directly into the psyllid hemocoel; and artificial diet membrane feeding in vitro. We have used our artificial diet system for screening candidate dsRNAs for B .cockerelli. Psyllid acquisition of the artificial diet was confirmed by food dye and Cy3-labelled dsRNA visualization. Both are obviously apparent in the psyllid gut tissues. When the dsRNAs for psyllid sequences were added into the food, we saw increased mortality of B. cockerelli for some sequences at higher concentrations of dsRNA. Four of ten sequences caused substantial psyllid mortality as compared with GFP dsRNA controls. Consistent and specific down-regulation of endogenous mRNA also was revealed by qPCR, and corresponding siRNAs were detected by Northern blot analysis. These latter two analyses demonstrate RNAi activity in psyllids. Second, we are using a TMV-based plant expression system which is efficient for producing dsRNAs/siRNAs for insert psyllid sequences in tomato plants. So far, decreased survival of B .cockerelli was detected for 2 from 6 sequences after 10 day feeding on plants. We are in the process of analyzing psyllids for RNA hallmarks of RNAi activity. Direct intrathoracic injection of dsRNAs into the psyllid hemocoel is also being used to test candidate sequences. Survival for injected psyllids ranges from 50 ‘ 60% three days post injection. We are using injection only as a positive control for comparison with the other two approaches, and as an alternative approach to identify candidate dsRNAs for subsequent analysis by oral feeding. In conclusion, we developed an applicable system for screening of psyllid sequences for RNAi effects, and efforts for transgenic plants are also underway. We have cloned the promising sequences into plant expression vector to produce dsRNA/SiRNA by transgenic approaches, and we will use these vectors to get transgenic tomato.
Objective 1. Localization of Liberibacter asiaticus (LAS) in the hemolymph and other tissues and organs of the Asian citrus psyllid (ACP). [A] Using Fluorescence In situ hybridization (FISH): We continued our studies to improve the sensitivity and specificity of several FISH protocols to localize LAS in hemolymph smears and dissected organs of ACP, and in leaf sections from HLB-infected plants as positive controls. So far, LAS has been detected in the hemolymph, filter chamber and midgut of LAS-infected ACP both from laboratory colonies and infected field trees, but not in healthy controls. It has been also detected in leaf sections from HLB-infected citrus plants but not in those from healthy ones. [B] Using quantitative RT-PCR on dissected insect organs: We tested different RT-PCR protocols for detection and quantification of LAS in dissected salivary glands, alimentary canals and other parts of individual ACP adults either from our laboratory colony or collected from field HLB-infected citrus trees. In field collected psyllids, the proportion of infected salivary glands (47%) was significantly lower than those of the alimentary canal (72%) or other body parts (79%). A similar trend was found with ACP adults reared as nymphs in our lab. on HLB-infected citrus. Interestingly, the relative titer of LAS, compared to psyllid genomic DNA in each ACP sample, was significantly higher in both the salivary gland and alimentary canal compared to that in the rest of the insect body. These results strongly suggest that the salivary glands constitute a major transmission barrier to LAS in the psyllid vector, and that LAS may replicate or accumulate in both the alimentary canal and salivary gland of ACP. [C] We continue to use a combination of transmission electron microscopy (TEM) and RT-PCR to compare the ultrastructure of ACP adults that have never been exposed to infected plants with those collected from HLB-infected citrus and are PCR-positive for LAS. This is providing a very useful library on the ultrastructure of the alimentary canal, salivary glands and other organs of ACP as well as its bacterial symbiotes, and will form the basis for future studies on LAS pathogen-vector interactions in ACP at the cellular and tissue levels. Objective 2. Clarification of various acquisition and transmission parameters between ACP and LAS. [A] We have started two large experiments to compare between young (2nd-3rd-instar) nymphs and adults having various acquisition access periods (AAP) on HLB-infected citrus plants. Previous reports have indicated that ACP nymphs are more efficient in LAS acquisition than adults, and that LAS probably multiplies in ACP nymphs but not adults, but the effects of various AAPs on nymphal and adult stages in this regard have not been carefully studied so far. Our experiments are thus designed to study the effects of various AAPs on both acquisition and transmission of LAS in ACP, as well as on LAS replication in both nymphs and adults. [B] Percentages of LAS-infected psyllids tended to be lower during late spring or early summer with no other obvious seasonal trends during 2008-2009 at a USDA grove in Fort Pierce, FL. A general decline in percentages of infected psyllids appears to have taken place during 2009-2010. No seasonal differences in percentages of infected psyllids or titers of LaAS in psyllids have been found with respect to psyllid sex or color morph. Among infected (CT<36) psyllids from the grove, an average of 18% (range 0 - 64%) psyllids transmitted LAS to healthy seedlings in monthly experiments, with no seasonal trends identified. Transmission rates are being investigated for each sex and for each of the three color morphs.
A series of research greenhouse research experiments have been completed that show that a number of commercial formulations of plant growth regulators (PGRs) (GA biosynthesis inhibitors) reduce Asian citrus psyllid fitness. Specifically, we found that two of the PGRs tested (prohexadione calcium and mefluidide) reduced ACP fecundity and survivorship while two others, uniconazole and paclobutrazol, reduced fecundity or survivorship, respectively. These results have been summarized in a manuscript that has been submitted to the Entomological Society of America for publication in a peer reviewed journal. A follow-up study to determine the duration of efficacy of these treatments has recently been completed using one of the PGRs (prohexadione calcium). The data from that study are currently being analyzed. An experiment is underway to determine what changes are occurring within plants that elicit the observed changes in psyllid fitness. Large container grown trees have been treated with Apogee and tissue samples are being harvested at 3 day intervals. The samples are being analyzed for nutrient, free amino acid, phenolic, and flavonoid contents. The final samples from that study will be collected during the week of October 18. Sample extraction and analysis will then be completed in the subsequent weeks and analysis of the data undertaken. Results of this study will be used to develop further studies to determine the basis for the observed effects. New work is being developed to determine whether the effects of PGRs on psyllid fitness correspond with changes in either acquisition or transmission of Candidatus Liberibacter asiaticus (Las). The first step in preparing for that line of research is to develop a population of Las-infected trees that can be used in acquisition studies. Greenhouse grown Las-infected trees used as infected budwood source material has been confirmed Las+ by PCR . Budwood from these trees has been harvested and used to graft inoculate a population of citrus trees. Acquisition studies will proceed as soon as the new plant material has been successfully infected. We anticipate that occurring early in 2011. In the interim, a population of ACP is being maintained and increased for use as soon as the plant material is ready.
Objectives of this project include 1) refinement of sampling methods, 2) testing the influence of adult density and shoot infestation on precision of estimated means and distribution of population within blocks, and 3) evaluation and integration of methods for assessing psyllid density, shoot density, and infestation rates into a user friendly system accessible to consultants and managers. Accomplishments are described for each objective. 1) We reported previously on comparisons among tap sampling, sticky traps and sweep net sampling methods. These results showed that at low density, the tap method was more efficient in detecting ACP than the sweep net ,although no significant difference was observed in numbers detected and sampling time at high density. For those comparisons ACP adult density averaged 0.5 or less per sample according to either method. Additional comparisons were made in two commercial groves in RCB designed experiments using different treatments to manage the psyllid. In the first grove, both methods provided the same statistical separation between treatments in July. There were <0.5 adults per sample in two treatments and the control and >1 adults per sample in one treatment. At <0.5 adults per sample, there was no significant difference between the two methods for adults or time per sample with an average of 0.3 ' 0.1 adults and 8 ' 0.2 sec, respectively. There also was no difference between the two methods in adults or time per sample at >1 adults per sample with an average of 1.4 ‘ 0.2 adults and 9 ‘ 0.4 sec, respectively. A month later in plots where adult numbers the previous month were highest, the tap detected 0.8 ‘ 0.1 adults per sample, significantly more than 0.2 ‘ 0.2 adults per sample using sweep net. However, time to conduct two samples did not differ and averaged 7.6 ‘ 0.4 sec per sample. In the other three treatments, number of adults and sampling time did not differ between methods, averaging 0.1 ‘ 0.0 adults and 7 ‘ 0.1 sec respectively. In the second commercial grove, sampling was conducted in Sept. and there was no difference between two methods to detect an average of 0.2 ‘ 0.1 adults per sample using 4 ‘ 0.4 sec with tap sample, significantly less than 7 ‘ 0.2 sec using sweep net. 2) We are collecting more data to compare the tap, sweep net, and sticky trap methods. Data will be subjected to regression analysis using a bootstrapping procedure and precision targets of 0.25 and 0.01 SEM:mean will be estimated to determine the number of samples required using these methods for routine monitoring and analytical modeling respectively. We have already reported some of these estimates from previous studies in earlier reports and in several of our presentations and workshops. An extension (EDIS) document describing ACP sampling techniques (ENY857/IN867) is in-press. and we are working on a peer review publication 3) A rapid system that includes tap samples, flush inspections and natural enemy evaluations along with forms and spreadsheets is available on our website swfrec.ifas.ufl.edu/entlab/ and was published in Citrus Industry. Several of our collaborators and many growers are using tap method to sample for ACP to help make treatment decisions in citrus. We also provide tap sampling kits to growers in our workshops or on demand. We are also working on the development of a website where growers will be able to enter their sampling data on the incidence of pest and disease in order to assess the need and recommendation of appropriate treatments. Stansly, P., A. Arevalo and J. Qureshi. 2010. Monitoring methods for Asian citrus psyllid. Citrus Industry 91(4) 20-22.
The goal of this project is to determine if infection of Asian citrus psyllid (ACP) by Candidatus Liberibacter affects the response of ACP to its citrus host plants. This information should provide us with important insights regarding disease spread. In this project we evaluated if ‘healthy’ (not carrying Candidatus Liberibacter) psyllids are attracted more to HLB-infected versus healthy citrus trees. We are also determining whether this behavior changes after the ACP vector becomes infected with the pathogen. Behavioral experiments indicated that HLB-infected citrus plants are more attractive to adult ACP than healthy plants in two-choice olfactometer experiments. Also, more ACP were attracted to HLB-infected plants than to healthy plants in open-air choice experiments. However, subsequent dispersal of ACP adults to healthy plants following their initial choice indicated that final setting preference was for healthy rather than diseased plants. We are are testing two hypotheses to explain the initial movement of ACP to infected plants and subsequent dispersal to healthy plants. 1. Yellow color of HLB-diseased plants due to chlorosis and yellowing of shoots may attract the ACP initially but psyllids then move to healthy plants after briefly feeding on the plant or 2. HLB-infected plants produce deceptive volatile compounds to attract ACP adults to facilitate the spread of bacteria. Settling experiments with HLB-infected and healthy plants in complete dark conditions produced similar results to the ones under full light conditions suggesting that initial movement of psyllids to HLB-infected plants is not due to the yellow color but to some other factors. Head space analysis of volatiles from HLB-infected and healthy citrus plants indicated that these plants had significantly different chemical profiles. For example, HLB-infected plants produced significantly more methyl salicylate (MeSA) than healthy plants while healthy plants produced higher amounts of methyl anthranilate (MA). Furthermore, HLB-infected plants were lower in pH than the healthy plants. MeSA is known to attract natural enemies and to repel herbivores and low pH is not suitable for insect development. Therefore, these two factors could partially explain the final settling preference of psyllids on healthy plants. Our ongoing research on this includes determination of response of psyllids to MeSA and MA compounds and nutritional analysis of HLB-infected and healthy citrus plants to determine the effects of nutritional status of plants on psyllid behavior.
Recently we identified several sulfur chemicals from guava that repel Asian citrus psyllid (ACP) in the laboratory, but are difficult to formulate into controlled release devices for field use because of their high volatility. As we continue to work on formulating these sulfur compounds into devices that will have practical application, we have also investigated several potential “of-the-shelf” essential oils for their repellency against ACP. These were chosen based on their known repellency to many insects and based on their perceived similarity to guava in chemistry. ACP generally rely on olfaction and vision for detection of host cues. Certain plant volatiles and plant-derived essential oil products are known to repel several insect species and are considered minimum-risk pesticides. We examined the effect of five essential oils previously reported to have activity against various insect species on ACP behavior in a two-port divided T-olfactometer in the laboratory in an effort to identify an effective natural repellent and/or insecticide for ACP. 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, .-caryophyllene, .-pinene, .-gurjunene and linalool did not repel ACP adults compared with clean air. Chemical analysis of the headspace components of coriander and lavender oil by gas chromatography-mass spectrometry revealed that .-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 3 oils ranged between 0.16 to 0.25 ‘g/ACP adult while LC50 values for rose and thyme oil ranged between 2.45 to 17.26 ‘g/insect. Our current efforts are focusing on quantifying the airborne concentrations of these essential oils found to have behavioral activity against ACP that are required to induce the effect. Our current results suggest that garlic chive, lavender, and coriander essential oils should be further investigated as possible repellents or insecticides against ACP. Also, these repellents may be useful in organic citrus production, which currently has few available tools for management of ACP. We have also developed a method with which to sample and quantify the airborne concentrations of sulfur violates directly in the field. We are perfecting this method so as to be able to directly quantify the airborne concentration of DMDS in the field that is associated with our SPLAT treatments. We believe this will help us understand why certain applications of DMDS show effectiveness in suppressing ACP populations while others do not. Our field results with DMDS released from SPLAT have been mixed. While some trials appeared to show reductions of ACP populations, others did not. We have almost completed a large investigation of four new SPLAT formulations of DMDS and will have that information compiled soon.
Collection of field data for the methyl salicylate (MeSA) study was concluded on 10/5/2010. Approximately 3300 traps samples and 1300 sweep net samples were collected over the course of the season from the three locations investigated (unmanaged, minimally managed and intensively managed groves). About half of the sweep samples and 1/3 of the trap samples have been examined for the presence of Asian citrus psyllids, parasitic wasps, coccinellid beetles, lacewings, ants, spiders and other predatory insects. In all groves, psyllid populations remained low throughout the season (even in the unmanaged grove). The overall average number of psyllids per trap was <1 for the intensively managed grove, ~1/trap for the minimally managed grove and up to 1.5/trap for the unmanaged grove. This represents the number of psyllids caught/trap per 2 week intervals. For the samples processed, there have been no significant differences between psyllid numbers or in the number of beneficial insects captured between plots treated with MeSA and control plots. We were successful in maintaining the flush collected so that psyllid development could be completed; however, only 4 parasitoids were recovered throughout the study. During this season, psyllid populations did not increase in the spring and early summer as in previous years in the citrus locations investigated, perhaps due to a combination of effective area wide management of psyllids with insecticides, winter freezes, atypical spring flush cycles and record high temperatures during the summer. Similarly, the low number of psyllids present in our study sites may have caused similarly low numbers of beneficial organisms captured in sweep net samples and sticky traps. Although MeSA may have initially attracted beneficial organisms, when suitable hosts were not found, they may have left our field plots. The remainder of our samples need to be processed for us to conclude whether MeSA had an impact on psyllid populations and populations of beneficial organisms in our trials this year. We are initiating psyllid behavioral studies with MeSA in the laboratory. MeSA has been reported as a repellent for several insect species and this will be tested in a two-choice olfactometer investigation. Additionally, psyllid responses to plants sprayed with an ethanol: MeSA solution will be tested in the following experiments. In one experiment, psyllids will be released in a large screen cage containing sprayed and unsprayed plants and the numbers of psyllids settling on each plant type over time (1 hour, 24 hrs, 3 days, 5 days and 7 days) will be recorded. In a second experiment plants will be sprayed with MeSA and an equal number will be unsprayed. Ten male and 10 female adult psyllids will be caged on new flush of each plant. After 7 days, the cages will be removed and the number of surviving adults as well as eggs and nymphs will be counted and compared.
The monthly immunomarking study to investigate the seasonality of Asian citrus psyllid (ACP) dispersal and the potential impact of abandoned citrus on nearby managed citrus has continued since the previous report. We now have 16 months of continuous data. Psyllid populations within the study area, which had been much lower this summer than last, have remained low through August and September. During the August trial, 6 psyllids were captured, with one psyllid moving from the interior of the abandoned grove into the interior of the managed grove plot. Six ACP were trapped in September, though there was no movement recorded between grove plots. Through ELISA and PCR we continue to confirm that HLB-infected psyllids are moving from abandoned citrus into nearby managed citrus. In evaluating the dispersal range of ACP, we previously presented data from June of 2010. To recap, in June a total of 179 adult ACP were captured, and 19% carried the protein mark. When we further evaluated the June study, we found that daily wind direction was not significantly correlated with psyllid dispersal. To determine if dispersal is guided more by flush availability than wind direction, we repeated the experiment in July. On the final day of the July experiment, we calculated relative flush abundance by randomly placing a square cube made of PVC pipes and fittings into the tree canopy and counting all of the flush emanating from within the cube. Five trees were selected at each trap site, and each tree was sampled twice in this manner. Psyllid populations were high in the studied area in July, and sub-sampling was necessary. We sampled a total of 541 captured adult ACP, and 18% were positive for the protein mark. ACP from both months that tested positive for the protein mark were subjected to PCR analysis to test for the presence of the greening pathogen. Protein-marked, HLB-infected psyllids were found on traps 2 km away from the marked area in both trials. Our results indicate that ACP can move at least 2000 m within 11 days, that wind is not significantly correlated with ACP dispersal, and that movement is positively correlated with the presence of flush.
Insecticides are currently the basis of Asian citrus psyllid (ACP) management programs and the number of annual insecticide applications has increased significantly. We continue our investigations of insecticide susceptibility and resistance among field populations of adult and immature Asian citrus psyllid in Florida. Our goals are to monitor resistance and develop effective psyllid management programs that mitigate escalation of resistance development. Five geographically discrete populations of ACP displayed a range of susceptibility levels against twelve tested insecticides. In 2009, one or more field populations of ACP exhibited higher LD50 values to fenpropathrin, imidacloprid, malathion and thiamethoxam compared with the laboratory susceptible population. The highest level of resistance was displayed by the La Belle population with a resistance ratio or RR (LD50 value of field population/LD50 value of laboratory susceptible population) of 38 and 13 to imidacloprid and thiamethoxam, respectively. Three populations displayed a moderate level of resistance to malathion (Ft. Pierce: RR = 5.4, Lake Alfred: RR = 5.0, Groveland: RR = 3.7). The Vero Beach population displayed a moderate level of resistance to fenpropathrin with a RR value of 4.8. In 2010, three diagnostic doses corresponding to 50, 75 and 95 percent mortalities determined from the laboratory susceptible population in 2009 were chosen to assess the susceptibility of field populations in 2010. Susceptibilities of field populations were compared with the laboratory susceptible population as in the previous year. In general, percent mortality of adult ACP from each field population was lower than that of the laboratory susceptible population for all three diagnostic doses tested. This indicates that resistance levels continue to increase from year to year. The susceptibility levels of immature ACP from four field populations to five insecticides were determined. The LC50 value for carbaryl obtained from the laboratory susceptible population was significantly lower than from the highest value obtained from one of the field populations representing a resistance ratio of LC50 [RR50] of 2.88. For chlorpyriphos, the highest LC50 of 8.31 observed from a field population was significantly higher than the laboratory susceptible population (2.58), representing an RR50 of 3.22. The synthetic pyrethroid, fenpropathrin, yielded a range of LC50 values from 0.15-0.57, with the lowest value obtained from the laboratory susceptible and highest from the Groveland population. Two of the field populations tested exhibited significantly lower susceptibility to imidacloprid than the laboratory susceptible population exhibiting RR’s of 3.81 and 2.27, respectively. Two of the populations tested showed significantly lower susceptibility to the microbial insecticide, spinetoram, than laboratory the susceptible population with RR50’s of 2.98 and 5.88. The current results suggest that ACP have developed varying levels of resistance to major insecticides currently registered for its management. Elevated levels of detoxifying enzymes in these populations may be underlying mechanisms of this resistance. Although none of these current resistance levels should result in product failures in the field, continuing elevation of resistance among Florida populations of ACP needs to be stopped. Rotation of insecticides is critical to mitigate this emerging problem.
.-aminobutyric acid (BABA) is known to induce resistance to microbial pathogens, nematodes and insects in several host plant/pest systems. The present study was undertaken to determine if a similar effect of BABA occurred against the Asian citrus psyllid (ACP) in citrus. A 25 mM drench application of BABA significantly reduced the number of eggs/plant as compared with a water control, whereas 200 and 100 mM applications of BABA reduced the numbers of nymphs/plant and adults/plants, respectively. A five mM foliar application of BABA significantly reduced the number of adults but not eggs or nymphs when compared with a water control treatment. BABA induces resistance in plants through a number of physical and biochemical mechanisms. Accumulation of pathogenesis-related (PR) proteins is one of the responses elicited by application of BABA to plants. Expression of systemic acquired resistance (SAR) in plants as a result of BABA application is related to elevated expression levels of the PR-1, PR-2 and PR-5 genes. The objective of this experiment was to determine whether exposure of citrus plants to ACP adult feeding alone or in combination with BABA treatment induces expression of the PR-2 gene. Significant variation in the expression level of the PR-2 gene was observed among treatments. Significantly higher expression of PR-2 was observed in plants exposed to 500 mM of BABA in combination with ACP adult feeding when compared to the control or other treatments tested. In citrus, SAR induction was explained by the high expression of the PR-2 gene, which in turn relates to a higher resistance of citrus against canker (Francis et al., 2009). The results of the present study show that the PR-2 gene was upregulated by more than 150-fold in citrus treated with BABA in combination with ACP adult feeding compared to the control or citrus treated with BABA or ACP feeding alone. These results suggest that PR proteins, or at least one PR protein in citrus, accumulates as a result of the combined effect of BABA and ACP feeding. Our results corroborate findings of another study, where green peach aphid, Myzus persicae, feeding resulted in the activation of defense-related genes, including PR-1 and BGL2 on Arabdopsis (Moran and Thompson, 2001). Lack of elevated PR-2 gene expression in the BABA treatment could have occurred because the concentration of BABA tested may have not been sufficiently high to cause gene expression. Alternatively, the interval between BABA treatment and our assay may have not been long enough to cause PR-2 gene upregulation. Therefore, optimizing the concentration and priming period of BABA is needed to further elucidate the mechanisms imparting induced resistance in citrus after treatment with BABA. Our results show that all three developmental stages of ACP were negatively impacted by BABA through induction of host-plant plant resistance in citrus. Induction of host-plant resistance may be a viable alternative or additional tool for ACP. BABA could potentially be applied as part of a tank-mix with other pesticides, including various plant activators and fungicides. At this stage, however, additional investigations are needed to optimize a cost effective and efficient dosage of BABA for optimal management of ACP under field conditions. In addition, further work is needed to evaluate other commercially available products, such as 2,6-dichloroisonicotinic acid (INA) and benzothiadiazole (BTH), for inducing resistance in citrus against ACP. The effect of BABA and other SAR inducers on other insect pests or diseases of citrus is also needed.
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