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.
More T. radiata wasps originally imported from Vietnam, China, and Pakistan were produced by colonies established at DPI-Gainesville and Southwest Florida Research and Education Center and released in Florida citrus. That is the major focus of two (number 1 and 3) of the three objectives of this project. From Pakistan, south China, and North Vietnam colonies established at DPI-Gainesville, 26,861, 16,248, and 12,940 wasps were produced and released, respectively, that increased the number of total released wasps to 48,079, 49,495, and 38,544, respectively. Parasitoids from the Pakistani colony were released at the SWFREC experimental grove in Collier County and from south China and N. Vietnam were integrated with biweekly sprays of horticultural oil in replicated trials at two commercial groves, in Glades and Charlotte Counties, respectively, the latter being organic. A total of 1,196 adults of D. citri and 58 adults of T. radiata (4.6% parasitism) emerged from feral nymphs collected in August at SWFREC and reared in the laboratory. No mummified nymphs were recovered on sentinel plants placed at these locations during July or August but 8% of nymphs were mummified among those recovered on sentinel plants placed in a neighboring commercial grove in July. Samples of feral nymphs collected in September from the Glades grove and reared in the laboratory produced 880 adults of D. citri and one adult of T. radiata (0.1% parasitism) from oil-treated plots and 741 adults of D. citri and and 25 adults of T. radiata (3.3% parasitism) from untreated plots. During the same month, nymphs collected from treated and untreated plots in the Charlotte grove produced 103 and 51 adults of D. citri, respectively, but no parasitoids. No mummified nymphs were recovered on sentinel plants placed in July in treated and untreated plots at both locations. The colony at SWFREC of the previously established strain of T. radiata produced 11,855 wasps that increased the previous total to 56,645 produced this year and used to maintain the colony at OrangeCo, conduct laboratory experiments, and release in experimental, organic and conventional groves including the ones mentioned above. These wasps were also released in the treated and untreated plots of an experimental block of a commercial grove in Collier county where treated plots received insecticides and nutritional sprays. The feral nymphs collected in August and reared in the laboratory produced 792 adults of D. citri and three adults of T. radiata (0.4% parasitism) from treated plots, and 764 adults of D. citri and one adult of T. radiata (0.1% parasitism) from untreated plots. In September, 448 and 366 adults psyllids but no parasitoids emerged from nymphs collected from treated and untreated plots, respectively. We are collaborating with California Department of Food and Agriculture, USDA-ARS, USDA-APHIS and Florida Department of Ag & Consumer Services (Division of Plant Industry) to arrange a meeting this November in Florida to discuss mass production and release of T. radiata for biological control of the Asian citrus psyllid. Publications: 1) Mann, R.S., J. A. Qureshi, P. A. Stansly and L.L. Stelinski. 2010. Behavioral responses of Tamarixia radiata (Hymenoptera: Eulophidae) to volatiles emanating from Diaphorina citri Kuwayama (Hemiptera: Psyllidae) and citrus. Journal of Insect Behavior (In press). 2) Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. Journal of Economic Entomology. 102: 2325-2332.
The objectives of this study are: 1) to develop a series of flexible stochastic models to predict the temporal increase and spatial spread of citrus disease. The models were initially characterized for citrus canker but have subsequently been extended to HLB. They can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies both in plantations and State-wide. 2) Test various control methods under field conditions to evaluate effects and collect data to parameterize models A SEIDR model (Susceptible, Exposed (latently infected but not yet infectious), Infectious but not yet symptomatic/detected, Detected and infectious and Removed trees) was developed. Using Markov-chain Monte Carlo methods, and extensive data from South Florida for successive snapshots of the occurrence of symptomatic detected trees in known populations of susceptible trees, we have been able to estimate the transmission rates and dispersal kernel for HLB. Current work is focused on the differential effects of host age on epidemiological parameters as well as variability across the plantation. From these it is possible to allow for uncertainty in the parameters as well as variability over time and through space. We used Baysian methods to infer posterior densities on the model parameters. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. A front-end (a web based version of the model) was developed for non-researcher users has now been nearly finalized. Via this web tool, both residential and commercial citrus scenarios can be tested and a wide variety of epidemiological and climate/weather variables have been included and are user selectable and changeable via sliding controls. Various disease control/mitigation parameters can be selected and simulations can been visualized to see the effect of these various control strategies. This web tool runs simulations one at a time and is highly instructive to growers and regulators. It is based on a more formal analytical model that can run thousands of simulations based on the same parameters and make more statistically valid predictions for regulatory intervention strategy building and regulatory/industry decision making. Currently the spread of HLB is being modeled in 10 contiguous blocks in SG, chosen to encompass both young and mature trees, since host age is likely to be an important consideration for HLB. The blocks are also relatively isolated from the surrounding outbreak. The outbreak is noticeably clustered with only a small number of isolated cases. The model continues to be improved upon to capture more and more of the true features of the data and the disease. The model is being extended to estimate spread, should HLB be introduced into new areas such as TX, CA or AZ. Field plots were established at the USDA, ARS Picos Farm to examine the effects of insecticide control and roguing in various combinations, both to evaluate treatment effects and to collect epidemiological data for model development. As with previous trials conducted in south Florida, to date there is no statistical differences among various control strategies. We interpret these results to support the hypothesis that control must be applied on an area-wide basis to see significant benefit from control efforts.
This project was designed to examine the potential disease control of citrus huanglongbing (HLB) by interplanting citrus with guava. In Vietnam guava has been shown to be an effective deterrent to HLB, slowing the disease and keeping plantings alive for up to 15 years that normally succumb in 2-3 year. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring ~1 year. Both nursery and field citrus trees are assayed for HLB every 60 days, and have been assayed multiple times. Psyllid populations are also being monitored continuously every 2 weeks within interplanted plots to document any repulsion of the vector due to guava. Results: Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus an unprotected nursery, have were established with disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) in June 2009 and were located in the protected and unprotected plots. The guava trees were grown to appropriate size as indicated in Vietnam prior to outplanintg. To date HLB appears to be progressing more slowly in nursery plots interplanted with guava that non interplanted plots. Citrus/guava interplantings: 3 commercial plantings with multiple replications were established. This required 1 year to grow the guava transplants and a second year to become established in the field before interplanting with citrus per Vietnam protocols. One trial was established in a commercial orchard with collaborators in Southern Gardens Citrus, but two years of freezes and replanting prompted the grower/collaborator to abandon the planting. A second was established in grove in Martin county but the plantation was sold and removed before data could be collected. A third trial planting was established at the USHRL Picos Farm in Fort Pierce. The Picos plot was interplanted with citrus in August 2009. Severe frosts during 2008/2009 and again during 2009/2010 winters affected the USHRL plots and caused a delay in the experiment. To date, several plants have been identified in the USHRL plots after multiple assays. The majority of these positives have occurred as the result of an edge effect. That is, treatment plots closest to an existing HLB+ planting of Valencia were affected. To date, there is no significant difference among the treatments. However this data is insufficient to draw any conclusions. Research was initiated on using a Y-tube olfactometer to investigate guava volatiles as repellents of the psyllid. Initial research involved calibrating the equipment and ensuring there was no bias in the behavior of psyllids under the assay. In an attempt to find a reliable control plant to test guava against, the following have been studied as known attractants: 1) whole lemon plants, (2) C. macrophylla shoots, (3)whole C. macrophylla plants, (4) starved psyllids with C. macrophylla shoots, (5)old psyllids with C. macrophylla shoots, and (6) old psyllids with lemon shoots. The best response was 1 week old psyllids with C. macrophylla shoots, although psyllid attraction to this odor source was weak. Research with white guava (germplasm from Vietnam) as a repellent alone or in combination with citrus will be initiated once the assay using citrus is refined.
A trunk cutting, herbicide spray applicator device was constructed, mounted on a small tractor and tested to kill HLB infected trees. In two tests, 6 cuts were better than 3 and full strength imazapur (Arsenal) worked better than the product diluted 50%. The best treatments killed over 95 % of the canopy in each test, but tree death was relatively slow requiring more than 1 month to show significant tree decline. During the testing process, improvements in the machine provided more reliable cutting and spraying. A third test has been applied for a final evaluation. BASF Chemicals has expressed willingness to obtain a label for their formulation of this herbicide as applied in these tests. Both the University of Florida Office of Technology Transfer and Chemical Containers Inc. were contacted regarding the possibility of patenting this equipment and the treatment procedure. All of this information was presented to the CRDF Committee at a meeting in Lake Alfred, FL. At this time there is no indication that Chemical Containers is interested in commercial development of this equipment. This is probably because the foliar nutrition cocktail mixes appear to be maintaining HLB infected trees in a productive state for several years and fewer growers are scouting and removing infected trees. Additional tree killing chemicals are being tested. The results will be reported in future.
This is the annual report of this project. The outcomes are as follows: 1) A method was developed for evaluating the responses of free-flying ACP to combinations of olfactory and visual cues within the controlled environment of a greenhouse. Tests measured the trapping rate of ACP on scented and unscented greenish-yellow sticky traps (ACP traps, AlphaScents, Inc.). Petitgrain oil, an essential oil distilled from sour orange leaves, was used as a test odorant because it contains substantial amounts of linalyl acetate and linalool, two of the primary volatiles emitted by the foliage of Meyer lemon, a favored ACP host tree in south Texas. ACP were presented with an array of either unscented or scented traps in a no-choice test. The trap array consisted of six rows of traps with two traps per row. Each trap measured 34 cm long x 2.5 cm wide. Scented traps had a rubber septum loaded with 1 mL. petitgrain oil stapled to the front. The traps were positioned upwind of a screened release cage with 1000 ACP collected from a local orchard. To determine the accumulation rate of ACP on the traps, a census of each trap was made at 15-, 30-, and 45 minutes intervals following their release. A total of five replicated tests were conducted for each treatment. Psyllids accumulated more quickly on the scented- versus unscented traps in the initial 30 minute interval following their release from the cage. During this time, the number of ACP caught on the scented traps increased by an average of 52% while on the unscented traps the average increase was 35% (t = 2.3609; P = 0.046). The accumulation rate of psyllids on the scented traps was twice as high as that on the unscented traps between the 30- and 45minute interval (18% for scented, 9% for unscented), but this difference was not statistically significant t = 1.8032; P = 0.71). This test may be useful for screening combinations of olfactory and visual stimuli and for selecting those that show the most promise for further testing in orchards. A drawback of the test is that it requires large numbers of pysllids, many of which fly to the ceiling following their release and do not respond further to the test cues. Pysllids collected from orchards appear to be sensitive to environmental stressors (i.e., drought, heat). A summer long drought in 2009 curtailed tests at the beginning of August because the psyllids ceased to respond to test cues.
The objective of this 2 year project was to provide direct assistance to growers on the east coast in developing and evaluating their psyllid management programs. Dr. Pasco Avery was hired at the IRREC to provide direct grower support for this program. During the past 2 years, Dr. Avery and the personnel hired to work in his program aided growers on the east coast by helping to assess psyllid population dynamics under differing management regimes implemented in both organic and conventional citrus operations. This helped growers to get a better understanding of what does and does not work in terms of psyllid management. In addition to the direct extension type assistance provided, Dr. Avery also evaluated the use of the fungal pathogen Paecilomyces formosoroseus for control of the Asian citrus psyllid which growers indicated was of interest due to need for additional control measures due to difficulties meeting pesticide MRL requirements for export to foreign markets. Studies were conducted in both and field conditions to better understand what potential this fungal pathogen holds for managing ACP. The following is a list of publications and presentations by Dr. Avery resulting from this work: Avery, P. B., W.B. Hunter, D.D. Hall, M.A. Jackson, C.A. Powell and M.E. Rogers. 2009. Novel delivery of the biocontrol fungi Isaria formosoroseus for managing the Asian citrus psyllid and reducing spread of citrus greening ‘huanglongbing’ disease. Fla. Entomol. 92 (4): 608-618. Avery, P. B., Wekesa, V. W., Hunter, W. B, Hall, D. G., McKenzie, C, L. Osborne, L. S., Powell, C. A. and M. E. Rogers. Antifeedant and lethal effects of the fungi Isaria fumosorosea on the Asian citrus psyllid Diaphorina citri. Biocontrol Science and Technology (submitted). Avery, P. B., Hunter, W. B, Hall, D. G., Jackson, M. A., Powell, C. A. and M. E. Rogers. Potential of a new biopesticide for managing Asian citrus psyllid. Indian River Citrus Seminar ‘ Ft. Pierce, FL, January 29, 2009. Avery, P. B., Hunter, W. B, Hall, D. G., Jackson, M. A., Powell, C. A. and M. E. Rogers. 2009. Broad spectrum potential of the biopesticide, Isaria fumosorosea for managing insect pests of citrus. 42nd Annual Meeting of the Society for Invertebrate Pathology. Avery, P. B., Hunter, W. B, Hall, D. G., Jackson, M. A., Powell, C. A. and M. E. Rogers. 2009. Investigations of the feasibility for managing the Asian citrus psyllid using Isaria fumosorosea. Proceedings of the International Research Conference on Huanglongbing: Reaching Beyond Boundaries ‘ Orlando, FL, December 1-5, 2008 – published in American Phytopathological Society on their Plant Management Network (PMN) website. This grower assistance project ended in spring 2010 with Dr. Avery taking a postdoc position with Dr. Lance Osborne at the UF Apopka REC. Dr. Avery will continue to work under Dr. Osborne on fungal pathogens which will likely include continuing research into the feasibility of managing ACP using fungal pathogens.
This is the final report of the proposal (FDACS Contract Number 58-1920-9-925 [40]). Our research objectives were: (1) Devise and perform alternative methods (microinjection and membrane uptake) to complete Koch’s postulates using a pure culture of bacteria isolated and cultivated in our laboratory and healthy psyllids as a transmission tool; and, (2) following successful inoculation or loading of the psyllids, we will complete Koch’s postulates. Short term cultivation of a strain of ‘Candidatus Liberibacter asiaticus’ (Las) from Taiwan (B239) was accomplished in our laboratory using two different published methods, and determined to be Las by conventional and real-time PCR and sequencing. We prepared stretched parafilm membrane sachets containing sucrose solutions in 1XTE buffer in which the cultivated bacteria were suspended. Sucrose solutions ranged from 5-20%, however, most experiments were conducted with 10% sucrose. The titer of the bacteria in sucrose was roughly assessed using realtime PCR (Ct values ranged from 20 to 38). Membrane sachets were suspended over 15 ml or 50 ml conical tubes within which 15-20 healthy adult psyllids and 5th instar nymphs were allowed to probe the membranes. After 48 – 72 hr acquisition feeding, psyllids were transferred onto sweet orange seedlings and the remaining fluid in the sachets was assayed by real time PCR. Psyllids and nymphs were allowed to feed on the sweet orange seedlings for 14 days after which they were removed, and assayed for Las by real time PCR. From more than 30 membranes attempted, nine test membranes, each containing 250 ‘ls of culture in 0.25 ‘ 0.5 ml sucrose solution, were successful in uptake of bacterial culture by the psyllids. The orange seedlings were observed for symptom development and assayed three months post-inoculation by real time PCR. Although no symptoms were observed at 3 months, Madam Vinous sweet orange seedlings inoculated with infectious culture had Ct values ranging from 19.5 to 43.8. Typical HLB leaf symptoms were observed by six months. A second approach used direct microinjection of the bacterial cultivation fluid into the hemolymph of adult Diaphorina citri. Psyllids were immobilized with a low velocity stream of C02 and approximately 0.01’l of bacterial culture (Ct value of the bacterial suspensions ranged from 32.9 to 38.3) was injected into the abdomen of each adult psyllid. Psyllids were allowed to recover and then placed on young sweet orange seedlings for inoculation feeding for 14 days. Surviving psyllids were captured, assayed by realtime PCR for the presence of Las, and plants were observed for 3 to 6 months for symptom development. Sweet orange plants inoculated in this manner also became infected, and showed symptoms typical of HLB. Our primary objective was to complete Koch’s postulates by transmitting a pure culture of Las using the psyllid vector. Although a stable pure culture was not attained, we did obtain short term cultivation of a fastidious bacterium which reacted to Liberibacter primers/probe in conventional and realtime PCR assays, produced positive infections of healthy sweet orange seedlings, and could be re-isolated. Our research helps to cement the causal relationship of Las to HLB and provides important insights into the nature of the pathogen-vector interaction.
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