Objective 1 is to conduct a field evaluation of nutritional sprays for control of HLB and HLB symptom expression and yield. The field study was set up May 2010 in Southern Grove, Hendry Co., FL. Six treatments were located in 4 plots of 150 trees per treatment (interior 10 trees in each block were identified for PCR, leaf nutrition sampling, tree health and yield evaluation). Treatments were 1) non-treated check; 2) Nutri-Phite sprayed 4 times bimonthly; 3) N-Sure sprayed bimonthly; 4) Agra Sol Mn/Zn/Fe plus Nutri-Phite plus triazone urea sprayed bimonthly; 5) Keyplex 1400 DP plus Nutriphite plus triazone urea sprayed bimonthly; 6) Wettable powder nutrients (Diamond R #2) plus Nutri-Phite P+K sprayed bimonthly. The materials were applied to both sides of the tree in 125 gallons per acre with an airblast sprayer driven at 2 mph to obtain thorough coverage. Four disease ratings have been taken so far and a slight decline in tree health has been observed, but no significant treatment effects have been observed. There were no significant treatment differences in yield at the first harvest, after the initiation of treatments the previous April. The second harvest was just completed and analyses are currently underway. Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application, Rep 1 using Hamlin sweet orange trees inoculated with HLB and treated bimonthly with the nutritional sprays treatments 1, 2, 3, and 5 from objective 1 has finished. Monthly monitoring of Infection rate and disease development did not show obvious treatment differences except a possible increased rate of decline in treatment 2 compared to all other treatments. Trees that were only PCR+ in root tissue showed an unexpectedly fast decline across treatments. After pruning trees at 6 MPI for canopy management, sampling at 7 MPI showed a slight reduction in titer in the new flush of all treatments except treatment 1, where no Las was detected until 8 MPI. This suggests that treatments 2,3, and 5 may potentiate movement of Las to new flush where psyllids are most likely to feed and acquire Las. Sectioned midrib samples were observed by light microscopy at 6 and 8 MPI and 9 MPI. At 6 MPI reduced phloem plugging and necrosis was observed in treatments 3 and 5, however these treatments had some symptomatic leaves without detectable Las. These leaves had abnormal starch buildup preferentially in phloem tissue instead of mesophyll cells. At 9 MPI there was significant variation in plugging between midribs within a treatment even with highly similar symptoms and Las titer. All treatments had a full range of phloem damage observable in midribs from similarly symptomatic leaves ranging from severe plugging and collapse to apparently healthy phloem. Rep 2 has been inoculated and monthly samplings of leaf and root tissue are underway. Root samples are split for qPCR Las quantification and starch analysis for a quantitive measure of phloem function throughout the plant. Microscopy will be continued, however the high variability of phloem plugging and collapse even within the same midrib from a symptomatic leaf makes interpretation of results difficult. We will pursue methods to quantify phloem plugging in microscopic images to improve interpretation that does not involve selecting representative images, which can be prone to bias. We are also pursuing other methods to quantify phloem function throughout the tree, since leaf to leaf variability in phloem plugging is high.
The host specificity testing for Tamarixia radiata, a parasitoid of the Asian citrus psyllid sourced from the Punjab of Pakistan has been completed. A 60 page Environment Assessment Report was prepared for USDA-APHIS and submitted for evaluation in November 2011. Safety tests conducted by Dr. Raju Pandey in Quarantine at UC Riverside clearly demonstrated that this parasitoid posed no undue risk to California’s environment, other species of insects, or humans. A 60 page Environment Assessment Report on Tamarixia that summarized the results of these studies was prepared by Mark Hoddle and Raju Pandey for review by USDA-APHIS. On 7 December 2012, APHIS issued a permit (P526P-11-04159) authorizing the release of Tamarixia from Quarantine for establishment in California for the biological control of ACP.On 20 December 2011 at 11:00am, 12 glass vials containing 186 female Tamarixia and 95 male Tamarixia (total 281 parasitoids) were opened to release the parasitoids in the Biocontrol Grove at UC Riverside. The eight colonies in Quarantine from which these parasitoids were sourced for release were tested using DNA analyses to ensure that they were free of the bacterium that causes HLB. All tests were negative for HLB indicating that the parasitoids were free of this bacterium. The UCR Biocontrol Grove is a repository for natural enemies that have been imported for the biological control of citrus pests (e.g., scales, mealybugs, whiteflies, etc) in California over the last 50+ years. With the releases of Tamarixia in the Biocontrol Grove, one more natural enemy is being established here to combat an invasive pest that threatens California’s agricultural prosperity. Since this initial release ~ 2,000 Tamarixia have been released in urban areas in LA where ACP infestations are highest. Two recoveries of ACP nymphs with Tamarixia emergence holes have been made in Bell Gardens, LA County. This is a very promising result given the relatively low numbers of parasitoids that have been released at ~15 sites during the winter in southern California. More details on the Tamarixia program at UCR can be found at these websites: http://cisr.ucr.edu/blog/news/first-release-of-tamarixia-radiata-in-california-for-the-biological-control-of-asian-citrus-psyllid/ http://cisr.ucr.edu/blog/invasive-species/tamarixia-radiata-release-video/
This is a one-year project. The overall objectives are to rapidly screen and evaluate chemical compounds for the control of citrus HLB using a graft-based screening method. We have made significant progress in the project in 2011 as summarized below: 1. Screening criteria: A total of seventy-four files were received in May 10 and Sept. 15, 2011. Based on the rank of an expert panel and the mode of action of the compound, 60 compounds from 41 of these files (ranked higher than 0.33) were purchased and tested. Compounds from 33 files were not tested when their ranks were lower than 0.17. Fifteen additional compounds were suggested to be tested by our team. The treatment concentrations of each compound were decided based on previous use of the compound. If the phytotoxicity of a compound was observed, the compound will be retested at a lower concentration. 2. Screening chemotherapy compounds against Las bacterium: A graft-based screening method was used based on our previous studies. The primary results showed that more than 60% of the scions survived and grew when treated with all forty compounds in the first round, except actidione (cycloheximide) at 50 mg/L (0%) and benzyl isothiocyanate at 50 mg/L (54.2%), which were retested at the lower concentrations of 25 mg/L. From Sept. 2011, the leaf samples from the seventeen treated compounds were taken for determining the Las bacterial titers by qPCR. The primary results showed that Ampicilin (Amp) and Rifampicin (Rim) were effective in reducing the Las bacteria to undetectable levels in HLB-affected citrus; Rifamycin (Rif) and Carbenicillin (Carb) were highly effective with lower bacterial titers and transmission rates; The other 13 tested compounds were not very effective in eliminating Las bacterium, but partly suppressed the Las bacterium, including Gentamycin (Gent), Kasugamycin (Kasu), Kanamycin (Kana), Colistin (Col), polymixin B (PMB), Hygromycin (Hyg), Isonicotinic acid hydrazide (INH), Rifaximin (Rix), Cycloserine (Cys), Amikacin (AMK), Quinoline (QUI), Chloramphenicol (Chlo) and Vanomycin (Van). The other grafted material will be tested soon. The twenty compounds in the second round have been grafted and will being tested later. 3.Problems and suggestions: As the contested files were available in September and all providers didn’t submit the contested compounds, some compounds such as peptides in Folder 9932744_044 and lead compounds in Folder 9932744_066 and 9932744_070 could not be purchased and tested. Scions treated with all other compounds have been grafted in December 6, 2011, so we hope to finish this project by June 6, 2012.
This project consists of 5 studies about factors that influence acquisition and inoculation of Candidatus Liberibacter asiaticus (LAS) by th Asian citrus psyllid (ACP), Diaphorina citri. This knowledge is basic to understand the pathogen transmission process and determine more efficient management strategies to manage Huanglongbing (HLB). The results of the first 3 studies on effects of vector development stadia and duration of acquisition access period (AAP) (study 1), leaf age (study 2), pathogen titer and symptom expression in infected citrus (study 3), were shown in the earlier reports of this project. The project is progressing as planned. Studies 1-3 are concluded and most experiments related to studies 4 and 5 were already set up. This report describes partial results of study 4, in which we are investigating the relationship between duration of the inoculation access period (IAP) and transmission efficiency of CLAS by ACP. First we set up an experiment to determine when ACP starts inoculating the pathogen after acquiring it from source plants (latent period), which is important to study the effects of insecticides on transmission. Groups of 3rd-instar nymphs and 1-week old adults obtained from our healthy laboratory colony were confined on LAS-infected sweet orange plants for a 48-h acquisition access period at 25oC. Next, 25 insects of each age group were individually and serially transferred to healthy sweet orange seedlings (test plants) for successive inoculations access periods (IAP) of 48 h at 25oC, until 37 days after the beginning of the acquisition. To assess successful inoculations, TaqMan’ Real-time PCR detection assays with LAS-specific primers were performed on DNA extracts from test plants 10 months later. First transmission events were observed after 11 and 13 days from beginning of the AAP by 1st instar nymphs and adults, respectively. This experiment was repeated and the first transmission event was observed after 9 days from the beginning of acquisition by nymphs, whereas no transmission was detected at anytime following acquisition by ACP adults; first symptoms in inoculated test plants were observed at 5 months after inoculation. In a second latency experiment, we used a longer AAP (96 h) of 3rd-instar nymphs and 1-week old adults on LAS-infected plants and subsequently transferred larger numbers of insects to test plants, in order to increase transmission rates and obtain a better estimate of the minimum latent period. Ten insects of each age group were serially transferred to healthy sweet orange seedlings for successive 48-h IAPs at 25oC, until 33 days after beginning of the AAP. The first transmission event occurred at 11 days after the onset of acquisition by nymphs for most groups tested (14 out of 15 inoculated plants were RT-PCR positive and symptomatic). For ACP groups that acquired LAS as adults, first transmission was detected at13 days from beginning of acquisition, but for only 1 out of 15 groups tested. After the first transmission event, transmission by each group of insets was intermittent, following a random or aggregated pattern. Overall, the results indicate that the minimum latent period of LAS in ACP is approximately 10 and 13 days at 25’C, after acquisition by 3rd-instar nymphs and adults, respectively. It also confirms that the pathogen is transmitted more efficiently by nymphs, as shown in study 1.
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. To date, we have cloned sequences at least 400 bp in length from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. In a side project, we have cloned and characterized two novel IP-free SUS1 promoter alleles (CsSUS1-1 and 2) from Citrus sinensis cv. valencia and have found them to drive phloem-specific expression in both Arabidopsis and tobacco when fused to a reporter gene. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, we evaluated the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). In this objective, Gateway-based vectors were used to express the selected insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We successfully generated Gateway vectors that will result in constitutive (35S promoter) or phloem-specific (CsSUS1 promoter) expression, respectively, of M. persicae-specific Coo2, GSTS1 and S4e dsRNA, as well as a control derived from GFP. Our results suggest that the M. persicae-specific dsRNA expressed in planta has a negative effect on both the lifespan of the insects and the number of offspring generated. In the fall of 2010, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. To date, we are in the process of transforming and regenerating citrus with the D. citri-specific gateway vectors for evaluation and use by the Florida citrus industry. Initial attempts to transform and regenerate Citrus sinensis ‘Valencia’ and ‘Hamlin’ containing reporter gene constructs were successful. Currently we have completed transformation of citrus callous tissue using gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. We are in the process of regenerating transformed lines, and are preparing to generate additional lines with the other transcript/promoter combinations. In summary, we have cloned a number of transcripts from both D. citri and our model organism, M. persicae, and have analyzed a subset of derived dsRNAs to test their effect on M. persicae using in vitro assays (objective 1). We have also cloned and characterized several novel phloem-specific promoters from C. sinensis, and have evaluated their expression patterns. We have also created new Gateway-derived vectors bearing a native citrus phloem-specific promoter, for use in RNAi in our M. persicae model and evaluated them in planta (objective 2). Finally, we’ve now generated similar vectors specifically designed against D. citri (objective 3), and are now generating transgenic lines expressing D. citri dsRNA for evaluation.
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. To date, we have cloned sequences from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, we evaluated the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). In this objective, Gateway-based vectors were used to express the selected insect dsRNA (Coo2, GSTS1 and S4e) either constitutively (35S promoter) or in a phloem-specific manner. Our results suggest that the M. persicae-specific dsRNA expressed in planta has a negative effect on both the lifespan of the insects and the number of offspring generated. In the fall of 2010, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. To date, we are in the process of transforming and regenerating citrus with the D. citri-specific gateway vectors for evaluation and use by the Florida citrus industry. Initial attempts to transform and regenerate Citrus sinensis ‘Valencia’ and ‘Hamlin’ containing reporter gene constructs were successful. Currently we have completed transformation of citrus callous tissue using gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. We are in the process of regenerating transformed lines, and are preparing to generate additional lines with the other transcript/promoter combinations.
1) Evaluation of screens impregnated with insecticide barriers. As explained in previous reports, the experiment is being conducted in two farms located in the Sao Paulo State. On the farm located in Sao Manuel 21 evaluations were performed. In both areas (with and without screen impregnated with insecticide barriers)13 insects were collected. In Descalvado 17 evaluations were performed, in a total of 90 and 94 psyllids collected in areas with and without barrier, respectively. On both farms, about 80% of the catch occurred from August to October, this probably happened due to favorable climate conditions to D. citri, observed in this period. Due to the no difference in the psyllids capture in areas with and without barrier, sample screens from both farms were taken to determine the insecticide amount present. Initially the screen showed 4 g of deltamethrin / kg of the screen. After 8 (Sao Manuel) and 5 (Descalvado) months in the field, the insecticide amount was detected: 0.28 and 0.53 g deltamethrin / kg of the screen, respectively. In this same period, samples were also taken from the screen to perform a bioassay in the laboratory to determine whether the screens were still effective against D. citri. After a 24-hour contact with the screens, it was observed an efficiency (Abbott, 1925) of 63 and 80% to the screens from San Manuel and Descalvado, respectively. The D. citri capture assessment using yellow sticky traps are being performed and they will be maintained until the experiment completes one year. New assessments for inseticide quantification (deltamentrina) and effectiveness of the screens are planned. 2) Evaluate the treatment impact of plants with systemic insecticides on the Ca. L. asiaticus trnsmission by starved psyllids. The application of insecticides has been performed and the transmission trials are underway.
The Asian Citrus Psyllid, Diaphorina citri Kuwayama, is the vector of the bacterium Candidatus Liberibacter asiaticus that causes citrus greening or huanglongbing (HLB) worldwide and in the United States. Despite the prominent role of this psyllid species, little is known about closely related species of psyllids and nothing about phylogenetic relationships of ACP within the genus Diaphorina. The genus Diaphorina comprises about 70 described species and additional species that are known to psyllid taxonomists, but have not been formally named yet. ACP was described from Taiwan in 1908, but other species of the genus are broadly distributed throughout the Mediterranean and tropical climates of the old world. More than 30 species are known from South Africa, but Diaphorina species also occur in other areas of Africa, Europe, the Near and Middle East, and South East Asia. Psyllids are generally very host-plant specific, meaning that species within one genus often only target a closely related group of host plants. This is very different in the genus Diaphorina, where species target at least 17 different plant families, among them Rutaceae (citrus and relatives). Three of the described species are known to be associated with Rutaceae (D. citri, D. punctulata, D. auberti), but there are additional undescribed species recorded from this plant family as well. We currently do not know if Diaphorina psyllids invaded citrus and its relatives once or multiple times and we also do not understand from which other plant groups this invasion happened. In summary, the patterns of host plant relationships, but also the degree of invasiveness of different Diaphorina species and the potential to vector bacteria in the Liberibacter complex are unknown. Collaborators Daniel Burckhardt (Museum of Natural History, Basel), David Ouvrard (Museum National d’Histoire naturelle, Paris), and Ian Millar (Plant Protection Institute, Pretoria) conducted field work in Israel, the Philippines, and South Africa and collected ~20 species of Diaphorina together with host plants. PCR was performed, genes sequenced at the UCR Genomics Core Facility, and phylogenetic trees were generated. Current results show that the genus Diaphorina is monophyletic, i.e. it is derived from one common ancestor that gave rise to all species contained in this genus. We found that ACP is a relatively basal species within this genus. Based on this analysis, ACP is closely related to Diaphorina lycii Loginova that is found in North Africa, the eastern Mediterranean, and east to Mongolia. Other than ACP that is restricted to feed on Rutaceae, this species is associated with different plant species that belong to the nightshade family (Lycium spp.). The current analysis includes one additional species of Diaphorina that occurs on Rutaceae, an undescribed species from South Africa that feeds on Diosma hirsuta L. The analysis indicates that this species is distantly related to ACP indicating that feeding on citrus and its relatives evolved at least two times independently within the genus Diaphorina. Including additional species of Diaphorina in this analysis will eventually reveal the complex pattern of host-plant evolution within this genus of psyllids.
This is a cooperative research project between Co-PIs Joseph Morse, Jim Bethke, Frank Byrne, Beth Grafton-Cardwell, and Kris Godfrey. One objective is to coordinate with researchers working on chemical control of ACP in Florida, Texas, Arizona, and elsewhere. Towards that end, Morse and Godfrey participated in the Second Citrus Health Research Forum in Denver in October 2011. After substantial discussion with the involved agencies (San Diego Ag. Commissioner’s office, CDFA, CRB, CPDPC, HLB Task Force Science Advisory committee, and others) on 10-17-11 we obtained a permit (#2847) from CDFA to rear ACP at the Chula Vista Insectary and another permit (QC1324) for the movement of plants for ACP colony maintenance. Permit #2847 clearly notes experimental protocols and procedures so that this work will be done as safely as possible to minimize any chance of ACP escape. We thank the many people and agencies that reviewed the experimental protocols and suggested additions and changes. To initiate the ACP colonies, we collected insects from an infestation in Boyle Heights on Oct. 27, 2011. Several hundred eggs were collected by cutting 3-4 inch terminal growth from an ornamental planting of Murraya. The terminals were placed in water in plastic vials then double bagged and sealed in a zippered ice chest for transport. The terminals with the eggs were transported to the Chula Vista Insectary and placed in contact with potted plants in cages in the facility. At the time of this report, we now have 5 cages with adults emerging. We intend to produce large colonies of insects that we can use in pesticide trials in the near future. The Chula Vista Insectary was inspected again by a CDFA representative and a San Diego County entomologist on Nov 23, 2011, and we received the go ahead to continue studies. One of the ACP permit protocol requirements for the Chula Vista Insectary is to test all plants and a sample of the insects from the colonies for HLB on a consistent basis. We cannot spare any insects until we have an established colony, but we have made contact with the CRB Diagnostic Laboratory in Riverside, and we will begin a program of testing all plants within the Chula Vista Insectary soon. Insect colonies struggle during the winter months, but we fully expect to have enough insects to run preliminary pesticide trials to confirm our methods by early spring. One initial goal is to test a variety of organic pesticides and other biologicals for efficacy and persistence. This will require lots of insects and plants. We will be applying pesticides using a backpack sprayer at full pressure on plants with and without insects. The idea is to assay for mortality on treated insects and to cage insects on treated plants over time to determine when those pesticides are no longer effective based on plant residues.
Valencia trees of three age categories, 1-years-old, 5-years-old and 8 to 10-years-old were selected for the study. All trees are in separate block and in close proximity (less than half a mile to each other) and are gown on the same soil series (Immokalee fine sand) with similar soils characteristics. An initial treatment was applied to all one-year-old trees to evaluate soil sampling, extraction and analytical procedures. An Imipacloprid solution was applied in circles about one meter is diameter with the citrus trees at the center. We will repeat the experiment three times: Spring 2012, Summer 2012 and Spring 2013. The initial application was used to evaluate soil and tissue sampling, extraction and analytical procedures. Briomaide was added to the imidacloprid solution to characterization water movement with subsequent irrigations. Soil samples were collected using bucket augers from five different depths (0-15 cm, 15-30 cm, 30-45 cm, 45-60 cm, and 60-75cm) for describing the basic soil chemistry and soil physics: organic carbon content, pH, cation exchange capacity (CEC), particle-size distribution, soil moisture release curve, and saturated hydraulic conductivity. It was determined that a 1:1 soil to acetonitrile solution extraction procedure worked well for Florida conditions is the following: 10 g of equivalent dry soil are weighted in centrifuge tubes, and then added with 10 ml of a mixture of HPLC grade acetonitrile and water (80:20). The tube is extracted in a horizontal shaker for 2 hours. The extract is centrifuge at 8000 rpm for 20 min, and filtered using Whatman 42 filter paper. Then the extract is analyzed in an Agillent HPLC-UV, with 50 .L of injection volume, 1 mL per minute of flow rate, a mobile phase of acetonitrile and water (60:40) and a wavelength of 270 nm. The Imidacloprid peak is conspicuous at 2.7 min. of retention time. Most chemical determination of Imidacloprid in plant tissue involved a long series of extraction and clean-up steps after extraction from the tissue. Many methods rely on liquid-liquid partitioning to isolate the chemical from cell walls and sap. During this study a method of extraction will be develop to analyze Imidacloprid concentrations in citrus tissue. The Asian Citrus Psyllid likes to feed on young shoots of recently expanded leaves (flush) for oviposition and nymphal development. On a weekly basis, all trees (n=10) per plot will be sampled for flushing patterns, psyllid adults and nymphs, and beneficial insects, the latter a significant component of psyllid management in Florida. All new shoots will be counted or estimated using a quadrant frame made from PVC pipe, to sample a volume of 7500 cm3 (50 x 50 x 30 cm) of tree canopy. The square frame will be randomly placed on the outer tree canopy about 1-2 m aboveground and flush will be counted to a depth of 30 cm within the specified area.
Laboratory and field studies were conducted to examine the behavioral responses of male and female Asian citrus psyllid (ACP) to their cuticular extracts and to identify an attractant for ACP. In olfactometer assays, more male ACP were attracted to 1, 5, or 10 female cuticular extract equivalent units than blank controls. The results were confirmed in field studies in which clear or yellow traps baited with 10 female cuticular extract equivalent units attracted more males than clear traps baited with male cuticular extract or unbaited traps. Analyses of cuticular constituents of male and female ACP revealed differences in relative amounts of certain compounds. Dodecanoic acid, present in greater amounts on females than males, elicited antennal responses from male and female ACP as measured by gas chromatographic-electroantennogram detection. Dodecanoic acid was also attractive to males, but not females, in laboratory olfactometer assays. Our results suggest that docacanoic acid is an attractant for ACP, but its function’sex attraction versus aggregation’remains to be determined. In addition to the above exciting finding, we had a breakthrough in finally developing an effective gas chromatograph coupled with electroantennogram (GC-EAD) system for determining how ACP antennal neurons respond to chemicals. This has allowed us to specifically narrow down five more potential pheromone components. We were able to purchase three of these from standard chemical manufacturers and we have been able to synthesize the two others. Behavioral testing of these is currently taking place. Also, using this new GC-EAD system, we are identifying the specific plant volatile attractants for ACP. We should have an attractive lure for ACP developed in the near future.
The goal of this project has been to determine if infection by Candidatus Liberibacter affects the response of Asian citrus psyllid (ACP) to its citrus host plants to understand a critical component of disease spread. In this project we evaluated if healthy psyllids are attracted more to HLB infected or healthy trees. Also, we determined whether this behavior changes when the ACP vector becomes infected with the pathogen. We conducted a series of behavioral experiments to investigate whether HLB-infected citrus plants are differentially attractive to ACP as compared with healthy citrus plants. We also examined if psyllids known to be infected with the pathogen behaved differently from uninfected controls in response to both healthy and HLB-infected plants. Our preliminary results indicated that HLB-infected citrus plants are more attractive to ACP adults than healthy plants in two-choice olfactometer experiments. More ACP were attracted to HLB-infected plants than to healthy plants in open-air cage experiments. However, subsequent dispersal of ACP adults to healthy plants following their initial choice indicated that final settling preference was for healthy rather than diseased plants. We hypothesized that initial movement of ACP to infected plants and further dispersal to healthy plants may be explained by production of deceptive volatile compounds by HLB-infected plants to attract ACP adults in the field to facilitate the spread of bacteria as occurs with apple phytoplasma Candidatus Phytoplasma mali, responsible for apple proliferation disease. Ca.P. mali hijacks the apple trees to produce specific chemical that attracts the plant-sap sucking psyllid vector to infected trees. This is a major factor for facilitating disease spread in apple. Alternatively, the yellow color of HLB-diseased plants due to chlorosis and yellowing of shoots may attract the ACP initially but psyllids move to healthy plants after sampling the phloem of diseased trees. The movement to new plants could be due to poor nutritional status of HLB infected plants. It is known that ACP adults are attracted to yellow color; therefore, initial attraction of ACP adults to diseased plants may be due to chlorosis of leaves caused by HLB. Also HLB-infected plants are deficient in zinc, iron, manganese, calcium, sulfur and/or boron and hence the subsequent movement of psyllids to healthy plants could be due the poor host suitability of HLB-infected plants. 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, we recently discovered that HLB-infected plants produce significantly more trans-.-ocimene than uninfected counterparts. Importantly, we also found that trans-.-ocimene is highly attractive to psyllids. Thus, the current working hypothesis is that infected plants initially produce large quantities of trans-.-ocimene to attract psyllids and then subsequently produce large amounts of MeSA to repel them and this mechanism facilitates spread of disease. We are currently verifying this hypothesis and continuing research on the effects of nutrient deficiencies on pysllid behavior. The ultimate goal of this research is to gain understanding how to develop citrus varieties that are not attractive to ACP.
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. Also, we have found that 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. This has allowed us to precisely measure the concentrations of repellent chemicals needed in the field to affect psyllid behavior which is helping guide development of practical release devices. Our field results with DMDS released from SPLAT in 2010 were mixed. While some trials with the initially developed formulation appeared to show reductions of ACP populations, others did not. However, we have now completed analysis and processing of data from an investigation of four new (advanced) SPLAT formulations of DMDS that were designed for longer field longevity. Two of these four formulations produced very good results, suppressing psyllid populations better than the previous formulations and longer than was previously achieved. These two formulations lasted longer than four weeks, but their full potential could not be investigated because the trial was unfortunately interrupted by a pesticide application. We plan to further investigate these two formulations in 2011 and hope to replicate these results.
The objective of this project is to evaluate methyl salicylate dispensers to determine whether their deployment in citrus can enhance biological control of Asian citrus psyllid. Locations were selected for the field trials with the commercially available methyl salicylate (MeSA) lure, Predalure (AgBio Inc.; Denver, CO). Our initial initial proof of concept research in a small plot setting evaluating the effect MeSA dispensers in small plots (35 tree) provided positive results. The treatments compared were plots treated with MeSA versusuntreated control plots; all treatments were replicated five times. Two dispensers were deployed per tree in April and populations of psyllids and their natural enemies were monitored through September. These data indicated that treatment of citrus plots with MeSA,increased populations of natural enemies such as beetle and fly predators of ACP and well as the ACP parasitoid, Tamarixia radiata. In addition, populations of ACP were lower in MeSA-treated plots compared with untreated controls. However, subsequent large-scale experiments in commercial groves did not reproduce the same results. During the 2010 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. 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. We are still processing samples from 2010 and thus have asked for a short extension of this project. 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.
The present investigation has provided baseline susceptibility data for several Florida ACP populations to commonly used insecticides and verifies reduced susceptibility to several insecticides among geographically separated populations. In general, reduced susceptibility to the insecticides tested was more widespread in the second year of the study. In 2009, there was reduced susceptibility to fenpropathrin, imidacloprid, malathion, and thiamethoxam, as compared with our laboratory susceptible strain, in populations from one to three sites. However, in 2010, ACP adults were less susceptible to each insecticide tested as compared with the LS (lab susceptible) population for one or more of the diagnostic doses tested. In 2009, populations from La Belle, Lake Alfred, Ft. Pierce and Vero Beach showed 7, 12, 13, and 18 fold decreases in susceptibility compared with the LS population, respectively, to chlorpyrifos. For imidacloprid, populations from Lake Alfred, Fort Pierce, Vero Beach, Groveland and La Belle displayed 8, 10, 10, 14 and 35 fold decreases in susceptibilities, respectively, compared with the LS population. For thiamethoxam, populations from La Belle and Groveland were 10 and 12 fold less susceptible, respectively, than the LS population. Surprisingly, results from present study on ACP mortalities as a result of the newly developed insecticide, spinetoram, even though its use in Florida began only in 2008, serve as an early warning for judicious use of this insecticide. Spinetoram is considered to serve as a replacement to organophosphate insecticides. Significantly lower adult mortality as a result of spinetoram was observed in three field populations at two of the diagnostic doses tested as compared with the lab susceptible population. In general, comparisons made at the LD75 and LD95 showed more resistant field populations against various insecticides than at the LD50 diagnostic dose. General esterase, glutathione S-transferase and monooxygenase levels were lower in adults and nymphs from the LS population than field collected populations, suggesting that insecticide resistance is positively correlated with levels of detoxifying enzymes. Differing levels of detoxifying enzymes and resistance among the various populations of ACP nymphs sampled could be a result of differential selection pressure imposed by insecticide spray schedules among the various sites sampled. Nymphs from the Winter Garden site exhibited the highest general esterase, glutathione S-transferase and monooxygenase levels which could be correlated with the relatively high resistance levels to carbaryl, imidacloprid and spinetoram. In at least one population of ACP nymphs, there did not appear to be a correlation between enzyme levels and resistance levels to chlorpyriphos, imidacloprid and spinetoram. This suggests that resistance in this population could be a result of other mechanisms. However, in the majority of cases, elevated enzyme levels were correlated with greater resistance levels. Use of enzyme inhibitors, such as piperonyl butoxide, diethyl maleate and triphenyl phosphate for cytochrome P450, gluthione S-transferase and carboxylesterase, respectively, may be useful in cases where increased enzymatic detoxification is contributing to resistance. We are currently using the above data to develop optimal rotation schedules and investigating binary mixture treatments for prevention of further resistance development.
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