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.
In our previous reports, we provided data showing RNA interference effects in potato/tomato psyllids (B. cockerelli) via standard injection and oral feeding of both dsRNAs and siRNAs. Homologs of actin, ATPase, Hsp70 and CLIC were shown to cause substantial psyllid mortality as compared with GFP dsRNA controls in oral feeding assays. The results led to a manuscript which is now under revision in PLoS ONE. Now we are concentrating our efforts on evaluating anti-psyllid RNAi effects in plants. In the long term this will include transgenic plants, but we are investigating using recombinant plant virus-based expression systems as tools for rapid evaluation. We have now evaluated three different virus vectors and five different host plants. These include Tobacco mosaic virus (TMV), Tobacco rattle virus (TRV) and Potato virus X (PVX) in different host species including tomatoes, tobacco, Datura stramonium, tomatillo and Nicotiana clevelandii. These plants were chosen as they are good hosts for the psyllid, but show differential virus effects. TMV systemic infections in tomatillo, tobacco and N. clevelandii develop very rapidly, within one week, and in tomato after 2-3 weeks. PVX gave consistent but weaker expression in tomatillo, tobacco, D. stramonium, and N. clevelandii, but not in tomatoes. TRV showed weak expression in tobacco and tomatillo, but not in and D. stramonium, N. clevelandii or tomatoes. Tomatillo grows very rapidly, is the good host for B. cockerelli, and systemic expression of TMV gave the highest expression for insert sequences. So, we focused on using the TMV-tomatillo-psyllid system. We used different feeding protocols, including whole plants, leaf cages and stem feeding. For RNA interference effects, knockdown of target mRNAs in dissected gut is more consistent than when using the whole insect samples, as we also observed after in vitro dsRNA oral feeding experiments. Reduced nymph numbers were observed in some experiments suggesting evidence for RNAi activity. We are also attempting to use a leaf-agar system, in which only the infiltrated leaf sections from test plants are transferred to agar plates for psyllid feeding experiments. In summary, our results so far show the possibility of inducing RNAi effects via plant virus expression systems, which could provide practical means to rapidly screen secquences for anti-psyllid RNAi, or could even be used in field conditins.
Our research included both laboratory and field experiments. In the laboratory, we used a controlled droplet generator to investigate the effect of droplet size on the mortality of Asian citrus psyllid (ACP). The technique involved spraying various commercially available formulations (e.g., Danitol, Dibrom, Lorsban) at different droplet sizes on psyllid infested potted trees and counting the number of ACP eggs, nymphs, and adults before spraying and at 3 days and 7 days posttreatment. The results showed that the smaller the droplet size, the greater is the mortality of all life stages of the ACP. For all chemistries, the results suggested up to 80% control of nymphs using sprays with droplet VMD of 40’100 ‘m. Danitol caused greatest toxicity to nymph stage. At day seven, naled was significantly more toxic than chlorpyrifos to eggs and nymphs at all droplet sizes tested. However, some formulations involving suspended particles could not be applied readily with our laboratory apparatus. In the field, we continued the low volume spray trials with insecticides in different chemical classes. The insecticides included insect growth regulators, organophophates, pyrethroids, neonicotinoids and one microbial insecticide. They were sprayed with at the recommended label rates or an experimental rate at spray volumes of ~5 gal/acre. Ambient weather conditions were recorded during applications and up to 28 days posttreatment. The temperature (19.5-38.2 ‘C), relative humidity (68%-95%), mean wind speed at application (0.9 -5.1 mph; 0.4-2.3 m/sec), total solar radiation (203-251 W/m2), rainfall up to day three (0.02″-2.3″; 0.5 mm-58.7 mm) and total rainfall at 28 days (5.0″-11.4″; 128 -290 mm) were measured. From the relative humidity data the delta-T statistic was calculated. The value of delta-T allows for the estimation of the evaporation time of an insecticide droplet and may have a significant impact for low volume applications where the droplet sizes are much smaller than high volume applications. Ideally, the value of delta-T should be between 2-8. Values outside this range indicate marginal spraying conditions and higher than 10 indicate conditions unsuitable for spraying. In our studies, the delta-T values during the applications ranged from 1.0 to1.8. Efficacy of each application was assessed by sampling populations of both adults and nymphs in treated plots compared with untreated control plots on days: three, seven, 14, 21 and 28 in a commercial citrus grove. Unbaited yellow sticky cards and tap counts onto white cards from multiple branches were used to determine the numbers of adults. Populations of nymphs were estimated by examination of individual flush terminals using a ranking scale. Regression analysis was used to determine if there was a relationship between the meteorological conditions measured during the trials and observed mortality of the psyllid nymphs and adults. The efficacy data for day three was correlated with the above canopy wind speed at application. There was a significant positive correlation, indicating that the most effective applications of residual insecticides (organophosphates and pyrethroids) occurred at lower wind speeds but not for insecticides from other classes. No correlation with the value of delta-T and efficacy was shown for all insecticides in all classes tested. The maximum daily temperature for the first three days was positively correlated with increasing efficacy for the residual insecticides tested but not for other classes. The cumulative solar radiation to day 21 post-treatment was negatively correlated with efficacy for the residual insecticides. There was no correlation between cumulative rainfall to day three and efficacy for all insecticides tested. It was unexpected that we found no relationship between rainfall and spray efficacy as other studies have indicated that post-application rainfall can significantly reduce the longevity of an insecticide application. Overall, the results showed that low volume application is effective for psyllid management in citrus.
Experiment 2 plots (within HLB-Wide-Area Management WAM) were harvested in August/11. The yield of this experimental area is still increasing year after year as expected for a 6 yr-old grove. At this harvest, plots without ACP control produced less (46.5 ton/ha) than plots with ACP control (57.2 t/ha for plots with insecticide spray every 28 days, and 58.3 t/ha for plots with insecticide sprays every 14 days). However this difference in yield in non-controlled ACP plots was not only due to the amount of HLB-eliminated trees in those plots, but because the amount of CVC-eliminated trees in those plots. The cumulative incidence of HLB in plots without insecticide was 7.3% and did not differ from 7.1% and 5.3% incidence in plots with insecticide sprays every 28 and 14 days, respectively. The cumulative incidence of CVC in plots without insecticide sprays was 4.8% and was significant higher than 0.6% and 0.5% incidence in plots with insecticide sprays every 28 and 14 days, respectively. The WAM of ACP has kept the ACP population density very low (almost zero) in the last two years even in the plots without ACP control, and the HLB progress rate still low (cumulative incidence from 6.3% in November/10 to 7.3% in October/11 for plots without ACP control; and from 4.5% to 5.3% for plots with ACP control). For Year 3, we will continue collecting data from Experiment 2 under regional management, analyzing the spatio-temporal disease and economics data from both experiments, and publishing the results on scientific journals.
PCR results during the past three years from the three sites where the treatments with the Boyd nutritional cocktail is being studied indicate that the incidence (number of trees infected) of HLB is increasing and that the amount of bacterial present is also increasing (lower ct values). The incidence of infected trees in the Hamlin orange trial at SWFREC has remained at 100% infection since the trial began in 2008. The incidence has increased in the two Valencia trials at grower sites from 79% to 100% and from 40% to 98%. The ct values in the Hamlin trial at SWFREC decreased from 28.01 to 24.99, and from 29.88 to 26.49 and 32.23 to 25.23 respectively, all statistically significant in the two Valencia trials in commercial groves. Yield per unit of tree volume continues to be treatments 2 (complete Boyd cocktail minus the SARs), treatment 9 (complete Boyd cocktail minus hydrogen peroxide, and treatment 1 (complete Boyd cocktail. These three treatments consistently are among the four highest yielding during the past three years. Treatments 4, 5, 6, and 8 which do not contain the foliar applied nutrients Mg, Mn, Zn, Mo, and B are among the lower yielding trees. We are well into the 4th year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test individual contributions of micro-nutrients + systemic acquired resistance inducers, and insecticidal control on: (1) ACP , (2) on incidence and titer of Can. Libericacter asiaticus (CLas), and (3) fruit yield and quality. While we are continuing with the experiment, results from the first 3 years are being written up for publication. Since our last report we have sprayed insecticides twice in designated plots: Agriflex at a rate of 5oz/ac + 2gal/ac of 435 horticultural oil on 19 July, and Dimethoate 4E (16oz) + 1gal 435 oil on 12 September The nutritional (‘Maury Boyd’) mix was applied on 29 July. Psyllid populations have been significantly lower (P < 0.0001) on insecticide treated trees throughout the growing season (16 June (0.06, 0.44 +/- 0.05 SEM), 30 June (0.04, 0.47 +/- 0.05 SEM), 14 July (0.40, 3.35 +/- 0.29 SEM), 28 July (0.10, 0.62 +/- 0.06 SEM), 11 Aug (0.00, 1.11 +/- 0.08 SEM), 25 August (0.03, 0.87 +/- 0.07 SEM), 8 Sep (0.24, 1.43 +/- 0.12 SEM), 22 September (0.05, 0.80 +/- 0.07 SEM respectively). Tap and flush sampling have been modified to include more complete counts of nymphs (all stages) and eggs on flush. Results of PCR for resets showed 0 and 4 positive HLB trees for January and July assessments, respectively. We are collecting samples from mature and reset trees this month for HLB detection. Leaf samples were collected 20 September from each plot for macro/micro nutrient analysis. A bucket trap was dispensed in the grove (7 October) to identify all leafminer species attracted to the CLM lure, and a fall citrus leafminer damage assessment using a modified Horsfall Barratt scale is planned for later this month when a flushing period is present.
This project has 5 objectives: (1) evaluate efficiency of ACP control techniques in cooperation with growers, (2) develop efficient monitoring methods for ACP, (3) accelerate testing of new chemistries and techniques for ACP management, (4) evaluate the economic component of the comprehensive program, and (5) provide an information bridge between researchers, growers, and industry. The following is an update of ongoing field and laboratory experiments. Experiments are underway to 1) compare selection rates for resistance using mixture (AgriFlex) versus rotation of its two components, and 2) evaluate stability of insecticide resistance and number of generations required to return tolerant populations to susceptibility. Estimates of dose-response relationships of ACP adults to the two insecticides and pre-mix were as follows: commercial grade thiamethoxam (Actara 25WG, LC25 6.48, LC50 15.36) and abamectin (Agri-mek, LC25 1.16, LC50 2.79) and a pre-mix (Agri-flex, LC25 0.26, LC50 0.91). The plan is to evaluate rate of resistance induction in caged psyllids using the LC25 dose, using discriminating dose bioassays to monitor response to 5 treatments: the premix, rotation, each insecticide alone, and a check. Later, insecticides will be withdrawn and return to susceptibility evaluated. Citrus leafminer (CLM) incidence and damage has been increasing, possibly due to increased use of broad spectrum insecticides to control ACP. Canker is also on the rise and growers are demanding assistance with management of CLM, thought to exacerbate the disease. We tested the hypothesis that heavy damage from CLM typically seen in late spring and early summer could be mitigated by controlling the first generation of the year in the spring flush. Aerial applications have not been used often for CLM control, so we also initiated a trial in July with 2 treatments of Intrepid (ground vs. aerial) compared to Delegate applied by air and results are described in the report for project 210. Intrepid (aerial/ground) applications showed the least damage of leaves compared with Delegate or untreated. Finally, we are initiating a colony of CLM to rear the parasitoid Citrastichus phyllocnistoides and support a program for monitoring CLM susceptibility to key insecticides. We began monitoring CLM using pheromone traps dispensed in a natural preserve (Okaloacoochee Slough State Forest) at distances of 2.0mi, 3.0mi, and 4mi (3 traps at each distance) from citrus to determine the furthest distance traps will catch citrus leafminer. We have found what we believe to be 4 different species attracted to the pheromone lure. We are in the process of identifying all the leafminer species caught by CLM pheromone lures in groves and in the slough through use of bucket traps and plant samples. Representative moths will then be keyed to species by a taxonomist and subject to DNA bar coding by a molecular biologist. A botanist has been contacted to identify the host plants. We are collaborating with the DPI CHRP program to evaluate results of the cooperative (area wide) ACP management program, in SW Florida, now the Gulf CHMA which uses a sampling protocol of 50 taps/block. The objective is to provide growers with tabular or graphical representations of results from this area.
Low volume (LV) aerial and ground sprays have become an important method of application in Florida citrus. We continue our evaluations of LV application of 435 horticultural mineral oil (HMO) which has shown promising results the last 3 years. This report documents updated results from the fifth trial begun on February 2011 in a 10.9 acre plot of ‘Valencia’ orange in Lee County. The efficacy of low volume spray spray of 435 horticultural spray oil (HMO) with the Grower Standard and an Untreated control are being compared. This trial is the first with sufficiently high ACP populations to assess the ability of LV oil sprays to provide suppression. Five sprays of 435 oil (13, 29 Jul @ 4gal/ac; 30 Jun 2, 22 Sep @ 2gal/ac) have been completed since the last report. Plots designated for the Grower Standard received 20 oz of Dimethoate + 2gal 435 oil on 30 June and 4oz of Delegate + 4gal 435 oil on 29 July. Mean adult psyllid populations over the last 3 months were 0.35 +/- 0.037,0.19+/-0.024 and 0.031+/-0.009 for the Grower Standard, Oil, and Untreated Check respectively. These values were all statistically different. Thus, the low volume oil treatments are suppressing ACP but not as effectively as the Grower Standard. Canker has worsened in the grove, so we added citrus leafminer (CLM) assessments to determine if treatments are providing some control. Pheromone traps were placed in the grove on 24 May a second damage assessment of leaves using a modified Horsfall Barratt scale will be conducted in late Oct. CLM control requires precise application timing on emerging flush to be effective, so there is considerable interest in aerial application to manage this pest. Therefore, we evaluated ground and aerial applications of the insect growth regulator methoxyfenozide (Intrepid 2F), and a ground application of spinetoram (Delegate WG), an analog of spinosyn A which is derived from actinomycete bacteria. Both products are selective with low vertebrate toxicity and widely used for CLM control. Treatments were applied 7 July in an 94 acre block divided into 12 large plots using a replicated complete block design with 4 treatments and 3 replicates. Pheromone traps were used to monitor CLM populations within all plots before and after application. Intrepid 2F treatments, regardless of application method, showed significantly less damage than the Delegate treatment or the untreated check on 20 July. On 29 July, significantly less damage was observed in response to the Intrepid2F ground and Delegate treatments compared to the untreated check. An assessment 2 months later showed less CLM damage in the Intrepid ground and Delegate plots, with trap captures lowest for Intrepid ground applications. Therefore, significant control was obtained with the aerial application of Intrepid, but not as much or as enduring as with the ground application.
Two 3-year field experiments are being conducted in two commercial orange blocks in Hendry County (southwest Florida). One of the groves is planted with ‘Earlygold’ oranges and the other with ‘Valencia’ oranges. Average HLB incidence estimated in both groves at the beginning of the experiment based on PCR analysis of a random sample of 160 trees was 98% in ‘Earlygold’ and 42% in ‘Valencia’. Experimental design is randomized complete block with 4 replicates and 4 treatments: (1) No insecticide, (2) Calendar applications, in order to drive vector populations close to 0, (3) nominal threshold of 0.2 psyllids per tap, and (4) nominal threshold of 0.7 psyllids per tap. Calendar applications are being applied every month. The insecticides used for these applications in the last three months were abamectine (Agri-Mek SC) at 3.5 fl oz/ac and horticultural mineral oil 2% in July, malathion (Gowan Malathion 8F) at 2.5 pt/ac in August and fenpropathrin (Danitol 2.4 EC) in September at 16 fl oz/acre. No sprays were used for treatments (3) and (4) during this period because nominal thresholds were not reached. No ACP adults have been found in treatment (2) in both blocks along the summer. ACP levels in the other treatments were (0.02 ‘ 0.01, 0.03 ‘ 0.01 and 0.05 ‘ 0.01 ACP adults per tap in treatments (1), (3) and (4) respectively for the ‘Earlygold’ block, and 0.08 ‘ 0.01, 0.010 ‘ 0.004 and 0.04 ‘ 0.01 ACP adults per tap in treatments (1), (3) and (4) respectively for the ‘Valencia’ block). Significant differences among treatments in the ACP cumulative numbers since the beginning of the experiment are being kept in the ‘Earlygold’ block with the lowest ACP populations in treatment (2) (F = 7.21; df = 3, 15; P = 0.005). Due to the low ACP populations in the ‘Valencia’ block, no significant differences have been found yet in the ACP cumulative numbers among treatments (F = 0.71; df = 3, 15; P = 0.5618). To evaluate the incidence that ACP calendar sprays have on the beneficial arthropod fauna two new exclusion experiments on ACP infested flushes were conducted in July and August respectively. As in the first experiment performed last May, mortality due to predation on citrus cohorts was high in July, with no differences between treatments (79.40 ‘ 7.42% and 90.74 ‘ 2.6% in treatments (1) and (2) respectively). In the experiment conducted in August, mortality due to biotic factors was lower. Again, no differences between treatments were found (63.30 ‘ 5.52% and 57.69 ‘ 10.57% in treatments (1) and (2) respectively). On the other hand, a new study has been initiated to evaluate how ACP sprays affect to CLM natural enemies. In this study, during each major flushing period 40 flushes per ACP threshold treatment are selected and the predation and parasitism rates are evaluated. In the first experiment, that was conducted in June-July, predation rates were 94.2 ‘ 1.94%, 92.63 ‘ 3.27%, 91.03 ‘ 2.6287.47 ‘ 5.15% and 87.47 ‘ 5.15% in treatments (1), (2), (3) and (4) respectively. According to these results it doesn’t seem to be yet a negative effect of the ACP sprays on CLM predation. On the other hand, parasitism rates only accounted for 1% of the CLM cohort mortality. The incidence of the ACP sprays on phytoseids abundance is also being evaluated in the two blocks. Significant differences on their cumulative numbers are being found between the calendar spray treatment and the rest of the treatments (t = -2.71; df = 11; P = 0.0204 and t = -2.42; df = 11; P = 0.0338 for the ‘Early’ and ‘Valencia’ blocks respectively). These results would indicate that calendar applications may are having a negative effect on these predatory mites.
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