The following is a summary of Dr. Avery’s activities since Ocotber 2009: 1) Packers of Indian River, Ltd. – Ft. Pierce: Monitored psyllids along the border using yellow sticky cards. Data collected was used to produce a distribution graphic for assessing the psyllid population dynamics throughout the year for the grower. Graph showed that the population peaked in March and August. 2) IMG Citrus ‘ Fellsmere: Assessed and compared the abundance of psyllids in orange and grapefruit trees managed by the same chemical spray program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitored inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. Overall, results indicated that there were more psyllids found on orange than grapefruit trees with similar flushing patterns; especially with the tapping samples. There was some fluctuation towards the later part of the year possibly due to aerial sprays conducted either near or on site. 3) Pine Ranch, Inc. ‘ Lorida:Assessed the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps, tap and flush sampling. Generally the population was low, but the numbers of psyllids may be influenced by the abandoned grove near the monitoring site. 4) River Country Citrus, Inc. ‘ Okeechobee: 1.Assessed the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps, tap and flush samples on young trees. Results indicated that very low numbers were present on young (2 yrs old) trees using Nuprid’. 2.Assessed and compared the abundance of psyllids in orange and grapefruit trees managed by the same oil spray program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. Overall, results indicated that there were more psyllids found on orange than grapefruit trees with similar flushing patterns. 5) Lindsey Groves ‘ Vero Beach: Assessed and compared the abundance of psyllids in orange and grapefruit trees managed by the same organic approved program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. Overall, results indicated that more psyllids were found on oranges than grapefruit with similar flushing patterns. 6) Premier Citrus ‘ Vero Beach: Assessed and compared the abundance of psyllids in orange and grapefruit trees managed by the same chemical spray program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. Overall, results indicated that there were no differences in psyllid abundance between orange and grapefruit trees with similar flushing patterns. 7) Laboratory and Field Research at IRREC: Assessed persistence and efficacy of PFR 97’ against the adult psyllid in the field on small trees; experiment was replicated 3 times. Overall, results showed fungal spores were viable and infectious against the adult psyllid up to 28 days post-spray. The effects of two formulations of Isaria fumosorosea Wize (blastospores and conidia) on feeding, honeydew deposits, and mortality of adult Diaphorina citri Kuwayama (Hemiptera: Psyllidae) was assessed in bioassay arenas. Psyllids infected by either formulation had reduced feeding as indicated by significantly fewer honeydew deposits [8 and 9 times less after 5 and 7 days, respectively] with mortality reaching 100% in fungal treatments compared to 0% in the controls after 7 days. Blastospores caused a significantly higher mortality than conidia in the first 3 days. The potential of auto dissemination of Pfr 97 by psyllids in the field is still being investigated.
The objectives of this project are establishing baseline toxicity data of various insecticides to field populations of the Asian citrus psyllid (ACP). Determining the resistance and cross-resistance development potential in field populations of the ACP and determining the mechanisms of insecticide resistance as part of insecticide resistance management program. In this quarter, using Ellman’s biochemical assay, the baseline data for acetylcholinesterase [AChE, the target site for organophosphate (OP) and carbamate insecticides] sensitivity to inhibition by OP and carbamate insecticides have been generated for 5 insecticides that are labeled for psyllid control in Florida citrus along with paraoxon (parathion) which was included as a standard insecticide. The tested insecticides were active forms of chlorpyrifos (chlorpyrifos-oxon), dimethoate (omethoate), malathion (malaoxon), aldicarb (aldicarb sulfoxide) and carbaryl. The toxicity data of a laboratory strain which was established in 2005 and has not been exposed to insecticides were used for comparison. Biochemical assays were conducted on AChE of five psyllid populations collected from one grove each in five counties (Hendry, Indian River, Lake, Polk, and St. Lucie) for determining the baseline AChE sensitivity levels and to compare them with laboratory strain. In general, AChE of all five psyllid populations collected from five Counties showed decreased sensitivity to inhibition to all the tested compounds compared with laboratory strain with few exceptions. The decrease in sensitivity of AChE to inhibition by different insecticides ranged from 1 to 3-fold for Hendry County, 1 to 3-fold for Indian River County, 1 to 3-fold for Lake County, 1 to 2-fold for Polk County and 2 to 4-fold for St. Lucie County. For Hendry County psyllid population, the highest decrease in sensitivity was observed for malaoxon and carbaryl (3-fold). For Indian River County psyllid population, the highest decrease in sensitivity was observed for aldicarb sulfoxide (3-fold) followed by chlorpyrifos-oxon and omethoate (2-fold). For Lake County psyllid population, the highest decrease in sensitivity was observed for carbaryl (3-fold) followed by chlorpyrifos-oxon (2-fold). For Polk County psyllid population, the highest decrease in sensitivity was observed for chlorpyrifos-oxon (2-fold). For St. Lucie County psyllid population, the highest decrease in sensitivity was observed for malaoxon and omethoate (4-fold) followed by chlorpyrifos-oxon (3-fold), paraoxon (3-fold), aldicarb sulfoxide (2-fold) and carbaryl (2-fold). Psyllid population from Polk County is still highly sensitive to most of the OP and carbamate insecticides tested except for chlorpyrifos-oxon when compared to laboratory strain. These data corroborates with insecticide susceptibility data obtained from bioassays and will serve as a baseline for monitoring resistance development at the target site (AChE) for OP and carbamate insecticide in ACP field populations. Further work to screen psyllid populations collected from 4-5 different locations in Florida is under way for determining the baseline sensitivity of AChE and monitoring resistance levels at the target site (AChE). Selection of one field population each for imidacloprid and chlorpyrifos resistance is in progress. Thus far, selection of 7 generations for imidacloprid and 3 generations for chlorpyrifos has been completed. Further selection of future generations and determining the mechanisms of resistance by toxicological and biochemical studies are in progress. These colonies will be used for further studies on determining the resistance and cross-resistance development potential in psyllids and mechanisms of resistance.
As of our last report on October 15, 2009, work has continued on the objectives of this proposal as outlined. However, during the winter months, psyllid populations decline to low levels in the field and also are unreliable for completion of under greenhouse conditions. Thus no new significant results on this project have been produced since the last report. Based on the results of our previous studies, multiple field trials are planned for spring of 2010 to apply the results of greenhouse studies to typical commercial citrus growing conditions. Effects of host plant nutrition on psyllid fitness: In previous experiments we demonstrated that the two most important essentials, Nitrogen and Potassium, have a significant effect on psyllid fitness. Varying levels of high and low Nitrogen and Potassium rates were applied to ‘Valencia’ orange plants grown under greenhouse conditions. Results of these experiments demonstrated that high nitrogen levels resulted in an increase in psyllid weight, an increased level of egg production and a shorter developmental time. Thus, higher nitrogen fertilization above what is needed by citrus plants may lead to an unnecessary increase in psyllid populations due to excessive use of nitrogen fertilizers. Conversely, addition of increased levels of Potassium fertilizer resulted in decreased fitness of psyllid populations. In field trials scheduled for spring of 2010, we will test varying combinations of Nitrogen and Potassium fertilizers under conventional growing conditions to determine the optimal rates of fertilizers to maintain tree health while minimizing psyllid reproduction rates. We will also be comparing these fertilization regimes to those being used by growers as part of an HLB/nutritional management approach to disease management. In our previous work we have also demonstrated that boron (0.25% Borax) applications have a significant negative effect on psyllid populations in the greenhouse as a result of increased adult mortality. Thus, we will also be examining the effects of 0.25% Borax applications on psyllid populations alone and in combination with varying levels of Nitrogen and Potassium fertilizers during filed trials in spring 2010. Effect of host plant species on psyllid fitness: Of the various citrus species tested thus far for effects on psyllid fitness, we have previously reported that Cleopatra mandarin is the most promising prospect for true resistance to Asian citrus psyllid of all the commercially available citrus varieties used today in citrus production. To convince ourselves that our results are real and not due to some unforeseen experimental error, we have conducted multiple trials using new Cleo plants to prove without a doubt that these results are indeed real…its real! Thus, during 2010, two trials are planned to further examine the resistance of Cleo to ACP. First, a series of 40+ genetically distinct Cleo hybrids planted at the UF/CREC will be challenged by caging psyllids on flushing terminals to determine whether any differences in susceptibility to ACP exists. If differences are found, then further investigations will be continued by the plant improvement team to determine whether these traits can be bred into currently available citrus varieties. In our second series of experiments conducted in the CREC teaching/demo grove with varying combinations of scion and rootstock combinations, we will investigate whether scion grown on Cleo has any advantage with regards to psyllid tolerance when compared to the same scion material grown on different rootstocks.
In the first experiment that was conducted to know the efficiency of systemic insecticides to control the Asian Citrus Psyllid (ACP), Diaphorina citri, and its effect on transmission of the bacteria Candidatus Liberibacter asiaticus, indicated that imidacloprid (Confidor 700 GrDA), 0.35 g AI/plant and thiamethoxam (Actara 250 WG) 0.25 g AI/plant, applied in the nursery tree bags, before planting, was efficient to control ACP until 60 days after application. The time to cause 100% of ACP mortality was between 5 to 7 days after the confinement of adults in treated plants. However, researches using electrical penetration graph (EPG) showed that in plants treated with imidacloprid and thiamethoxam, after the first feeding on phloem, the adults do not do more probing. We carried out the first PCR of the plants in this experiment and the results were negative, no plants have been detected the presence of the bacterium L. Ca asiaticus. No transmission results yet. We started the second experiment to determine if the systemic insecticides are effective until 90 days after application and its effect on transmission of the bacteria. In this experiment, the time to reach 100% mortality ranged from 3 to 7 days for both systemic insecticides tested (imidacloprid and thiamethoxam). The insecticides were effective up to 90 days after application. The results of PCR carried out for the ACP, in some periods, were positive for 100% of the samples, consisting of 10 insects tested, but in the confinement held at 46 days after application, in any sample was detected the presence of the bacteria. No acquisition in this period. In bioassays performed at 75 and 90 days after application, the percentage of positive samples was 50 to 70% and 10 to 40%, respectively. We started the experiment 2, the difference from the experiment 1 is the application of varying doses of the systemic insecticides and confinement of the ACP in plants treated only 7 days after application. To thiamethoxam (Actara 250 WG), the doses tested were: 1, 0.5, 0.1 and 0.05 g/nursery tree and imidacloprid (Provado 200 SC) were: 1.75, 0.9, 0.2 and 0.08 mL/nursery tree. Using electrical penetration graphs (EPG) techniques, we are studying the probing behavior of ACP. In the preliminary results we observed that the time feeding on phloem, in plants which were applied the systemic insecticides imidacloprid, was 7.58 and 6.95 minutes to 35 and 95 days after application, respectively. When applied thiamethoxam the results were 13.28, 15.41, and 17.50 minutes, respectively to 20, 35 and 95 days after application. In the control, the average time feeding on phloem was 129.79 minutes. For both insecticides, all specimens died in two days after those times of phloem sap ingestion. After first probing and ingestion of phloem sap, the insect stopped feeding and no more probing was observed. In foliar application, were tested imidacloprid (Provado 200 SC) and lambda-cyhalothrin (Karate 50 SC). Until 14 days after application, adults of ACP probe the citrus trees, but did not reach the phloem and died in 24 hours. In the test plant, approximately 80% of the ACP adults reached and sucked on phloem for 2 hours, in average, in a 5-hour record test.
The relationship between the Asian citrus psyllid (ACP) and Candidatus Liberibacter asiaticus (Las) continues to be studied to optimize disease management strategies. In laboratory studies of Las acquisition by ACP, nymphs reared on infected plants were more likely to acquire the pathogen than adults. Acquisition by nymphs ranged from 60-100%, whereas acquisition by adults only reached 40% after 5 weeks of feeding on infected plants. Transmission of Las from parent to offspring (transovarial) occurred at a rate of 2-6% in ACP eggs, nymphs, and adults. Experiments to determine the rate of Las transmission to healthy citrus by ACP were assessed one year after inoculations. Transmission of Las by individual ACP ranged from 4-10%, depending on amount of time Las(+) ACP spent feeding on plants, while groups of 100 or more ACP transmitted the pathogen at a rate of approximately 88%. The proportion of Las(+) adult ACP decreased over time when held on healthy plants. Due to the low rate of acquisition and long time period required for successful transmission by adult ACP, experiments to determine the latent period require for Las replication and transmission by ACP have not produced any Las(+) plants after more than one year of incubation following inoculation. Collectively, these results indicate that healthy adult ACP are poor potential vectors of Las but may, after continuous access to Las followed by oviposition on infected plants, give rise to highly infective nymphs. In subsequent experiments designed to evaluate the fitness of Las(+) ACP reared on infected citrus compared with healthy ACP reared on healthy citrus, Las(+) ACP produced significantly more eggs although adult longevity was reduced. Ongoing studies have thus far indicated no differences in the fertility of infected versus healthy ACP. These results underscore the importance of controlling ACP nymphs to reduce secondary disease spread within a grove. Additionally, oviposition on infective citrus plants, rather than adult acquisition feeding, appears the most important component of the Las transmission cycle. Preventing oviposition on Las-positive plants may provide significant disease control if included as part of an integrated pest management (IPM) strategy. Investigations continue into the seasonality of Las(+) ACP. During 2009, monthly sampling at 5 study sites across the state showed that % Las(+) ACP fluctuated throughout the year, with a season-long average of <1-5% LAS(+) ACP at our central Florida study sites. At our Homestead site where 100% of the trees are Las(+), fluctuations in %Las(+) psyllids were present but the overall % infection rate was much greater (64%). Comparing the results from years 1 and 2 of this study, the months with greatest % Las (+) ACP were not consistent between years. However, we hypothesize that plant flushing patterns, as influenced by climatic conditions, may be a key factor influencing shifts upward in %Las(+) ACP. In 2010, this study will focus specifically on plant-related factors.The effects of insecticides on the feeding behaviors of Las(+) ACP were examined using an electrical penetration graph (EPG) monitor with waveforms produced characterized based on the studies of Bonani et al (2009) and Youn et al (unpublished). For ACP feeding on plants with imidacloprid levels ranging from 7.8-20.7ppm, there was significant reduction in non-probing behaviors (np), stylet penetration (C), phloem salivation (E1), phloem ingestion (E2) and xylem ingestion (G) thus suggesting that pathogen transmission should be significantly reduced on imidacloprid treated plants. However, for aldicarb treated plants, the only statistically significant difference found was an increase in (E2) on aldicarb treated plants. These results suggest that aldicarb applications will not prevent pathogen transmission prior to causing mortality of adult ACP. Plants used in EPG studies are currently being held for subsequent Las analysis to compare actual rate of pathogen transmission between treatments. We are currently determining the level (PPM) of imidacloprid that is required in a plant (degradation studies) to provide effective disruption of psyllid feeding behaviors responsible for pathogen transmission.
1: Localization of Liberibacter asiaticus (Las) in internal organs of Asian citrus psyllid (ACP). In order to study the cellular interactions of Las in its psyllid vector, the following four methodologies have been used for localization of this bacterium in dissected organs and hemolymph smears of ACP and in leaf sections and extracts of HLB-diseased plants: A. Immunofluorescence confocal laser scanning microscopy. Two polyclonal and several monoclonal antibodies prepared against Las membrane proteins have been tested at various dilutions (1/20 to 1/400) and incubation times (3-27 hrs). Some of the monoclonal antibodies produced positive fluorescent labeling in the phloem area in sections of HLB- infected citrus leaves and in hemolymph smears of psyllids collected from HLB-infected trees, but not in whole-mount insect organs. These results are encouraging but appear to indicate that such antibodies may be slow to penetrate whole insect organs. Efforts are now underway to resolve this issue with various permeabilization procedures or by immunolabeling of paraffin sections rather than whole organs. B. Fluorescence In situ hybridization (FISH). The oligonucleotide primer tested so far is based on the following sequences (20 bases) of Las, tagged with Alexa Fluor 488: TCGAGCGCGTATGCAATACG. Several FISH protocols have been tested on dissected organs of ACP and on leaf sections and extracts from HLB-diseased plants. Green fluorescence, indicating Las, was detected in the filter chamber and midgut of field-collected ACP, but not in healthy controls. It was also detected in leaf sections and plant extracts from HLB-diseased plants but not in those from healthy plants. We are continuing to refine the FISH procedure and to test two other recently-designed Las primers . C. Quantitative RT-PCR of dissected insect organs. We tested two different RT-PCR procedures for detection of Las in dissected salivary glands, alimentary canals and other parts of individual ACP adults. In two successive experiments, using DNeasy Kit for extraction of DNA, Las was detected in 17-33% of the salivary glands, 27-47% of the alimentary canals, and in 47-53%of the rest of the body of psyllids collected from HLB-infected trees in Fort Pierce, FL. during November and December 2009. To our knowledge, this is the first direct demonstration of Las (using PCR) in the alimentary canal and salivary glands of ACP. D. Transmission electron microscopy (TEM) We are currently using a combination of TEM and RT-PCR, comparing the ultrastructure, including the bacterial fauna, of psyllid adults that have never been exposed to infected plants with those collected from HLB-infected trees and tested positive for Las with RT-PCR. Fixation and embedding of samples have been done, while thin-sectioning and TEM observations are underway, providing the base for TEM-immunolabeling studies. 2: Clarification of various acquisition and transmission parameters between ACP and Las. [A] Limited success has thus far been achieved using an artificial diet-in vitro feeding method for Las detection in live ACP. Further improvements in this technique are continuing. [B] Among adults field collected from Las-infected trees, averages of 37% females and 38% males have tested positive by PCR. No differences in infectivity among the three color morphs of adult ACP have been noted. [C] To assess the incubation period of Las in young citrus with respect to testing PCR-positive, groups of field-collected adult ACP were caged on individual citrus plants for 5 days and then assayed individually for Las. Plants that had been infested by Las-infected ACP were then PCR assayed weekly. Among trees infested by at least one infected ACP, 10%, 25%, and 50% tested positive within 4, 6 and 31 weeks, respectively. No relationship was found between the number of infected ACP per plant and the incubation period of Las. [D] There was no evidence of sexual transmission of Las between males and females of ACP. [E] In an acquisition study, greater percentages of females acquired Las than males.
USDA test 1. A new block of young, HLB’free citrus (Valencia on Carrizo) was planted on May 1, 2008 in a grove with substantial levels of HLB. Three psyllid control treatments (programs) are being compared in this planting: 1) a monoculture of citrus receiving monthly insecticide applications; 2) citrus interplanted with orange jasmine with a relaxed insecticide program for the citrus and orange jasmine not treated with insecticides; and 3) citrus interplanted with orange jasmine with a relaxed insecticide program for the citrus and regular applications of imidacloprid to orange jasmine. For plots with jasmine, a jasmine plant was planted between each citrus tree along some rows in each plot. Treatment 2 is being studied because psyllids may be strongly attracted to jasmine and killed, reducing numbers of psyllids that go to citrus. Treatment 3 is being studied because psyllids may be strongly attracted to jasmine thus reducing numbers of psyllids that go to citrus, and population levels natural enemies of the psyllid may be enhanced by having jasmine in the vicinity of citrus. None of the trees tested HLB positive just before planting in May 2008. By one year later, a mean of 0.9, 0.6, and 0.6% of the trees had been identified as infected under treatments 1, 2 and 3, respectively. During August 2009, a mean of 9.8, 3.9, and 4.2% of the trees had been identified as infected under treatments 1, 2 and 3, respectively. The rapid increase in incidence of HLB in the plots between May and August 2009 was attributed to increases in psyllid infestations during June and July, particularly in one Treatment 1 plot that was adjacent to older citrus. During 2009, a total of $148, $268, and $118 per acre worth of insecticides (materials only) was applied to treatments 1, 2 and 3. The large cost of treatment 2 was due to two applications of Bayer Feed and Protect to jasmine. USDA test 2. A new block of young, HLB-free citrus (Valencia on Carrizo) was planted during December 2008. Three treatments (psyllid control programs) are being compared in this planting: 1) citrus receiving monthly insecticide applications; 2) citrus under a relaxed insecticide program; and 3) citrus treated once every three weeks with spray oil. There are three replications of each treatment. None of the trees tested positive prior to planting. All of the trees were treated just after planting with imidacloprid. A hard freeze on Jan 22 damaged almost every tree, and a number of the trees died. These were replanted during February. All trees were treated with imidacloprid during February, April and May giving time for the trees to recover. The experiment was to officially began in June, but five trees tested positive in June. We removed these and replaced them with healthy trees during early August and resampled every tree, confirming that no tree tested positive. As of today, we have no results to present for this planting. UF test. An experiment was initiated to evaluate the ability of systemic insecticides to protect a new planting of citrus from psyllids and consequently HLB infection. We planted 112 Hamlins in two rows at 15 ft spacing on 03 Mar 2009. Half of each row was considered a replicate and divided into two main plots, treated and untreated. Treated plots were split into two subplots on 13 March, one receiving a liquid formulation of imidacloprid (Nuprid 2f @ 32 oz/acre ‘ 0.5 lbs a.i./acre) and the other receiving a solid formulation of the same (Suscon 13 @ 10 lbs/acre ‘ 0.5 lbs a.i./acre). On 11-Sep 2009, leaf samples were obtained from all 112 Hamlin trees and were sent to the HLB laboratory at the IFAS ‘ SWFREC facility in Immokalee Florida. Results from this testing showed no significant treatment effects (Chi square = 3.61, P = 0.182). At that time, 10 of the 56 Control or untreated trees tested positive for HLB whereas 10 of the 28 trees treated with the granular slow release imidacloprid were positive and 6 of the 28 trees treated with the liquid formulation of imidacloprid were positive for HLB.
Sampling – A stratified random sampling procedure was used to assess spatial dispersion of adult ACP according to yellow sticky trap sampling and stem tap sampling procedures, and optimum sample sizes and allocation were determined for both sampling procedures. Trap sampling and tap sampling indicated that adults were aggregated among trees, within individual strata and among strata across the block of trees studied. Regardless of whether trap sampling or stem-tap sampling is used, sampling should be conducted throughout a block because there can be significant variation across a block of trees in numbers of psyllids. Validation sampling indicated that, for commercially-acceptable precision levels at means of 1 or more adult psyllids per sample, 30 stem-tap samples or 28 sticky trap samples are required in a block of trees up to ten acres in size. Larger numbers of samples were required at lower densities. Biological Control – (a) Release of three new biotypes of Tamarixia from Asia were initiated in Florida. It is too early to gauge establishment. (b) The insect pathogen Hirsutella citriformis may sometimes provide significant levels of adult psyllid mortality. Adult psyllid cadavers mycosed by this fungus were most common in a group of mature trees during the fall and winter from October through March. Near absence of cadavers mycosed by the fungus during May ‘ June may be due to environmental conditions unfavorable to the fungus. In collaboration with the University of Florida, a method of culturing the fungus on media was developed. We are in the process of verifying pathogenicity of the culture. A study is planned to evaluate a number of citrus chemicals for toxicity to the fungus. (c) Releases of the parasitoid Diaphorencyrtus from south China. Over 9,000 wasps have been released in four counties in central and southwest Florida over the last three years (Collier, Hendry, Lake and Orange Co.). About 4,000 were released during 2009. Wasps were recovered in low numbers from commercial and research citrus groves in Immokalee during 2008, but no recoveries have been made during 2009. Studies are being continued on the biology of the parasitoid. Seasonal HLB profile in adult psyllids ‘ In this study, psyllids are collected fortnightly from diseased trees in a grove. Over all sample dates, an average of 91.7 (SEM 5.9) % of the trees tested HLB positive (mean CT 23.9, SEM 0.3) in the sample area. From Feb 2008 through Nov 2009, a mean of 33% (SEM 1.8) psyllids tested positive, with a minimum of 3% on one sample date and a maximum of 85% on another sample date. An average CT value of 25.2 (SEM 0.2) was recorded for infected psyllids over the entire study period. No definite trends in seasonality of HLB in adult psyllids have yet been identified over the course of this nearly two-year study with respect to percentages of psyllids infected or titers of the bacterium based on RT-PCR CT values. Beginning June 2008, once a month psyllids from the infected trees were caged individually on citrus seedlings for 7 days. After the 7 day period, the psyllids were tested for HLB and seedlings infested by an infected psyllid were assayed 6 months later for the pathogen. Over all study dates, an average of 57% psyllids tested HLB positive; an average of 25 (SEM 9.7) % infected psyllids transmitted the disease, with means per sample date as low as 0% and as high as 75%. It is too early to determine if there are any seasonal trends in transmission rates. The study was expanded during fall 2009 to include identifying the sex and body color of psyllids in the individual transmission experiments.
Since an effective control of the Asian citrus psyllid (ACP) requires a large component of chemical insecticides, we have continued our research on different pesticide chemistries to further optimize low volume technologies used in the HLB management. These efforts have been conducted with a view to strengthening the case for low-volume registration of test formulations to the EPA, in addition to demonstrating the effectiveness of low-volume technology to the citrus industry. The work has involved both laboratory and filed studies. In the laboratory, we have used a controlled droplet application apparatus to generate different spray droplet sizes to investigate the effect of droplet size on the mortality of ACP using various commercially available pesticide formulations that are labeled for citrus applications.The technique involved spraying different droplet sizes on psyllid infested potted ‘Swingle’ trees (8-inch) and counting the number of ACP eggs, nymphs, and adults before spraying and at 3 days and 7 days post-treatment. By varying the pesticide concentration, the amount of sprayed active ingredient was kept constant for all droplet size ranges. The pH of all the formulated products were measured to ensure label compliance and best spraying practice. To date, our results have shown that the smaller the droplet size, the greater is the percent mortality of all life stages of the ACP. The formulations examined have been: Danitol 2.4EC (synthetic pyrethroid), Lorsban Advanced (organophosphate), and most recently Dibrom 8E (organophosphate). For all chemistries, the results suggest up to 80 percent control of nymphs using droplets in the range of about 40 ‘ 100 ‘m median diameter. Danitol and Dibrom produced this effect by day 3, with Lorsban taking until day 7. The egg and adult stages showed similar trends, but with greater levels of control. Nonetheless, due to the particle size of the pesticide ingredients in available formulations, not all products are suitable for implementation into this sensitive and precise system. Three other formulations have been screened for experiment this spring against the glasshouse culture: EverGreen (natural pyrethroid), AgriMek (abamectin), and Knack (pyriproxifen). In their present formulations, both Movento (spirotetramat) and Provado (imidacloprid) are not compatible with the laboratory droplet applicator. However, due to the importance of these active ingredients in the citrus industry, a dialogue with the appropriate formulation chemist at Bayer has begun with a view to using suitable formulations in the near future. Field trials of a number of candidate formulations were conducted in commercial groves using a truck mounted and modified Curtis Dyna-Fog’ ULV applicator delivering 1-2 gallons per acre. The results showed that low-volume applications were effective in controlling ACP adults and nymphs. In the case of Delegate (spinetoram) and Lorsban, results against nymphs were comparable to that of high volume applications, except Delegate without crop oil. Also in this study, the low-volume application gave identical control against adults as the standard volume, with psyllid numbers remaining minimal for the 21 days of the study. The use of low-volume applicators in a citrus grove is a nascent technology and various refinements to the application technique have been made throughout the year, such as: reflective flagging tape for night time applications, installation of a throttle controller and use of a 50 mesh screen on the in-line formulation pump. Given the importance of droplet size rage in label limitations, droplet sizing technique of the ULV applicator was further refined in field experiments. The work involved measuring the air output of sprayer at various spatial distances and determining the most suitable location for sampling the spray cloud. Using a droplet sizing instrument, the volume median diameter of the generated droplets were about 53’3 micron at 5-ft distance. A revised protocol for use of the droplet generator and a manuscript for publication in the J. of Economic Entomology have been prepared. A poster outlining the results and experimental procedures was presented at the recent ESA meeting in Indianapolis.
The goal of the present project is to establish economic thresholds under different juice price scenarios that optimize returns on investment when a nutrient/SAR package is being applied in groves with moderate to high incidence of HLB. Two 3-year field experiments were initiated in two commercial orange blocks in Hendry County (southwest Florida). One of the groves is planted with ‘Early Gold’ oranges and the other with ‘Valencia’ oranges. Average HLB incidence estimated in both groves based on symptoms is more than 70%. 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 will be applied approximately every 1-2 months and consist of a rotation of insecticides recommended for managing this pest. Adult psyllid populations are being monitored every two weeks by tap sampling. Flushing patterns and flush infestation is being estimated by assessing the number of new shoots per tree and evaluating the proportion infested with psyllids. Negative incidence of spray applications on natural enemy populations will be evaluated by counting beneficials in tap samples and flush observations. At the beginning of the experiment, average psyllid infestation in the ‘Early Gold’ block was estimated at 0.26 ‘ 0.04 adults/tap. On July 30, treatments (2) and (3) were sprayed using the spinosyn spirotetramat (Delegate) at 4 oz/ac plus 5% 435 horticultural mineral oil. The treatment drove psyllid populations in sprayed plots down to 0.01 ‘ 0.01 and 0.03 ‘ 0.01 adults per tap in treatments 2 and 3 respectively. One month and a half later, populations were still low: 0.01 ‘ 0.01 and 0 adults/tap in treatments 1 and 2 respectively, but also low (0.07 ‘ 0.03 and 0.21 ‘ 0.06 adults/tap) in treatments (1) and (4) respectively. An additional spray with the organophosphate Dimethoate at 24 oz per acre has been scheduled in treatment (2) for the second week of October. Cumulative psyllid numbers are below threshold in all treatments though greater in treatments 1 and 4 (2308 and 1062 adults/tap respectively) compared to treatments 2 and 3 (227 and 843 accumulated adults/tap respectively) as a consequence of the spray in late July. Comparisons between sprayed and unsprayed treatments have not yet revealed negative effects on beneficials. In the ‘Valencia’ block, average psyllid infestation was estimated at 0.06 ‘ 0.01 adults/tap at the beginning of the experiment. Treatment (2) was sprayed on September 28 with the organophosphate Dimethoate at 24 oz per acre. Cumulative adults/tap previous were similar among treatments (93, 91, 49 and 58 adults/tap for treatments 1-4 respectively, all below the cumulative number corresponding to pre-established theoretical thresholds (448 and 1568 adults/tap for the 0.2 and 0.7 thresholds respectively.
The goal of the present project is to establish economic thresholds under different juice price scenarios that optimize returns on investment in groves with high incidence of HLB. Two 3-year field experiments were initiated in different two commercial orange groves in Hendry County (southwest Florida), one block planted with ‘Early Gold’ orange and the other with ‘Valencia’ orange. Average HLB incidence estimated in both groves based on symptoms is more than 70%, and a recently completed PCR analysis of the ‘Early Gold’ block indicated greater than 98% incidence. Experimental design is randomized complete block with 4 replicates and 4 treatments: (1) No insecticide, (2) Calendar applications to drive vector populations to the lowest possible level, (3) a nominal threshold of 0.2 psyllids per tap, and (4) a nominal threshold of 0.7 psyllids per tap. Calendar applications will be applied approximately every 2-3 months and consist of a rotation of insecticides recommended for managing this pest. Adult psyllid populations are being monitored every two weeks by tap sampling. Flushing patterns and flush infestation is being estimated by assessing the number of new shoots per tree and evaluating the proportion infested with psyllids. Negative incidence of spray applications on natural enemy populations is being evaluated by counting beneficial insects in tap samples, suction samples and flush observations. Treatments (2) and (3) were sprayed on July 30 in the ‘Early Gold’ block, using the spinosyn spirotetramat (Delegate WG ) at 4 oz/ac plus 5% 435 horticultural mineral oil. Treatment 2 was also sprayed with the organophosphate Dimethoate at 24 oz per acre the second week of October. Treatment (2) in the Valencia’ block was sprayed on September 28 with the organophosphate Dimethoate at 24 oz per acre. The Delegate spray on 30 July In the ‘Early Gold’ block drove psyllid populations in sprayed plots (treatments 2 and 3) down to a cumulative average of 0.01 ‘ 0.01 and 0.03 ‘ 0.01 adults per tap respectively. Density of ACP adults has remained close to zero in both treatments to date and ACP numbers in treatments (3) and (4) have not exceeded their nominal thresholds of 0.2 and 0.7 ACP/tap. At the end of December, Cumulative ACP numbers were significantly lower in the treatment that received two sprays (2) compared to unsprayed treatments (1 and 4) (P = 0.0062; df = 2,15; F = 7.725) with no other differences among treatments. The ‘Early Gold’ block was harvested the second week of December. As yet no significant treatment effect on yield has been observed (P = 0.3582; df = 2,15; F = 1.112). Neither is the correlation between the yield loss and adult ACP cumulative numbers significant, although a trend is evident (r2 = 0.1390; P = 0.1549; df = 1,14; F = 2,261). Monzo, C., Arevalo, H.A. and Stansly, P. A. 2011. Thresholds for vector control in young citrus treated for symptoms of HLB with Nutrient/SAR Package. Poster presentation, 2011 International Research Conference on HLB, Orlando 10-14 Jan 2011.
This research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllid feeding. Both protein-based and RNAi strategies were tested by feeding psyllids artificial diets. To support the artificial diet assays, we optimized the diet composition by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period. Using this approach we identified suitable buffers and optimal diet pH during the feeding period. In separate experiments, Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from the Asian Citrus psayllids that was identified in our laboratory were added to artificial diets on which psyllids were allowed to feed. TMOF was tested at a concentration of 10 ‘g/’L and CPI was tested at a concentration of 3 ‘g/’L. Since psyllids take in very small amount of the artificial diet, in nl quantities, the amounts of the tested proteins are physiological rather than pharmacological. After 10 days of feeding, 100% mortality was observed in psyllids feeding on diets containing TMOF or CPI, whereas, 40% mortality was found in psyllids feeding on the control diets. The earliest significant effect of added TMOF to the diet was observed at 4 days of feeding. TMOF caused ~15% mortality whereas the mortality of the control group was less than 5%. CPI fed psyllids caused a significant higher mortality than the controls after 7 days of feeding. CPI was tested at lower concentrations that were used for TMOF, because of limited availability of the purified protein. Because of these results we plan to purify more CPI for subsequent testing. In the fourth quarter of the grant period we plan to expand this work and study dose effect and optimal concentrations, as well as, potential for synergistic effects when both TMOF and CPI are fed together to psyllids in the same diet. Continued success of this strategy will allow us to develop transgenic citrus expressing these proteins in the phloem making citrus resistant to adult and nymph psyllids. A third polypeptide was also tested at concentrations similar to TMOF, this polypeptide prevented psyllids from successful eating the artificial diet causing starvation and death. We plan to continue these studies and further characterize the polypeptide and feed it in concert with TMOF and CPI to be able to find out the combined effect on the Asian citrus psyllids. In parallel to these studies, we synthesized dsRNA molecules targeting 11 different psyllids essential genes encoding three different classes of proteins (alpha-tubuliln, V-ATPase, and Cathepsins). The DNA encoding a fragment of each of the 11 genes was synthesized and cloned into appropriate vectors. Primers used for dsRNA synthesis using the Megascript kit from Invitrogen was used to produce double stranded RNA (dsRNA) for RNA inhibition (RNAi) studies. We produced dsRNA for two alpha-tubulins and two V-ATPases. Initial feeding studies with alpha-tubulin dsRNA and V-ATPase dsRNA caused ~60% psyllids mortality as compared to only ~30% mortality for psyllids fed a control diet containing an equal amount of dsRNA not specific to the psyllid. Influence of dsRNA on cognate psyllid transcripts will be tested in psyllids that remain alive at 8 days. In summary we: 1. Prepared dsRNA molecules against alpha-tubulin and V-ATPase and fed them to Asian citrus psyllids showing that they are very effective causing high mortality. 2. Showed that physiological amounts of TMOF, a new polypeptide that was recently discovered in our lab and CPI that were fed to psyllids by an artificial diet caused 100% mortality.
The purpose of this proposal is to identify and develop attractants, both pheromone and host-plant based, for the Asian citrus psyllid (ACP). The intent is to develop a highly effective attract-and-kill control system for ACP with such attractants, as well as to develop highly effective monitoring traps to effectively evaluate ACP population densities to better determine the need for spraying. Thus far, in collaboration with USDA colleagues, we have confirmed that virgin and mated male ACP are attracted to female ACP. These data suggest that female ACP produce an attractant chemical, potentially a pheromone, that attracts male ACP. Within the past few months, a female-produced sex attractant pheromone was identified in the potato psyllid by researchers in Washington, which is the first example of a psyllid pheromone. Second, we have proven that both male and female ACP are attracted to their host plant volatiles. The host plants tested were: ‘Duncan’ grapefruit, sour orange, ‘Navel’ orange, and Murraya paniculata. Responses varied by plant species and by psyllid sex and mating status. Generally, evidence of attraction was stronger in females and in mated individuals of both sexes relative to virgins. The presence of a visual cue typically enhanced attractiveness of olfactory cues; in no case did unmated individuals show evidence of attraction to host plant odors in the absence of a visual cue. In behavioral assays in the laboratory, we found that .-Butyrolactone is attractive to male ACP, but not to females suggesting that this chemical may be part of the female ACP pheromone blend. In collaboration with an industry partner, (Alpha Scents, West Linn, OR), we obtained custom-made release devices for .-Butyrolactone as well as dispenser for synthetic plant volatiles identified and developed by a USDA collaborator. In our initial field tests, results with .-Butyrolactone have been inconclusive. Although in one trial it appeared that this chemical increased catch of ACP on traps, the results were inconsistent in follow up trials. We are currently analyzing cuticular extracts of ACP to find further pheromone components because it appears that although .-Butyrolactone may be a component of the pheromone, it is not the only chemical responsible for attracting male ACP. This work is being conducted in collaboration with Stephen Lapointe from USDA-ARS in Fort Pierce. Also, we have evaluated a 5-component blend of synthetic plant volatiles as an ACP attractant based on our work with psyllid attraction to citrus. This blend and its associated dispenser is produced by Alpha Scents. We have shown attraction of ACP to these chemicals in the laboratory, but catch of ACP on traps in the field was not increased by this plant volatile lure in the field. We continue to work on refining this blend and its dosage in an effort to develop an attractive lure for the field. Concurrently with our work towards developing an ACP attractant, we have developed an attract-and-kill formulation for ACP with our industry partner and Co-PI Darek Czokajlo from Alpha Scents. We are working with a gel matrix with UV-protective properties that releases both the attractant and contains a small amount of pesticide. As ACP approach and touch the lure droplet laced with insecticide, they pick up a lethal dose of toxicant and die. We compared formulations containing 6, 14, and 22% imidalcloprid against Asian citrus psyllids in the laboratory. We found the the 14% imidacloprid formulation is superior to the 6% formulation, but that there was no added benefit of the 22% formulation. An optimized attractant is still needed before this formulation could be successfully employed for ACP control and this research is currently in progress. In separate trials working on a different attract-and-kill formulation consisting of an emulsified wax formulation (SPLAT, ISCA Technologies), we compared the insecticides Spinosad, Methoxyfenozide and Tebufenozide against the psyllid. We found that Methoxyfenozide and Tebufenozide are not effective with this formulation and that Spinosad is only marginally effective resulting in about 50% mortality. Our ongoing studies are focusing on testing more insecticides with the wax formulation so that more effective psyllid kill can be achieved.
The purpose of this project has been to develop an effective repellent for the Asian citrus psyllid (ACP). Our work was initiated by investigating the volatiles released by guava plants and their effects on ACP behavior. Interplanting citrus with guava, Psidium guajava L., was reported to reduce ACP populations and incidence of HLB. Therefore, we initiated a series of investigations on the response of ACP to citrus volatiles with and without guava leaf volatiles and to synthetic dimethyl disulfide (DMDS), in laboratory studies and in the field. DMDS was recently identified as a metabolite produced in large quantities by wounded guava leaves. Volatiles from guava leaves significantly inhibited attraction of ACP to normally attractive host-plant (citrus) volatiles. A similar level of inhibition was recorded when synthetic DMDS was co-released with volatiles from citrus leaves. In addition, the volatile mixture emanating from a combination of intact citrus and intact guava leaves induced a knock-down effect on adult ACP. Compounds similar to DMDS including dipropyl disulfide, ethyl-1-propyl disulfide, and diethyl disulfide did not affect the behavioral response of ACP to attractive citrus host plant volatiles in laboratory olfactometer assays. Our field experiments confirmed the results of our laboratory olfactometer assays. Deployment of synthetic DMDS from polyethylene vials (Alpha Scents) and SPLAT wax dispensers (ISCA technologies) reduced populations of ACP in an unsprayed citrus orchard for up to 3 weeks following deployment. Given that population densities were equivalent among plots prior to the deployment of DMDS treatments, we hypothesize that DMDS repelled adult ACP from treated plots. However, we cannot exclude the possibility that a proportion of the ACP populations in DMDS-treated plots may have been reduced due to direct intoxication. By the fourth week, there was no remaining DMDS in the dispensers, which likely explains why populations were once again equivalent in treated and control plots. Given the volatility of DMDS, one of the main obstacles to the development of a practical DMDS formulation for ACP management will be development of a slow-release device that maintains the chemical above a behaviorally active threshold for long periods. The polyethylene vials and SPLAT dispensers evaluated in these initial proof-of-concept investigations will likely not be economically practical for releasing DMDS for control of ACP in their current form. Both the number of dispensers required per acre (~200) as well as the amount of active ingredient required per three weeks (~3 kg) would likely be economically prohibitive for a hand applied dispenser. Furthermore, the dispensers evaluated in this study resulted in a ~2/3 decrease in field populations of ACP, which would be insufficient for effective control of this pest as a stand alone treatment. Another immediate logistical hurdle for developing DMDS into a practical psyllid management tool is the chemical’s strong and unpleasant odor. This may render field application difficult and potentially limit the use of DMDS depending on fruit harvesting schedules or proximity to urban areas. Ideally, a slow-release dispenser needs be developed that could achieve 150-200 d of behaviorally efficacious release. ACP populations are much more prevalent on crop borders and thus targeted applications of DMDS to those areas may be immediately useful with a dispenser that is not yet optimized. Our current efforts are focussing on further optimizing these dispensers to increase the duration of efficacy. ISCA has recently developed four new formulations of SPLAT dispensers that we will be evaluating in the early spring which show promise. Most recently, we have identified new compounds that are ACP repellents. In general, trisulfides (dimethyl trisulfide) inhibited the response of ACP to citrus volatiles more than disulfides (dimethyl disulfide, allyl methyl disulfide, allyl disulfide). Monosulfides did not affect the behavior of ACP adults. A blend of dimethyl trisulfide (DMTS) and dimethyl disulfide (DMDS) in 1:1 ratio showed an additive effect on inhibition of the response of ACP to citrus volatiles and was more effective than DMDS alone in the lab.
The purpose of this investigation has been to develop, evaluate, and optimize biorational management tools for Asian citrus psyllid (ACP) including insect growth regulators and antifeedants. In our first set of laboratory studies with insect growth regulators, we investigated the activity of the insect growth regulators pyriproxyfen (Knack), buprofezin (Applaud) and diflubenzuron (Micromite) on ACP eggs, nymphs and adults to evaluate its potential usefulness as a biorational insecticide for inclusion into an integrated pest management (IPM) strategy for ACP control. All three chemicals exhibited strong ovicidal and larvicidal activity against ACP eggs and nymphs, respectively, in age- and concentration-dependent manners. Fewer eggs hatched into nymphs at the higher concentrations tested (80-160 ‘g mL-1). A significantly lower percentage of early instar nymphs (first, second and third) survived and emerged into adults at the higher concentrations tested (80-160 ‘g mL-1) compared with late instar nymphs (fourth and fifth). Furthermore, all three chemicals exhibited transovarial activity by significantly reducing the fecundity of females and viability of eggs deposited by females that emerged from treated fifth instar nymphs. Topical application of each chemical to adults also significantly reduced fecundity and egg viability. Application of each chemical at 160 ‘g mL-1 resulted in the highest inhibition of egg hatch in younger eggs (0-48 h old) laid before or after treatment and strongest suppression of adult emergence from early instar nymphs compared with other rates tested. Each chemical also markedly reduced female fecundity and egg viability for adults that were exposed either directly or indirectly. Also adults emerging from nymphs treated with pyriproxyfen were deformed and died soon after emergence. The direct (ovicidal and larvicidal) and indirect (transovarial) effects of the IGRs against immature and adult ACP, respectively, suggest that integration of these insecticides as part of an IPM strategy should negatively impact ACP populations over time. In a separate investigation, we have been studying the sub-lethal effects of various insecticides. Given the broad use of imidacloprid for management of ACP, particularly on young trees, we investigated it’s possible sub-lethal effects first. Because of the variation in spatial and temporal uptake and systemic distribution of imidacloprid applied to citrus trees and its degradation over time in citrus trees, ACP adults and nymphs are exposed to concentrations that may not cause immediate mortality but rather sublethal effects. Our objective was to determine the effects of sublethal concentrations of imidacloprid on ACP life stages. Feeding by ACP adults and nymphs on plants treated daily with a sublethal concentration (0.1 ‘g mL-1) of imidacloprid significantly decreased adult longevity (8 d), fecundity (33%), and fertility (6%) as well as nymph survival (12%) and developmental rate compared with untreated controls. The magnitude of these negative effects was directly related to exposure duration and concentration. Furthermore, ACP adults that fed on citrus leaves treated systemically with lethal and sublethal concentrations of imidacloprid excreted significantly less honeydew (7-94%) compared with controls in a concentration-dependent manner suggesting antifeedant activity of imidacloprid. Sublethal concentrations of imidacloprid negatively affect development, reproduction, survival, and longevity of ACP which likely contributes to population reductions over time. Also, reduced feeding by ACP adults on plants treated with sublethal concentrations of imidacloprid may potentially decrease the capacity of ACP to successfully acquire and transmit the HLB causal pathogen. Pymetrozine is a chemical that is known to paralyze the muscles involved in plant probing in plant-sap sucking insects such as aphids and is known to prevent transmission of aphid and whitefly transmitted viruses. In our initial investigations, we found that at a 100 ppm dosage applied to citrus plants, pymetrozine inhibits acquisition of Liberibacter by psyllids on Valencia, Clepatra Mandarin, and Persian lime by approximately 50%.
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