We have constructed artificial mirco RNA interfering RNAs and are evaluating them as a strategy to deliver specific anti-psyllid RNAs to psyllid targets. Our belief is that these will be highly specific, and we know that miRNAs traffic in the phloem so should be available for phloem-feeding hemipterans such as psyllids. Seven amiRNAs targeting BC-ATPase were made and cloned into pBluescript SK, which are pAMIRA1, pAMIRA1c, pAMIRA2, pAMIRA2c, pAMIRA3, pAMIRA3c, and pAMIRA2PE. Four amiRNAs targeting GFP were made and cloned into pBluescript SK as well, which are pAMIRG1, pAMIRG1c, pAMIRG2, and pAMIRG2c. All 11 amiRNAs were then cloned into pGWB2, a gateway binary vector for expression driven by 35S promoter and into a viral vector with one extra stemloop from amiRA2 for amiRNAs of BC-ATPase and extra stemloops from amiRG1 and G2 for amiRNAs of GFP. The viral vector carrying amiRNAs were cloned into binary vector pCB301. We know that we can test RNA constructs on D. citri by in vitro feeding, but our hope is to also transiently express interfering RNAs in citrus such that they can move in the phloem and be acquired by feeding D. citri. We are utilizing a variety of approaches to test this possibility. So far we have performed agroinfiltration experiments on citrus and tobacco plants (the latter for the potato psyllid, Bactericera cockerelli). We used 35S promoter driven constructs and plant viruses in attempts to achieve gene expression. GFP assays failed to show detectable fluorescence in 5 different citrus types, but high level GFP expression was seen in tobacco.
We have constructed artificial mirco RNA interfering RNAs and are evaluating them as a strategy to deliver specific anti-psyllid RNAs to psyllid targets. Our belief is that these will be highly specific, and we know that miRNAs traffic in the phloem so should be available for phloem-feeding hemipterans such as psyllids. Seven amiRNAs targeting BC-ATPase were made and cloned into pBluescript SK, which are pAMIRA1, pAMIRA1c, pAMIRA2, pAMIRA2c, pAMIRA3, pAMIRA3c, and pAMIRA2PE. Four amiRNAs targeting GFP were made and cloned into pBluescript SK as well, which are pAMIRG1, pAMIRG1c, pAMIRG2, and pAMIRG2c. All 11 amiRNAs were then cloned into pGWB2, a gateway binary vector for expression driven by 35S promoter and into a viral vector with one extra stemloop from amiRA2 for amiRNAs of BC-ATPase and extra stemloops from amiRG1 and G2 for amiRNAs of GFP. The viral vector carrying amiRNAs were cloned into binary vector pCB301. We know that we can test RNA constructs on D. citri by in vitro feeding, but our hope is to also transiently express interfering RNAs in citrus such that they can move in the phloem and be acquired by feeding D. citri. We are utilizing a variety of approaches to test this possibility. So far we have performed agroinfiltration experiments on citrus and tobacco plants (the latter for the potato psyllid, Bactericera cockerelli). We used 35S promoter driven constructs and plant viruses in attempts to achieve gene expression. GFP assays failed to show detectable fluorescence in 5 different citrus types, but high level GFP expression was seen in tobacco.
We have previously shown that citrus engineered to produce dsRNA’s, matching the sequence of a specific Asian citrus psyllid (ACP) mRNA, induced ACP mortality when adult ACP fed on these plants. We have also shown that when psyllids containing the bacterium that causes citrus greening (Candidatus Liberibacter asiaticus,CLas) are fed on these plants for 10-15 days, the CLas is no longer detected in the psyllids. Furthermore, none of the nymphs hatched from eggs laid by CLas-containing psyllids contain CLas if fed on the dsRNA producing citrus. Replications of these experiments have been conducted during this reporting period. Furthermore, this research was now expanded to understand the molecular response occurring within the ACP as a result of specific dsRNA ingested. This was performed by conducting RNAseq analysis of the transcripts isolated from ACP that have fed on the specific dsRNAs. These experiments have been conducted and RNAseq data has been generated and is now being analyzed.
Incidence of citrus canker has been increasing in Florida due in part to increased pressure from citrus leafminer (CLM). It is likely that broad-spectrum insecticides for ACP management have impacted natural enemies of CLM leading to its rebound as a pest. The objectives of this study are: (1) Assess the use of pheromone traps for monitoring CLM populations, and development of a preliminary degree-day model, (2) evaluate effectiveness of early season and aerial applications to reduce CLM and canker, (3). Obtain baseline susceptibility to key insecticides, and (4) Use a diagnostic dose to monitor resistance in field populations of CLM exposed to intensive versus modest insecticide use. Objective 1: We have been monitoring adult CLM weekly using delta pheromone traps in 3 commercial groves under different management scenarios: Grove 1) sprayed at peak trap capture using different trap densities, Grove 2) sprayed based an ACP threshold or biweekly low volume (LV) sprays of horticultural spray oil (HMO), and Grove 3) sprayed at threshold or untreated. CLM damage is being assessed using a modified Horsfall-Barratt scale 1 to 8 and canker by percent incidence for individual trees, number of areas or ‘hits’ present in a tree, and proportion of diseased fruit. In May, leafminer damage and canker assessments resumed for 2013. Pheromone traps were deployed in citrus groves and non-citrus areas (OK Slough, 2011, 2012, 2013) to determine density of non-target species captured by the CLM lure. At this time, specimens are still being dissected for species identification. Canker and leaf damage assessments for spring were completed in May for four commercial groves, and data entry is pending. Likewise, pheromone trap data for May, June, and July are still in process of being counted and recorded. Four phenology models (206 degree days, 238dd, 265dd, and 398dd) are currently being analyzed for the best fit with Florida moth flight. Models were compared by looking at the differences in degree days between estimated model peak flight (50% generation time) and peak CLM flight of moth catch. The smallest difference in degree days between model and CLM peak flight represents the best fit. Thus far, there is no significant difference between models 206, 265, and 398dd, but least difference was seen comparing capture data from mature trees to predictions of the the 206 dd model whereas the 265dd model fit best with data from a grove with 10% resets. We are looking into the association with flush, and it appears both models may need to be further tested for another year. Objective 2: A 130-ac experimental block (Grove 4) divided into 4 replicated treatments: 1) Delegate applied early, 2) Delegate applied late, 3) Sprays following peak CLM flight, and 4) Rotations of OPs and pyrethroids. Delegate was applied early on 16 Apr at 4oz/ac with 2% v/v 435 citrus oil @ 90 gpa. On 29 Apr, 3lbs of copper, Dimethoate 1.5 pints (peak flight only plots), and Movento 16 fl oz (on Grower Standard and Delegate Late plots). Delegate Late will be sprayed in a couple of weeks Application in the peak-sprayed plots are finished this year, and their effect on late summer, fall CLM damage will help determine if just two precisely planned peak sprays are effective at controlling CLM the remainder of the year.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are useful for monitoring CLM populations and may catch thousands of moths per week. However, there is a lack of information on effective range of the traps which compromises their usefulness. Moreover, we have preliminary results showing that at least 3 other leafminer species are also captured, including Phyllocnisitis insignis, P. vigitegenella, and an unknown new species. Objectives of the proposed project are: (1) evaluate the effective range of standard delta sticky traps armed with CLM pheromone, (2) assess species composition of trap catches within and at defined distances from citrus groves, (3) calibrate traps with direct assessment of damage and canker incidence/intensity as tools to monitor success of CLM management programs, and (4) provide an analysis of the economic benefit of CLM management in citrus cultivars over a range of canker susceptibility. Funds for this grant were received on 5/10. Objective 1: Lure Degradation Study for Spring 2013 began on 20 May: Delta pheromone traps (ISCA) were dispersed in a 39ac block of Pineapple orange at Duda & Sons, Inc. located in Hendry County, Florida. Tree spacing is 12 x 25 feet with 24 plots (1.63ac each). Two commercial lures were tested 1) Alpha Scents (A) lure, and 2) ISCA (I) lure. Pheromone traps were dispensed at ~1ac positions at central locations in plots and data collected at 1 week intervals for comparison of trap catch. Traps with lures changed on a weekly basis served as a positive control. Trap durations throughout the block were random with 3 replicates. Four days after traps were dispensed, all aged traps for brand A caught the same number of moths as control, however, aged brand I traps all caught significantly fewer moths than the positive control. There were no significant differences between moth catches from traps aged 4 or 8 weeks. At 9 days no significant difference was observed in number of moths captured among the positive control for Brand A or brand I aged 4 or 6 weeks. Likewise, no significant difference at 14 days in moth catch amoung all traps of the A brand, whereas only week 4 of the I brand was similar to the positive control. At 30 days, aged I traps were significantly different than the positive control, while 4 and 8 week A traps were still catching moths similar to a new lure. Hence, during spring lures of either brand remained effective for up to 12 weeks. Lures are currently being aged for Fall 2013 efficacy testing. At with next peak flight we will start the mark and re-capture experiments. Objective 2: Bucket traps in the OK slough were re-armed with kill strips and lures on 28 June. Moths will be collected this week, separated by morph, and submitted for PCR testing for species identification in Gainesville. Objective 3: Bucket traps were dispensed 28 June, at a density of 1 per treatment plot in a commercial grove where we have been conducting a nutritional trial the past 5 years. Moths will be collected this week, separated by morph, and samples sent to Gainesville for species identification by bar-coding. In this grove, we have historically noted two species of moth captured. These results will help us correlate the true percentage of CLM present , its associated leaf damage and canker presence, and perhaps determine an economic threshold.
We have previously shown that citrus engineered to produce dsRNA’s, matching the sequence of a specific Asian citrus psyllid (ACP) mRNA, induced ACP mortality when adult ACP fed on these plants. We have also shown that when psyllids containing the bacterium that causes citrus greening (Candidatus Liberibacter asiaticus,CLas) are fed on these plants for 10-15 days, the CLas is no longer detected in the psyllids. Furthermore, none of the nymphs hatched from eggs laid by CLas-containing psyllids contain CLas if fed on the dsRNA producing citrus. Replications of these experiments have been conducted during this reporting period. Furthermore, this research was now expanded to understand the molecular response occurring within the ACP as a result of specific dsRNA ingested. This was performed by conducting RNAseq analysis of the transcripts isolated from ACP that have fed on the specific dsRNAs. These experiments have been conducted and RNAseq data has been generated and is now being analyzed.
This project evaluates the impact of psyllid control programs on non-target pests, beneficial insects and mites. Two large replicated blocks of mature citrus in two conventional groves of Hendry County and one each in Collier and Lee counties untreated or treated with foliar sprays of insecticides, horticultural oils, and nutritionals were sampled for ACP, citrus leafminer (CLM), citrus rust mites (CRM), citrus red mite, predatory mites, snow scale, chaff scale, Glover scale, purple scale, citrus blackflies, whiteflies, mealy bugs, as well as their natural enemies and fruit damage. The calendar and 0.2 ACP adults per tap sample threshold treatments in the block of ‘Early Gold’ in Hendry county, received Micromite 6.25 oz/ac + 435 oil (2%) on 29 April and Delegate 4.5 oz/ac + 435 oil (2%) on 11 June. In ‘Valencia’ block, calendar treatment received Danitol at 16 oz/ ac on 8 April, Micromite 6.25 oz/ac + 435 oil (2%) on 29 April and Delegate 4.5 oz/ac + 435 oil (2%) on 11 June. The treatment with threshold of 0.2 adults per tap sample also received applications of 8 April and 11 June and 0.7 adults per tap sample threshold received 8 April application only. All treatments received a nutritional spray on 9 April. These treatments provided significant suppression of ACP. In May, CRM averaged 0.01 and 0.59 per lens field (LF) in calendar and 0.2 ACP threshold treatments in ‘Early Gold’, respectively, significantly less than 3.4 per LF in untreated or 11.4 per LF in 0.7 ACP threshold treatment which also remained untreated, indicating that Micromite controlled both ACP and CRM. CRM populations were down in June averaging ‘ 2.7 per LF and no significant effect of Delegate observed. In ‘Valencia’ CRM averaged 1-3 per LF in April and there was no significant effect of Danitol. In May or June, CRM dropped to ‘ 0.3 with no effect of Micromite or Delegate observed. Predatory mites averaged ‘ 0.1 per LF in both blocks during three months and did not differ across treatments. Voliam Flexi at 7oz/ac applied to insecticide treatments suppressed ACP in a block of ‘Valencia’ in Collier county where insecticides, insecticides + foliar nutrition, nutrition only and untreated are being compared against ACP. CRM averaged ‘ 0.6 per LF, not signifcantly different between treatments but seen mostly in nutritional and untreated plots except June. Predatory mites did not differ between treatments in April or May but averaged 0.4 per LF in the insecticide only treatment in June significantly more than ‘ 0.05 per LF in the remaining treatments. Low ACE populations in a block of ‘Valencia’ orange in Lee county were compared among treatment with biweekly applications of 435 oil, a grower standard that did not require insecticides and an untreated check. CRM average dropped from 4.2 per LF in April to 0 per LF in June in the untreated plots with no significant difference observed between treatments. Predatory mites averaged ‘ 0.08 per LF again with no differences among treatments. A manuscript entitled ‘Microbial and Synthetic Insecticidal Sprays for Asian Citrus Psyllid and Citrus Leafminer Control in Oranges’ was presented at the annual meeting of the Florida State Horticultural Society and submitted for publication. Studies concluded that synthetic insecticides provided more psyllid reduction than microbials, and reduced leafminer for up to 17 days. New products tested and in the registration process for use on citrus will extend the range of tools available to control these pests. While the effectiveness of microbial and oil sprays tended to be short lived, they could still be useful to conserve natural enemies, for application on blooming citrus and in organic groves which prohibit synthetic products.
Transmission of CLas by the ACP can be affected by abiotic and biotic factors. Perturbations in temperature are shown to affect ACP reproduction and survival; moreover, it is likely that such fluctuations could also impact on ACP vector efficiency in transmitting CLas. Work has been completed in assessment of ACP acquisition of CLas under different temperatures. Week-old psyllids from laboratory colonies were subjected to temperatures ranging from 5-38oC for 2-4 day periods in controlled growth chambers and subsequently placed on Clas positive citrus plants for an acquisition access period of 2 or 4 weeks. All chambers were maintained at given temperatures ‘ 1’C, 50% RH ‘ 5%, and 14:10 (light:dark) photoperiod. To evaluate acquisition of CLas, we are quantifying CLas accumulation within the gut, hemolymph, and salivary glands of dissected psyllids using qPCR. In addition, a proportion of experimental ACPs in above treatments were used to assess the efficiency of Clas inoculation to healthy citrus plants by ACP. Preliminary analyses of these data suggest that Clas acquisition and inoculation is impacted by temperatures psyllid’s experience prior to CLas transmission. The endosymbiont Wolbachia is a potential agent for reducing competences of insect vectors. Field studies conducted over the past year indicate that Wolbachia infection rates differ among ACP populations in Florida. Preliminary analysis of field-collected psyllids suggests that more than one Wolbachia strain may be present in Florida psyllid populations. Currently, we are using multi locus sequence typing (MLST) to characterize the diversity of Wolbachia in Florida ACPs populations. In addition, we are continuing to propagate robust breeding lines of Wolbachia-free psyllids for fitness and transmission bioassays.
The 2013 harvesr of the 2012-13 Valencia crop will be the last data collected on yield as the project will end in December this year. We have collected five year harvest in the replicated Valencia trial in a commercial grove in southwest Florida evaluating the Boyd cocktail. The yield and juice data shows HLB infected trees have increased yield each year and are healthy, growing well, and maintaining production. Yield for the most productive treatments was about 2-boxes/tree on 8 year-old (7 to 8 foot) trees. The common ingredient in the most productive treatments during the past 5 years has been micro- and macro-nutrients, some also had phosphite (phos acid) and/or salicylate (salicylic acid). Yield has increased in all treatments each year. Juice maturity and quality factors don’t appear to be affected by HLB in trees receiving foliar nutritional sprays. Juice quality among treatments of percent juice/fruit (52.9-58.0%), acid (0.74-0.80), Brix (10.59-11.23), ratio at harvest (13.77-14.96), and pound-solids/box (5.88-6.40) have all been in the normal and acceptable range. While producing the 2012/13 crop the trees have increased production and maintained juice quality in spite of the 2012 January 4 freeze causing delayed spring growth and bloom, the drought with only 38 inches of rain (53 normal) in Immokalee in 2012, and the unprecedented fruit drop experienced industry wide was much less. We are well into the 5th year of a replicated experiment in a 12-acre commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test effects two factors: (1) micro-nutrients + systemic acquired resistance inducers, and (2) Asian citrus psyllid (ACP) chemical control on ACP populations on Can. Libericacter asiaticus (CLas) titer, and plant yield. Since our last report we applied Voliam Flexi @7oz per /ac on insecticide plots (10 Apr), and a foliar Boyd mix nutritional spray was used on 12 Apr . Psyllid populations for Insecticide treatments exceeded the 0.20 threshold only on 4 Apr. The treatment resulted in significantly (P< 0.05) fewer adult ACP than non-insecticide treatments on all dates sampled between 4 Apr and 26 June. Significantly fewer (P < 0.05) adult ACP were seen in the insecticide only and and combined nutrient + Ins treatments than the nutrient only and untreated control on dates 30 Apr; 16, 31 May; and 14 June. Significantly fewer nymphs (?) were seen during high flushing periods in insecticide only and insecticide + nutritent plots compared to nutrient only plots on 4 Apr and all treatments on 26 June. Mature trees and resets planted June 2010 were tested for HLB detection on 11 Apr. No significant differences in percent HLB between treatments were found for sampled adult trees (mean 96% - 100%) or resets (mean 79% - 86%). While there were no significant differences in Ct values for reset trees (Mean range 27.73 ' 29.40), mature trees with Ins+Nutr treatments had significantly lower Ct values (24.08) than Insecticide only (25.24) trees.
Objectives of this project include: 1) scale up production of Tamarixia radiata to levels that will allow better assessment of the potential impact of augmentation on ACP populations and ultimately the spread of HLB, 2) use genetic techniques to identify parasitoids recovered from the field to demonstrate establishment and effectiveness of released strains, and 3) pass on rearing technology to both private and public sectors to encourage production and an adequate supply of these parasitoids into the future. This quarter 74,130, 51,710, 97,547 and 84,988 T. radiata were produced in colonies from South China, Pakistan, North Vietnam and Florida strain previously established from Taiwan and South Vietnam and maintained at DPI, Gainesville. The colony at SWFREC produced another 58,484 “Florida Strain” T. radiata. Wasps from these colonies were used for research, to maintain the colonies and to release in conventional and organic citrus groves in Collier, Lee, Hendry, Lake, Indian River, Hillsborough, St Lucie, Polk, Alachua, and Hardee counties and also sent to Commonwealth of Dominica. In June, parasitism by T. radiata was evaluated using sentinel plants with an average of 54 nymphs per plant exposed for one week in a block of 12 acre ‘Valenica’ split into 16 plots to evaluate insecticides, nutritionals, insecticides + nutritionals and untreated treatments, where about 12,000 parasitoids were released between April-June. Parasitism averaged 6% (0-69%) and 5% (0-31%) in the release and no release plots, respectively, of untreated and nutritional treatments. In the nutritional and untreated plots with releases, parasitism averaged 4% (0-33%) and 7%, (0-69%) respectively, compared to 7% (0-31%) and 2% (0-8%), respectively, in the plots without releases. Parasitism of feral nymphs collected from untreated plots and reared in the laboratory averaged 3% (n=33) and 17% (n=35) in March and April, respectively. Nymphs collected from other treatments were not parasitized except 3% (n=82) in April in nutrition + insecticide treatment. At SWFREC where some 27,000 wasps were released, parasitism averaged between 0 and 18% in June in the blocks that get treated with insecticides as needed. Psyllid populations were generally low at other conventional groves in Hendry and Lee counties and parasitism was negligible. Xulin Chen, completed her Masters with thesis on ‘Manipulation, Rearing and Storage of Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) Parasitoid of Diaphorina Citri (Hemiptera: Psyllidae). She already started her PhD and will be working on functional response of T. radiata to different host densities of psyllid nymphs, host identification by this parasitoid including marking and chemical clues. We met will DPI and USDA collaborators and organic growers to evaluate Tamarixia performance in their groves. Locations for trials have been selected at Clermont, Ft Pierce, Sebring and LaBelle. Release and sampling plans are being finalized. The objective is to increase release rates high enough to either control ACP or determine that such is not feasible. Chen, X., Rohrig, E., and Stansly, P., A. 2013. Carbon dioxide anesthesia of Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) parasitoid of Diaphorina citri (Hemiptera: Psyllidae). Florida Entomologist 96(1): 246-248
The overall objective of this research is to develop a push-pull system for the Asian citrus psyllid (ACP) that can complement integrated management systems in young citrus plantings. Towards that goal, visual factors that affect psyllid takeoff into flight and landing are being examined. In our first study, we conducted laboratory studies on choice of ACP to different colored lights using high-intensity LED lights. Initially a comparison was made between time intervals of the day to determine if psyllids were more visually responsive during a particular time of day. There was a clear and consistent pattern of greatest responses obtained in early afternoon with lower responses in the morning, late afternoon and evening. A comparison of different colored LEDs of equal light intensity indicated that greatest attraction response was to yellow and green LEDs with no differences in response between males and females. Data collected in an additional study examining attraction responses to a range of 5 different equal light-intensity ultraviolet-emitting LEDs is currently being analyzed. In these studies ACP were classified by age, gender and abdominal color (blue/green, brown/gray, yellow/brown) to determine if responses to colored stimuli (LEDs) differed between different components of the psyllid population. One of our sub-objectives was to conduct a spectral analysis of flush tissue as a basis for optimizing visual attractants for ACP. This has been initiated with data collection currently underway. Preliminary results on the spectral reflectance (280-800 nm) from leaves indicate clear differences between flush tissue and older leaves for both Citrus sinensis (Ridge pineapple sweet) and Murraya paniculata maintained under optimal fertilization and watering schedules and long daylength photoperiod. This will be continued to include other citrus varieties and to compare leaves collected from groves with BLB but under different nutritional regimes.
As reported in the previous update, it was observed that after being held on plants expressing Peptides A, B or C, Asian citrus pysllids harboring Candidatus Liberibacter asiaticus (CLas) at rates between 20-80% appeared to be ‘cleared’ of the bacteria 15 days post-exposure and that F1 progeny were devoid of CLas. To corroborate this finding and to investigate it further, additional plants, control and experimental, were graft-inoculated. Approximately 50% of plants for controls tested positive in ELISA for CTV expression and those expressing peptides are currently being tested. Once full sets of plants are available, experiments will commence. We have concluded feeding inhibition assays previously reported and are currently conducting psyllid development and longevity assays to measure the response of psyllids to peptides A, B, C and D. Although all four peptides appeared to reduce psyllid feeding, peptide D was associated with the greatest significant reduction in psyllid feeding. Quantitative PCR is the method currently used to determine infection rates of CLas within the psyllid and host plant. While qPCR is the most sensitive method to detect CLas, it does have a finite limit at which it can detect any template. The report of false-negatives has wide-sweeping ramifications on the interpretation of the efficiency of D. citri as a vector of CLas, such as in the experiment described above. We optimized the reaction by using corrected forward and reverse primers, invoked the use of a standard curve to determine the dynamic range of the reaction itself, and employed a nested-PCR approach to pre-amplify potential CLas 16S template and used this reaction as a template in qPCR. This last step enabled us to find CLas in samples in which the initial titer was below the dynamic range of the qPCR reaction. We have validated that there are false-negatives being obtained by the old procedure, and we are currently performing experiments to estimate the error rate and to finalize the method to be employed in future experiments.
Our objective has been to determine how Asian Citrus Psyllid (ACP) behavior is affected by Las-infection in response to healthy versus diseased citrus trees. In previous experiments, we have determined that ACP adults initially settle on Las-infected plants as compared with nearby uninfected counterparts. We hypothesized that while the Las-infected plants are initially attractive to ACP, after prolonged feeding, the ACP experiences imbalanced nutrition and choose to seek a better host. In fact, we have proven that infected plants are less preferred by ACP for feeding as compared with uninfected citrus plants. Over the past year, we have conducted numerous settling bioassays to examine host selection behavior of ACP. We conducted binary choice tests between two plant types including: control vs. old infection, control vs. new infection, new infection vs. old infection, old infection without nutritional supplement vs. old infection with nutritional supplement, new infection vs. old infection with nutrient spray, and control vs. old infection with nutrient spray. All plants used in settling experiments were approximately four year old Hamlin sweet oranges. Control plants were uninfected, healthy plants in each case. Infected plants were either newly infected (<5 month since PCR detection) or old infected (>12 months since PCR detection). Nutrient-sprayed plants were old-HLB infected plants. Based on these experiments, we have found that newly infected plants are very attractive to ACP as compared with the other treatments tested. ACP prefer to settle on these plants over controls and old-infected plants. Old infected plants are not as attractive as newly infected plants when compared with controls. ACP initially settle evenly between control and old or nutrient sprayed plants but then choose to move to the infected plant over a seven day period. When ACP are given a choice between newly infected plants and nutrient sprayed plants, the nutrient sprayed plants appear to regain some of their attractiveness and ACP settle more evenly between these two choices of plants. In addition, we have conducted settling experiments between control and newly infected citrus in the presence of high amounts of methyl salicylate (MeSA) released from commercially available dispensers. This experiment was conducted because HLB infection causes citrus trees to release MeSA. Our results indicate that treatment of plants with high levels of methyl salicylate interferes with the ability of ACP to differentiate between infected and healthy citrus. Therefore, use of MeSA may be a promising method to prevent ACP from finding infected citrus trees to acquire the HLB pathogen. In the last quarter we have experiments in which ACP are pre-exposed to methyl salicylate prior to conducting settling assays as described above. Several dosages of MeSA were pipetted onto a cellulose matrix and placed into a heat resistant nylon resin bags, after which adult ACP were placed into the bags for one hour of exposure. After the hour of exposure, the ACP were released into cages containing one healthy and one young infected citrus plant. After 24 hours the number of ACP were counted on each plant. These results were compared to the numbers of untreated ACP settling on healthy versus young-infected plants. Our results indicate that the ability of ACP to choose between uninfected (healthy) versus infected host plants is impaired after pre exposure to high doses of methyl salicylate. These is further evidence that MeSA may be a tool for controlling spread of HLB by impacting the ACP’s ability to locate infected plants to acquire the HLB pathogen.
We have initiated this investigation with two laboratory experiments. The aim of the first experiment is to understand how communication between plants may affect psyllid host acceptance. Recent studies show that plants in the vicinity of herbivore-infested plants release inducted volatiles in the absence of damage. This indicates that plants communicate and that a pest-damaged plant causes its nearby undamaged neighbor to release these volatiles, which act as ‘SOS’ signals that attract natural enemies. This plant communication allows plants to ‘anticipate’ the arrival of herbivores (pests) and increase efficiency of induced plant defenses. The Las-pathogen (Candidatus Liberibacter asiaticus) induces the same response as Asian citrus psyllid (ACP) feeding. Consequently, we hypothesize that uninfected plants located nearby Las-infected plants may be ‘fooled’ and also release volatiles that attract vectors. This system may increase the spread of the pathogen that causes HLB disease. To test this hypothesis, replicates of two ‘Valencia’ plants were placed into glass domes, and sampled during three days to collect the volatiles from either a Las-infected plants versus the volatiles from uninfected citrus plants (control). Subsequently, these two plant treatments were placed in a cage for a choice test, and 50 ACP were released into each cage replicate. The number of ACP on each plant was recorded during 3 days after initiation of the experiment. Early evidence suggests that there may be plant to plant communication taking place, signaling infection by Las. The goal of the second experiment was to test the effect of drought stress on recruitment of natural enemies of ACP and the expression of Las-induced plant volatiles. ‘Swingle’ plants were submitted either to a control treatment, where water content was maintained at 90% per pot water capacity, or to a drought stress treatment where plants were deprived of water until the leaves began wilting and then were subsequently watered. Using laboratory olfactometer experiments, we tested if plants under drought stress recruit natural enemies differently as compared with non-stressed controls. We first demonstrated that well-watered plants infested with 50 ACP were more attractive to the parasitoid, Tamarixia radiata, than a well-watered plant without psyllids. This experiment confirmed that a citrus plant infested by psyllids releases volatiles that attract natural enemies. However, under drought stress, plants infested with 50 ACP were not more attractive than an uninfested drought stressed plant. This experiment demonstrated that under drought stress, herbivore-inducted volatiles are decreasing to such an extent that recruitment of natural enemies is impacted. Our next objective is to test if the attractiveness of HLB-infected plants is lowered by submitting them to a drought stress treatment. Finally, regarding the field portion of this project, we selected plots where an entire grove or grove portion has been replanted (2 plots) and where only a fraction of total trees (less than 50%) has been replanted within a setting of otherwise mature and bearing trees (2 plots). Collection of biotic and abiotic data, as well as, conducting ACP monitoring will be implemented this summer. Our objective is to determine the biotic and abiotic factors that impact grove colonization by psylids in this field study.
Our objective for this project has been to evaluate botanical compounds as repellents for Asian citrus psyllid (ACP) with the purpose of developing possible repellent formulations for use in the field. In previous quarters we reported on evaluation of five botanical oils as repellents for ACP. In particular, we have found that fir oil is an effective repellent of ACP. Based on this finding, we began working with the commercial pest management industry to begin developing dispensers for practical application in the field. Several types of dispensers and formulations were designed and so far one was developed for field testing. Specifically, in the last quarter a fir oil high-release device was formulated by Alpha Scents, Inc and we obtained a sufficient number of these for field evaluation. The test was conducted in replicated bocks of sweet orange resets in central Florida. Our goal was to conduct the trial within resets to evaluate the potential of this repellent device for young tree protection. Trees were treated with one dispenser per tree at a rate that was based on previous laboratory tests. Out intent was to treat plots with a rate that was effective in previous laboratory experiments. Identical nearby untreated plots served as controls. There were five replicates or treatment and controls. Treatments were applied to plots at random. Plots treated with the devices, as well as controls, were monitored weekly and the number of adult psyllids per tree was measured by tap counts. In addition, we counted the number of ACP nymphs and the number of active leafminer mines on flush when present. The results of this initial experiment suggest that the amount of fir oil released by these initially designed dispensers did not affect the host acceptance behavior of ACP or citrus leafminer sufficiently. Populations of both pests were not deterred sufficiently from infesting treated plants for practical effectiveness of the dispensers that were tested. In a separate test, we evaluated a commercial prototype product that incorporates several of the plant botanical compounds we evaluated previously into an oil for direct application to the crop. In this case, we did observe significant reduction of flush infestation by ACP as compared with control plots. We are pursuing development of products that incorporate these active ingredients into formulations that cover the leaf surface. Finally, we continue to work with ISCA Technologies on prototype product development. We are planning further evaluation of the various active ingredients we and others have discovered for incorporation in ISCA’s SPLAT technology. A field trial of this latest formulation is planned in Florida in the near future.
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