Yield and juice data from the fifth year (2008-2012) of the replicated Hamlin trial on station at SWFREC evaluating the Boyd cocktail ingredients shows that the trees are growing well and maintaining production. 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 fall fruit drop experienced industry wide, the 7 to 8 foot tall trees produced above 2 boxes per tree. The highest yielding treatment in each of the past 5 years has been treatment 2 (Boyd cocktail less SARs) with 2.5 boxes/tree. Not significantly different are treatments containing foliar applied micro-nutrients (Mg, Mn, Zn, Mo, B,) with a macro source of N and K as DKP + KNO3, and a Phosphite. Each year since 2008 the yield has increased in this trial. Treatments which do not contain the above foliar applied nutrients have been among the lower yielding trees showing nutrient deficiencies and considerable symptomatic leaves of HLB. Juice quality was down this year when fruit was harvested late December. Juice content, acid, and Brix were low and ratio was good. The 30-acre Valencia trial in a commercial grove will be harvested in April. 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 Delegate (5oz/ac + 1% Oil) and Danitol (16oz/ac + 1% v/v 435 Oil) on insecticide plots (8 Nov, 5 Dec, respectively). Psyllid populations for Insecticide treatments exceeded the 0.20 threshold on 3 occasions (1, 15, 30 Nov), which included two dates following the Delegate application. Insecticide treatments resulted in significantly (P< 0.05) fewer adult ACP than non-insecticide treatments on all dates sampled. All treatments resulted in significantly (P < 0.05) fewer adult ACP than the untreated control on dates 15, 30 Nov. Only in insecticide treated plots did we see significantly fewer ACP than the control on 18 Oct, 1 Nov, and 14, 26 Dec. Mature trees and resets planted (June 2010) were tested for HLB detection (24 July). While there was no significant (P < 0.05) treatment effect on percentage HLB for mature trees, Insecticide-only treated resets had significantly less (P < 0.05) HLB (0.94'0.04 SEM) than the other treatments. There were no significant differences in Ct values for mature (Mean range 22.59 ' 24.65) and reset (Mean range 29.20 ' 31.97) trees. October HLB results are still pending, and another collection date is scheduled for next week. During the past 3 months phloem studies were done on nutrient treated HLB trees at both the Southwest Research and Education Center and at the Orange Hammock in Felda, Florida. Studies included both cytology work as well as fluorescent dye translocation studies to determine if new phloem is being produced in those trees treated with additional nutrient materials. Visually the fluorescent dye translocation studies again verified that phloem uptake was better in the trees that received the supplemental nutrient materials. That is that the fluorescent dye was not translocated from the application zone with untreated leaves but was in the treated leaves. Microtiter plate assays again were used to quantify the amount of dye retained (or translocated) in the sites. Assays did not agree with the visual assessments during this time period due to sampling procedures and seasonal variability. The procedures have now been revised.
During fall 2012, we monitored 4 trials where pheromone was deployed to suppress citrus leafminer in collaboration with ISCA and grove managers to address pheromone carrier, timing, deployment pattern, and efficacy of mating disruption on leaf damage caused by citrus leafminer. 1. Winter and spring application of pheromone (2013). Cooperator: Packers of Indian River. Winter and spring pheromone applications in a factorial design with split plots in 4 blocks will be treated with rubber dispensers loaded with triene (834 mg/ha) in winter (February) and spring (April). Main plots (6.4 ha) and subplots (3.2 ha) will be 35% larger than last year. Trap catch and leaf mining will be evaluated. 2. Trials 1-2: Solid dispensers, Golden River Fruit Co., St. Lucie Co. We monitored traps in 0.14 ha plots treated with rubber dispensers (318 per ha) loaded with ‘natural’ 3:1 blend or triene only. Trap catch disruption was >95% for 18 wks. There were no differences in CLM infestation suggesting plots were too small for flush evaluation. A 68 ha plot was treated with rubber dispensers loaded with natural blend on 24 Aug. Trap catch disruption was >95% for 18 wks. There were no differences in infestation at 8 wks suggesting that female moths arrived in treated plots already mated, or moths mated in treated plot despite strong trap catch disruption. 3. Trial 3: Large plot rubber dispensers, Packers of Indian River, St. Lucie Co. An experiment was started in Sept. to compare disruption in 0.87 ha plots treated with rubber dispensers containing ‘natural’ 3:1 blend (330 dispensers/ha) or untreated. Plots were 4 times longer than wide. Trap catch disruption was >98% for 15 weeks. As above, there were no differences in CLM infestation. 4. Trial 4: Large plot rubber dispensers, TRB Groves, Charlotte Co. We continued monitoring a grower validation trial treated on 20-23 August (42 ha) with solid rubber dispensers loaded with pheromone (natural blend). Trap catch disruption was >99% for 11 weeks. 5. Manuscript near completion. We are near completion of a manuscript: ‘Attraction of a Native Florida Leafminer, Phyllocnistis insignis (Lepidoptera: Gracillariidae), to Pheromone of an Invasive Citrus Leafminer, P. citrella: Evidence for Mating Disruption of a Native Nontarget Species.’ The manuscript shows that a native leafminer is influenced in groves treated with citrus leafminer pheromone which in turn could influence populations of citrus leafminer if these two species share common natural enemies.
The 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 with Delta pheromone traps under 4 management scenarios: 1) sprayed at peak trap capture using different trap densities, 2) sprayed based an ACP threshold, (3) biweekly low volume (LV) sprays with horticultural spray oil (HMO) and, 4) untreated control. 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 a replicated trial in a 10 acre block of mature ‘Valencia’, significantly less leafminer damage was seen in Oct with LV-HMO (HB rating 2.25’0.13) than the grower standard (GS, 2.60’0.11) or UTC (2.60’0.14). In addition, the proportion of fruit with canker was significantly less in oil-treated plots (0.02’0.01) than the GS plots (0.04’0.01) demonstrating that oil was managing CLM and canker as well as GS. In another replicated trial evaluating results of threshold based insecticidal control of ACP, (P<0.05) fewer CLM were seen in the Oct 2012 flush on trees treated with insecticide compared to trees treated only with foliar nutrition. Untreated trees also had significantly less incidence for canker than the other treatments which corresponded to less flush. In a replicated trial on canker-susceptible 'Earlygold' orange, lower (P < 0.05) canker ratings (0.94'0.02 on a 1-8 scale) were seen in plots sprayed at a 0.2 ACP/tap threshold than the 0.7 threshold (1.08'0.10). In another experiment traps were placed in grapefruit at 1, 3 or 5/ac. More captures at lower densities would indicate greater effective range. We did find more moths (P < 0.05) in traps placed at 5ac compared to 3ac, although during Oct (11, 18, 25), there were more captures at 1 trap/ac than at 5 traps/ac indicating season variation in the effective range of the traps. Pheromone traps have also been deployed in citrus groves and non-citrus areas determine optimal spatial scale for trap distribution and to see if non-target species are captured. Collections of moths (755) and host plants (11) were made at 3 known distances from citrus groves in a State Forest. Moths and photos of mined host plants (2011, 2012) were sent to the Kawahara lab in Gainesville for identification. The CLM phenology and temperature data is currently being analyzed to determine degree days for growing season peak flights and generation time to develop the degree day model. Objective 2: Trees sprayed at peak trap capture had significantly lower incidence (P < 0.05) of canker, CLM damage was less compared with trees sprayed with Delegate earlier (1.14'0.05) or later (1.14'0.04). Objectives 3 and 4: CLM colonies are being rebuilt after a series of crashes in preparation for the baseline susceptibility studies to begin (Feb) followed by field testing of a diagnostic dose.
December 2012 Update: Activities of the CHMA program assistant (Brandon Page) Oct-Dec 2012: During this reporting period, the CHMA assistant provided support to the CHMA effort by attending various local CHMA meetings as well as statewide meetings. Specifically, Mr. Page attended nine (6) local meetings of growers or CHMAs. The locations or CHMA meetings during this reporting period inlcuded: Ft. Meade/Alturas CHMA, Hardee CHMA (2 separate meetings), NE Desoto CHMA, CREC Field day, and Hillbillies. Mr. Page also attended and participated in 2 regional meetings by representing the CHMA program at SunBelt Ag Expo (Moultrie GA) and the FL Ag Show (Balm). In addition to attending meetings, daily updates were made to the CHMA website based on communication with CHMA grower leaders. Work also continued on the mapping program that will generate TRS maps of psyllid counts for each CHMA. The program was completed in late December and after some additional testing will be made available online through the CHMA website in early 2013.
December 2012 update: In the multi-year field study designed to determine the effects of different pesticide management strategies on the ability to bring young trees into production, we previously reported that during the first 12 months after planting, the HLB infection rate in the majority of plots remained low. By treatment (season-long program), the HLB infection rates (from least to greatest) were as follows: rotation of soil-applied neonics + foliar insecticides = 0% HLB, rotation of soil-applied neonics + foliar applications of kaolin = 0% HLB, foliar applications of Kaolin only = 1.3% HLB, foliar insecticide applications only = 2.5% HLB, untreated control plots = 3.8% HLB, and soil-applied neonicotinoid applications only = 11.3%. The increased rate of HLB in the neonic only plots corresponded with a failure to applications to control psyllid populations. Resistance assays conducted with psyllids collected from these plots yielded a resistance ratio to neonicotinoids as high as RR=126. Coincident with the finding of localized resistance in these plots, the psyllid populations increased across the majority of plots and ultimately were controlled below detectable levels with the application of a broad-spectrum pyrethroid insecticide. Since that time, no additional product failures have been observed indicating that the resistant psyllid population had been successfully eliminated. However, the 2 month window of failed control across the entire block ultimately lead to a dramatic increase in HLB incidence across all treatments as later determined by PCR analysis of leaf samples collected from each tree during the 15 and 18 month HLB evaluations. By the 18 month HLB evaluations, the HLB infection rates (from least to greatest) were as follows: Kaolin only = 31% HLB, rotation of soil-applied neonics + foliar applications of kaolin = 35% HLB, soil-applied neonicotinoid applications only = 40% HLB, rotation of soil-applied neonics + foliar insecticides = 43% HLB, untreated control plots = 52% HLB,and foliar insecticide applications only = 53% HLB. While these results may appear discouraging, they demonstrate that if proper product rotation is followed and thus resistance is prevented, HLB infection rates in young trees can be maintained at low levels. A spatial analysis of HLB infection in relation to proximity to the initial plots where resistance development was documented has shown a significant trend in HLB infection rate by treatment applied. With the resistant psyllid population eliminated, we will continue to follow disease incidence in this grove to determine how much additional disease spread occurs, particularly with respect to those trees not yet infected.
The goal of this project is to determine the overwintering habits of Asian citrus psyllid (ACP), including determining alternative hosts, so as to understand how to improve dormant season control strategies for ACP. The dormant season is the ‘weak link’ in the seasonal phenology of ACP and thus the time when populations of psyllids can be affected most, when targeted appropriately. We have completed a detailed survey of ACP presence in Lake Kissimmee State Park (Polk Country, FL), where we captured ACP consistently throughout a season. In this forest, occurrence of citrus is particularly rare (only three tangerine trees), and no Rutaceae were found in the trapping locations or at nearby (‘500 m) areas to where ACP were captured. In addition to trap capture data, complementary laboratory no-choice feeding bioassays for ACP performed using Red bay, gallberry, and blueberry. These were selected as possible alternative hosts, given their presence in locations where ACP were captured. The results suggest that ACP may have a wider alternative host feeding range than previously thought, which may allow for significant dispersal even through dense forests in Florida up to 2.3 km from large-area plantings of citrus. For our next set of experiments, we have acquired a new, industrial-strength vacuum system for collecting insects in the field. During the next two months, we will sample two locations comprised of commercial citrus. Each will be divided into two comparison plots: one oriented north-south and the other east-west. We will sample the citrus canopy at three different heights and orientations at the four cardinals directions. In addition, we will employ a truck-mounted platform to sample the very tops of the canopies of citrus trees. Our objective is describe the two dimensional distribution of ACP within commercial grown citrus trees during winter. Hobo data loggers will be placed at these various locations to correlate temperature and relative humidity with ACP distribution. Another aspect of this project is to determine the population density (fluctuations) of ACP seasonally by monthly sampling of 10 citrus groves with varying management input (conventional, organic and abandoned) in order to understand the overwintering preference of ACP. Our results to date indicate that there is significant variability in the number of ACP occurring within these three different management scenarios. Our currently ongoing objective is to correlate these distribution differences among the various treatments with specific biotic and abiotic factors such as: citrus leaf nutrient analysis, descriptive habitat analysis (plant and insect biodiversity indexes), and ACP fitness analysis (survival and fecundity studies). These additional data should help us understand why differing population densities of ACP observed in the treatments we are investigating do not always correlate well with ACP management practices.
Previously, we have reported efficacy results for two insecticidal peptides expressed by the CTV vector used in bioassays with ACP and the brown citrus aphid (BCA). During those experiments, we observed significant variation in psyllid responses among plants expressing the same peptide. We expect that this variation may be due to differential expression of the peptide genes among the transformed plants; therefore,we are now developing peptide-specific qPCR assays to quantify peptide gene expression between and within plants. Once qPCR screening is complete, psyllid survival will be assessed in response to plant peptide titer. Since our last report, two new peptides have been identified in artificial feeding assays as effective in reducing the fitness of Asian citrus psyllids (ACP) by reducing insect survival and fecundity. These peptides have been introduced into the previously described CTV vector and introduced into citrus. During the past quarter, the majority of plants graft inoculated with these peptides, 85 and 100% respectively, have been identified as positive for the peptide-vector constructs during screening by enzyme-linked immunosorbant assays (ELISA). Following the completion of qPCR assays for these peptides, bioassays will be completed to assess the survival, fecundity, and development of ACP and BCA, and settling preference and feeding behavior of ACP.
Since our precious report, we have completed the hiring of personnel to conduct research on this grant, including two post-doctoral associates. One objective of this project is to determine points of the Asian citrus psyllid (ACP) life cycle that may be susceptible to control programs. Previous research has highlighted that environmental perturbation such as a drop in temperature, may affect psyllid ability to transmit Candidatus Liberibacter asiaticus (Las). Work has begun to assess ACP tolerance to cold shock and how periods of cold shock may effect ACP acquisition of Las. Psyllids from laboratory colonies are subjected to 0 ‘ 1’C, 5 ‘ 1’C, 10 ‘ 1’C, 27 ‘ 1’C, 30 ‘ 1’C, 34 ‘ 1’C periods for 1, 2 or 4 weeks in controlled growth chambers. All chambers are maintained at 50 ‘ 5% RH and 14:10 (L:D) photoperiod. Psyllids are then exposed to Las infected sweet orange for 2 or 4 weeks and later tested for acquisition of Las. In further experiments we will assess the onward vector capacity of these psyllids in inoculation experiments. These data will yield clues as the effects of perturbations in temperature on ACP and their vector capacity. In addition, we are assessing the effect of Wolbachia endosymbionts on ACP, and ACP acquisition and inoculation of Las. We have begun by collecting psyllids from 10 sites in Florida between Lake county and Miami Dade county, assessing both the abundance of ACPs and the rate of Wolbachia infection within these 10 populations. During investigations we have observed low abundance of ACP in 6 of 10 sites, therefore we have concentrated on two heavily populated sites from which to raise lab colonies for further experiments; one grove in Polk County and one in Miami Dade County. Previously we have found 70-80% Wolbachia infection these counties respectively. Using single pair crossing and qRT-PCR analysis of F1, F2 and F3 generations we are currently generating one Wolbachia positive and negative colony from each county. Once fully established we will use these colonies, together with existing colonies micro-injected with non-native Wolbachia, in bioassays to assess the effect of Wolbachia on ACP fitness. Experiments will include investigations of longevity, fecundity, development and viability of nymphs, and interactions between Wolbachia and Las within ACPs. These data will yield important clues as to a potential role of Wolbachia in controlling ACP populations and Las dispersal.
The questions to be addressed by this research project are 1) how does Imidicloprid move in the sandy soils of south Florida, 2) how long does imidicloprid persist in sandy soils, and 3) how much Imidicloprid leaches below the root zone of citrus trees. Isotherms relating soil Imidicloprid concentrations in soil solutions with Imidicloprid concentrations in the solid phase of sandy soil were conducted using Immokalee fine sand. Imidicloprid was found to penetrate to a depth of 45 cm and have greatest affinity in the 0-15 cm soil depth due to higher soil organic matter at that depth. These data were supported by soil partition coefficients (Kd) of 1.68 mg of Imidicloprid. These data suggest that Imidicloprid is leached rapidly from the soil if not taken up by the target plant. Data from two field studies in the spring and summer of 2012 indicated that Imidicloprid concentrations decreased by 90% in 10 days at the 0-15 cm depth by tree uptake and recommended microsprinkler irrigation. Initial analysis of soil samples from the spring study indicate that Imidicloprid concentrations decreased rapidly in the soil and was either taken up by the plants or leached within 5 to 10 days at recommended microsprinkler irrigation rates, however reduced irrigation following applications allowed for greater soil residual time and uptake. During these initial application studies, adult psyllid populations per tree decreased from approximately Imidicloprid concentrations reached the 15-30 cm depth in 5 days after application and persisted at that depth for approximately 15 days. During this initial application study, adult psyllid populations per tree decreased from approximately 1.7 for both treated and non-treated trees to 0.1 psyllids per tree for the treated trees at 22 days after application. Psyllid populations on treated trees were below 0.1 psyllids per tree from 22 to 50 days after application. These data would indicate great persistence of the insecticide in citrus tissue and is supported by elevated leaf tissue concentrations.
Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops (such as Bergera koenigii) to attract and kill. Objective 1. A bioassay was developed at SWFREC using a wind tunnel to determine ACP response to wind speed and direction. ACP are marked with fluorescent powders for easy observation and released into the wind tunnel. Preliminary results indicate slow movement downwind in preference to upwind in the absence of a host plant. In addition, two launch surfaces are being tested (glass petri dish and paper lined dish) to determine the best release method but results are still inconclusive. Objective 2. Lab trials at CREC indicate females avoid high densities of conspecific females, irrespective of whether direct contact is made with psyllid-infested plants. An olfactometer was used in the absence of light to demonstrate that the avoidance behavior was probably elicited volatile cues. A volatile collection system is being developed to identify this potential behavior modifying odorant. They are also in the process of establishing experiments to determine whether female egg laying or feeding may induce a systemic change in volatile release from plants that may affect subsequent settling and oviposition behavior of female and male ACP. Also in progress are bioassays to investigate a possible repellent effect of high ACP nymphal densities on ACP female movement and behavior. Preliminary results suggest little or no affect on ACP settling or movement; however, it appears that oviposition by female ACP is deterred on plants that are infested with high densities of nymphs as compared with non-infested controls. Flight mill trials to determine flight capability of ACP indicate that barometric pressure and season may be affecting ACP movement behavior with less vigorous flight being observed in winter compared to spring and summer. Objective 3. Monitoring ACP movement with sticky traps continues in a 15 acre abandoned Murcott grove in preparation for another attempt at mark-recaputre using fluorescent markers although no marked ACP were recovered during a spring trial. Objective 4. Yellow sticky traps set at three canopy heights are giving mixed results, with more ACP captures in the upper canopy of an old reset grove of ‘Valencia’ orange compared to a more even distribution at younger and more uniform grove of ‘Earlygold’. More ACP are captured at both locations on the east side of the trees than the west side, possibly indicating movement toward morning light. At both locations and a third along the Caloosahatchee river, ACP captures are greatest adjacent to a hammock or wind break indicating that these features may serve to arrest movement across the block. In ‘Earlygold’ grove, two sided traps set between insecticide sprayed and unsprayed blocks with low and high populations of ACP to evaluate the direction of movement do not indicate any predominance. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting was submitted to and is now under revision for the refereed journal Pest Management Science. Subsequent results indicate that the 5 ft strip of metallized mulch is being shaded by the canopy and no longer effective at repelling ACP on trees more than 5 ft tall. Trials to test different ways of utilizing UV reflectance to repel ACP are being planned.
This project began 11/1/2012 and our efforts are now ongoing. We have colonies of both psyllids (Bactericerca cockerelli and Diaphorina citri) to be used in our work and are now beginning efforts to test in planta RNAi effects and discover the optimal construct development.
Dead/preserved Diaphorina citri psyllids samples collected from Florida, China, Brazil, Taiwan and Pakistan have been received from collaborators. We have performed small RNA sequencing on four separate psyllid samples so far and examined these by small RNA, next generation sequencing to find known and unknown viruses capable of infecting D. citri. We are still in the process of bioinformatic analysis by using local scripts and commercial software on our small RNA datasets. Our analyses are far along for the Taiwan D. citri RNA sample, and less so for the others. We obtained 13 million reads for the Taiwan D. citri which decreased to 11 million after adapter and sequence error removal yielding small RNAs with an average length of 21.6 nucleotides. De novo assembly was then used to create larger contigs from these small RNAs, yielding contigs with an average size of 274 nucleotides. We performed search and align Genbank queries using BLAST and have identified putative viral sequences. So far we have confirmed the identity of different genomic segments of the Diaphorina citri reovirus from the Taiwan sample. Our contigs shared a sequence identity >95% with significant E-values with this reference virus. In order to confirm our sequenced-based discovery we designed specific primer pairs based on our in silico sequences and performed RT-PCR for all of the D. citri RNA samples. RT-PCR confirmed the presence of Diaphorina citri reovirus in the Taiwan, China and Florida RNA samples, but not in those from Pakistan or Brazil. We should mention here that we have found signature sequences for other viruses in the Taiwan sample but initial E-values were not significant. We are in the process of performing more computational analysis on these and the remaining samples. These data so far validate our approach for virus discovery and give confidence for our longer term objectives for using viruses as tools for D. citri management.
The purpose of this investigation is to identify/confirm new non-neurotoxic compounds that could be useful for management of Asian citrus psyllid (ACP). Using novaluron, a chitin biosynthesis inhibitor, we are developing a high throughput assay system to more efficiently screen novel non-neurotoxic compounds for efficacy against the ACP. Novaluron is used on food crops including apples, sweet potatoes, and brassicas and has been shown to be effective against several hemipteran species including sweet potato whitefly (Bemisia tabaci), greenhouse whitefly (Trialeurodes vaporariorum), pear Psylla (Psylla piri). It is an insect growth regulator (IGR) and is effective on growing stages of the insect when molting takes place. Because it is a non-neurotoxic insecticide that targets the chitin biosynthetic pathway, a pathway specific to arthropods and fungi, it is potentially a well suited insecticide to use in a rotation scheme to reduce the use of broader spectrum, non-specific compounds in the management against insecticide resistance. Currently, novaluron is being evaluated for its efficacy against ACP egg hatch, and nymph and adult emergence. We are developing high-throughput bioassays to evaluate these parameters that can then be utilized to test other non-neurotoxic compounds, specifically other IGRs such as tebufenozie and methoprene. For testing other non-neurotoxic compounds that are most effective if ingested by the adult stage of ACP, we are developing and refining an artificial feeding assay that will enable us to introduce the compounds to the insect, including amino acids, which are known to have a range of biological effects on insects such as disruption of flight metabolics and egg production. Also, we are testing lectins, which have been shown to disrupt insect gut function. Because there is a range of sub-lethal effects expected from these compounds, appropriate bioassays to detect biological consequence of the test compounds are being developed. For example, the ingestion of L-canavanine by female insects is expected to have an effect on egg production; hypothetically, resulting in fewer eggs laid or eggs laid that are less viable. The effects in this case will be determined by dissecting the ovary of treated insects and eggs counted in addition to determining viability of laid eggs.
The objective of this investigation is to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology. We have initiated this study with complementary field and laboratory experiments. Regarding the field experiment, we are still following the Keyplex’ program. The most recent treatment was applied on October 23rd. The next application (pre-bloom application) will be performed near the end of February. Starting this month we will also monitor the number of psyllids within each replicated treatment block with standard sticky traps and tap sampling. Complementary greenhouse experiments have been established and are in progress. We initiated choice test experiments with plant clippings placed in water and placed into custom-designed arenas (50 x 32 x16 cm; conditions within arenas: 23’C, 48% RH, 14:10 LD). Twenty ACP are placed within each arena and allowed to settle for 24 hours. So far, three two-choice combinations have been tested (HLB-infected versus uninfected plants, uninfected and nutrient-supplemented plants versus uninfected and non-supplemented control, and HLB-infected and nutrient-supplemented versus HLB-infected, but not supplemented control). The preliminary results indicate that ACP prefer to settle on nutritionally supplemented citrus particularly, the within a few days following the spray application. This trend seems to be more pronounced on HLB-infected than on uninfected citrus. We are also performing an experiment to determine the effect of nutrient treatment on ACP feeding. To determine whether nutritional treatments influence ACP feeding, leaf discs (60mm) are cut from the leaves of nutrient-supplemented or non-supplemented control plants and individually placed on 1.5% agar beds in 60 mm disposable petri dishes. The petri dish lids are lined with 60 mm filter paper discs, after which 10 adult ACP of mixed age and gender are released into dishes. Sealed petri dished are then inverted to collect honeydew droplets on the filter paper for 24 h. Honeydew production by ACP is an indirect measure of the amount of feeding on plants (i.e. production of honeydew is positively correlated with amount of feeding). HLB-infected and uninfected plants are tested separately. At the end of the experiment, filter papers are collected and treated with ninhydrin to stain honeydew droplets for subsequent quantification of the results. Nutrient supplemented and control (unaltered) treatments will be replicated 20 times. After 10 replications, it appears that there is more feeding taking place on HLB-infected and nutrient supplemented citrus compared with HLB-infected, nut non-supplemented controls. However, feeding is the same on uninfected plants, whether or not they were supplemented with the nutritional spray.
Our main objective for this project is to evaluate botanical compounds (plant volatiles and essential oils) as possible repellents of Asian citrus psyllid (ACP). The goal is to identify “off the shelf” treatments that are inexpensive and effective for field use. In the previous quarter, we reported on settling and field experiments with botanical oils as repellents or attractants for ACP. In this quarter, we have continued this research. We have continued to collect and analyze data from: (1) olfactometer bioassays indicating that fir oil is repellent and clove and camphor oils are attractive to ACP, (2) laboratory settling experiments with fir oil treated plants, and (3) field trapping experiments using baited yellow sticky cards. We are currently designing experiments to test fir oil as a repellent in field experiments. Based on our results to date, we are hypothesizing that fir oil may be best used to protect resets or newly planted groves that have a small canopy. Therefore, we are designing experiments to look at repellency in both of these scenarios. Furthermore, we will revisit the use of clove and camphor oils in various release devices to determine if release rate can alter the efficacy of these treatments as attractant lures. In conjunction with the field experiments outlined above, we will be conducting olfactometer assays to evaluate the repellency of fir oil to Tamarixia radiata, the main ecto-parasitoid of ACP. Next we will conduct parasitization assays in a caged arena with fir oil treated and untreated plants infested with ACP nymphs.
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