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. Once spring flush has reached full maturity (May), leafminer damage and canker assessments will resume 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. Preliminary results of species (5) attracted to the CLM lure were submitted (Feb 2013) for publication in Florida Entomologist. Moths captured in recent years were preliminarily identified to morph by examination of genitalia (Kawahara lab). In the OK Slough (2011), at 1 mi from citrus, preliminary morph separation showed 20% (n=5) of the species were identified as Morph A, 60% B, and 20% F; at 2 mi 35% (n=38) were A, 1% B, 61% C, 2% D; at 3 mi from citrus 48% (n=33) were A, 8% B, 42% C, and 2% F/E, which were large, accidental species; at 4mi 55% (n=15) were A, 5% B, and 40% C. For 2012, 100% at 1 mi (n=5) were A; at 2 mi (n=69) 96% were A and 4% C; at 3mi, 86% (n=135) were A, 13%C, and 1% D; at 4mi, 74% (n=27) were A, 7% B, 11% C, and 7% D. P. insignis was not captured in any commercial grove. For Grove 2 (67% A, 33% C) and Grove 3 (83% A, 17% C), Species A has been confirmed as P. citrella, Species B as P. insignis, and C could be 1 of 4 species, P. citrella, P. vitegenilla, or 2 new species. Likewise, Species D and E are unknown at this time and wait for dissection of genitalia and PCR processing. First peak flight this year occurred 14 Feb (LV Trial), 27 Feb (untreated), 12 Feb (Grove 3 treated), and Bucket traps will be dispensed at 1 ac densities in groves (May) to determine moth densities. Phenology (2011, 2012) from 5 groves [Groves 1 ‘ 3 plus Grove 4 (described in Obj 2) and Grove 5 (Unmanaged)] and temperature data is being incorporated to develop the degree day model and calculator for publication on our website (Estimate June launch). In Grove 2, Danitol (16oz/ac) was applied on 14 Jan, and HMO was sprayed at 2gal/ac on 19 Mar and 4 Apr. Obj 2: A 130-ac experimental block (Grove 4) was divided into 3 replicated treatments: 1) Delegate applied early, 2) Delegate applied late, 3) Sprays following peak CLM flight, and 4) Rotations of OPs and pyrethroids. The block was hedged 20 Feb. Mustang (4.3oz) was applied (24 Jan); Admire Pro (10oz/ac) and Imidan (1lb/oz) was applied on all plots 28 Feb. Peak-flight plots were sprayed on 13 Mar (Micromite 80WGS at 6.25oz/ac). Peak flights have occurred 15 Jan, 25 Feb, and 1 Apr 2013.
Effect of temperature: Environmental perturbations such as changes in temperature are known to affect ACP reproduction and survival. However, such changes also potentially impact on ACP’s vector competence of Las. Experiments are underway to assess ACP tolerance to temperature changes and how such changes effect ACP acquisition of Las. Psyllids from laboratory colonies are subjected to 5 ‘ 1’C, 10 ‘ 1’C, 15 ‘ 1’C, 22 ‘ 1’C, 27 ‘ 1’C, 30 ‘ 1’C, 34 ‘ 1’C, 38 ‘ 1’C periods for 1, 2, 3 or 4 days in controlled growth chambers. All chambers are maintained at 50 ‘ 5% RH and 14:10 (L:D) photoperiod. ACPs are exposed to Las infected sweet orange for 2 or 4 weeks, either before, during or after the change in temperatures. Acquisition of Las by ACPs is assessed by qPCR of dissected ACPs to quantify and localize the Las accumulation within the insect. In addition, ACPs subsequent vector capacity is assessed in inoculation experiments. To elucidate the role of temperature on the vector ‘ pathogen ‘ environmental interaction on gene expression, ACP heat shock protein expression during experiments will be quantified by qRT-PCR. Currently, we have identified several psyllid heat shock protein analogues and are developing assays to determine shifts in gene expression in response to variable temperature exposure. Wolbachia infected ACP colonies: The purpose of this project is to develop Asian citrus psyllids that are non-competent vectors of Candidatus Liberibacter asiaticus (Las) through manipulation of their facultative endosymbiont, Wolbachia. The first step in this process is to investigate the distribution of Wolbachia infection in Florida ACP populations, and to characterize the strains of the endosymbiont is present. Since the inception of this project, we have sampled ACP from approximately 20 sites throughout Florida. Analysis of these samples indicates that Wolbachia is present in all psyllid populations, with infection rates ranging from 86-96%. Molecular characterization of Florida Wolbachia strains in ongoing. To investigate the effect of Wolbachia on Las vector competence and psyllid life history, we are establishing Wolbachia-infected and Wolbachia-free insect cultures derived from single field-collected psyllids. Due to the high rate of Wolbachia infection in natural psyllid populations, only two of the isofemale psyllid lines attempted have been confirmed to be free of Wolbachia endosymbiont infection. Thus, to facilitate future bioassays, we are also establishing Wolbachia -free insect cultures using tetracycline to clear psyllids of native Wolbachia. Use of this antibiotic has proven effective in clearing a wide range of insects of Wolbachia infection. Previously, we have successfully eliminated bacteria from psyllids by direct microinjection of tetracycline into insects. For the current project, psyllid adults are reared on an artificial diet consisting of a 20% sucrose solution containing tetracycline (20 mg/100 g distilled water) using a membrane feeding system. Currently, we are confirming the Wolbachia -free status of infected psyllid lines. We expect to have isofemale lines fully established by 05/2013. Once established, cultures will be used in bioassays to evaluate the effects of differential Wolbachia strains and co-infections on D. citri fitness and Las transmission, and immune gene expression. In addition, to further understand the bacterial molecular manipulations of their insect host, we have begun investigating the effect of Wolbachia on the expression of host reproductive genes. We have identified two vitellogenin homologs and targeted six male accessory gland proteins homologs for analysis pending acquisition of sufficient psyllid RNA from Wolbachia+/- laboratory cultures.
The fifth year harvest this spring 2013 (2012-13 crop-year) of the replicated Valencia trial in a commercial grove in southwest Florida evaluating the Boyd cocktail was done in March. The yield and juice data shows the trees 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 most treatments each year. Juice quality among treatments of percent juice/fruit (52.9-58.0%), acid (0.74-0.80), Brix (10.59-11.23), ratio (13.77-14.96), and lb. solids/box (5.88-6.40) were all in the acceptable range. Juice quality factors test doesn’t appear to be affected by HLB. While producing the 2012 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, and the fall fruit drop experienced industry wide was much less. We have completed the 5th and last year of a replicated experiment in a 12-acre commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test the effects of 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 Movento (16oz/ac + 2% v/v 435 Oil) on insecticide plots (24 Jan). Psyllid populations for Insecticide treatments have not exceeded the 0.20 threshold (28 Jan ‘ 21 Mar). Insecticide treatments resulted in significantly (P< 0.05) fewer adult ACP than non-insecticide treatments on 7, 23 Feb and 3, 21 Mar. Insecticide and Nutrient+Insecticide treatments had significantly (P < 0.05) fewer adult ACP than the untreated control on dates 28 Jan, 7, 23 Feb, and 21 Mar, with all treatments less than untreated (P< 0.05) 8 Mar. Mature trees and resets planted (June 2010) were tested for HLB detection (19 Oct). All sampled adult trees were 100% infected regardless of treatment, and reset trees had no significant difference between treatments (Mean HLB % 75 ' 85). There were no significant differences in Ct values for mature (Mean range 24.19 ' 24.99) and reset (Mean range 25.17 ' 27.47) trees. 2013 Harvest (3 Mar) showed greatest yield in Ins plots (Ins+Nutr 70.5 kg/tree, Insecticide alone 63.0 kg/tree, Nutrition alone 57.9 kg/tree and Untreated 45.5 kg/tree). No significant treatment effects were found for acid (0.73 ' 0.80), brix (10.26 ' 10.86), ratio of acid to brix (2.59 ' 2.67), or lb solids per box (5.67 ' 6.18). During the past 3 months additional phloem studies were done on nutrient treated HLB trees at both the Southwest Research and Education Center and at the Orange Hammock grove in Felda, Florida. Cytology studies as well as fluorescent dye translocation studies indicate new phloem is being produced in those trees treated with additional nutrient materials. Visually the fluorescent dye translocation studies continue to verify that phloem uptake is better in the trees that received the supplemental nutrient materials. The fluorescent dye was not translocated from the application zone in untreated leaves but was in treated leaves. Microtiter plate assays used to quantify the amount of dye retained (or translocated) appear to be influenced by the seasonal variability in the plant growth activity.
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 Lee and Collier 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. Additionally, four replicated studies conducted at SWFREC included 39 foliar treatments of recommended and experimental insecticides evaluated against ACP, citrus leafminer and natural enemies were submitted for publication in the Journal of Arthropod Management Tests of the Entomological Society of America. The calendar treatment in each of the two blocks in Hendry county, ‘Early Gold’ or ‘Valencia’ received Diflubenzuron (Micromite) at 6.25 oz per acre in April. In May the ‘Early Gold’ block was sprayed with Abamectin (ABBA) at 15 fl oz per acre and ‘Valencia’ block with Spinetoram (Delegate) at 4.5 oz. The later was also used in the calendar treatment of ‘Early Gold’ in June. These treatments suppressed ACP and CRM compared to the untreated control in both blocks. Rustmites were also an issue at the ‘Valencia’ block in Collier county but were controlled by Movento at 16 oz per ac directed at ACP in May. CRM averaged 0.09 and 4 per lens field in the grower standard and biweekly oil treatments, respectively, in a block of ‘Valencia’ orange in Lee county, significantly less than 10 per lens field in the untreated control, indicating that both insecticidal and oil sprays reduced ACP and mites. These findings suggested that correct treatment choices for ACP control could also control CRM. However, this may not be the case with all insecticides. For example, rustmites increased 5-fold in the calendar treatment of ‘Valencia’ block in Hendry county which received an imidacloprid spray in July, compared to three treatments that received no spray. The cause of rustmite resurgence in treated blocks may have been reduced populations of predatory mites which appeared to be too low to provide significant suppression of CRM, indicating possible need for augmentation. We have found that the predatory mite Amblyseius swirskii provides significant reduction in ACP immatures under controlled conditions. These mites are commercially available and might be useful in reducing psyllids, rust mites, some other mites and additional insecticide sprays in citrus. A reduction of 44% and 50% in ACP nymphal cohorts in untreated and calendar treatments, respectively, and 80-90% mortality or disappearance of CLM larvae indicated that some generalist predators were still able to suppress these pests in treated environments. Treatments tested at SWFREC showed that most commonly used and experimental insecticides reduced ACP for 3-4 weeks or even more with some new products such as Apta 15 EC (Tolfenpyrad). However, most treatments lasted 1-2 weeks against CLM except Delegate 25 WG, Voliam Flexi and microbial products Grandevo and MBI 206 lasting 3 weeks thus need for more effective products that impact both ACP and CLM. Ladybeetles, spiders and ants were reduced in the plots treated with Portal 5 EC (fenpyroximate) and Apta 15 EC but not much with microbial products. Publications: Qureshi, J. A., B. Kostyk and P. A. Stansly. 2012. Registered and experimental insecticides for control of Asian citrus psyllid and citrus leafminer on mature orange tress. Proc. Fla. Stat. Hort. Soc. 125: (In press) Ferrer, C. M., J. A. Qureshi and P. A. Stansly. 2012. Insecticidal control of Asian citrus psyllid: effects on secondary pests and natural enemies. Citrus Industry. 93 (4): 12-16.
Objectives: 1) scale up production of Tamarixia radiata to levels to better assess the potential impact an augmentation program may have on the ACP population and ultimately the spread of HLB, 2) genetic techniques to identify parasitoids, and 3) pass on rearing technology to both private and public sectors. Production of T. radiata continued at Southwest Florida Research and Education Center (SWFREC), Immokalee and Division of Plant Industry (DPI), Gainesville. Colony of Florida strain maintained at SWFREC produced 157,264 wasps. An additional colony of the same strain and others from South China, Pakistan and North Vietnam produced 266,730, 236,887, 230,829 and 245,850 wasps, respectively. Wasps from these colonies were used for research, to maintain the colonies and to release in conventional citrus mostly in southwest Florida and organic citrus, mostly in North Florida. Releases were made in Collier, Lee and Hendry, Lake, Indian River, St. Lucie, Polk, Hardee and Hillsborough counties. About 42,900 wasps from all four colonies were also sent to Commonwealth of Dominica. Experiments comparing effects of host density on fecundity of T. radiata showed that fecundity increased with host density to a maximum of 11 to 12 eggs per day per female with no significant differences among host densities of 40, 50, and 60 nymphs of 4th instar. Due to insecticides use and Tamarixia releases psyllids and flush nymphal populations were generally low in the groves. Parasitism averaged 29% (n=246) in April in a block where releases were made at SWFREC Collier county compared to 9% (n=196) in block without releases. In May 17% (n=18) nymphs from release block were parasitized and none from block without releases (n=89). In July, 26% (n=325) nymphs were parasitized in a young block but no parasitoids emerged from sample collected in August (n=128). In a neighboring conventional grove untreated plots, parasitism averaged 17% (n=63) in April with releases compared to only 3% (n=36) without releases. No parasitoids recovered from plots treated with nutritionals only (n=69) and insecticides (n=21). In June 9% nymphs (n=149) were parasitized in untreated plots without releases and none in nutritionals (n=20) or insecticide treated (n=28). In December, 82% (n=11) and 76% (n=42) parasitism was observed in the untreated and nutritional only treatments. No parasitoids were recovered from nymphs collected in April from the untreated plots (n=70) or plots treated with biweekly applications of 435 oil (n=202) or grower standard insecticide (n=32) in a block of ‘Valencia’ in Lee county. Only 4% nymphs (n=130) were parasitized in June from oil treated plots and none from insecticide treated (n=82) or untreated (n=60). No parasitoids were recovered from 201 nymphs collected in May from plots under calendar sprays or untreated in a block of ‘Early Gold’ in Hendry county. However, parasitism averaged 36% (n=26) in untreated plots of ‘Valencia’ oranges. Parasitism rates of 28%, 39% and 6% in Polk county and 28% in Lake county were observed in July in blocks of organic citrus using releases of T. radiata. In August, parasitism rates averaged 24% in Hardee county, 39% and 9% in Polk county and 100% in Lake county. One symposium on Tamarixia radiata and one on management of ACP were organized at Florida Entomological Society and Entomological Society of America. Two new colonies of the biparental Diaphoencyrtus aligarhensis from Pakistan initiated and maintained at DPI and SWFREC and releases started in southwest Florida. Qureshi, JA., E. Rohrig and P. Stansly. 2012. Introduction and augmentation of natural enemies for management of Asian citrus psyllid and HLB. Citrus Industry. 93(6): 14-16.
Florida growers have reported that enhanced nutritional programs (ENPs) maintain productivity of HLB-infected trees. However, efficacy and sustainability of the nutritional approach for HLB disease management remains uncertain. Complementary studies of multiple ENPs and their individual components compared to the standard nutritional program (SNP) on nursery and field trees were initiated in 2010. Two independent nursery trials were initiated with final data collection finished and data analysis currently underway. From monitoring temporal bacterial movement and replication it was found that Candidatus Liberibacter asiaticus (Las) populations are similar for ENPs and the SNP. Minor differences in Las movement have been observed. Las invaded new flush tissue faster in ENP treated trees than SNP trees. Phosphite treatments have caused Las to favor early invasion of root tissue compared to other treatments. Preliminary analysis of the final takedown results from nursery trials shows a reduction in visual symptom expression with ENPs. However, there were no significant differences in the HLB-associated phloem plugging or reduction in leaf number, leaf size, and fibrous root mass. There is an observed trend that the enhanced nutrition may be increasing fibrous root loss compared to control trees, but further analysis is necessary. Results of final nutrient status of the trees is pending. The field site was chosen for its mix of healthy, presymptomatic, and HLB symptomatic trees to determine if observed differences resulted from effects on healthy or infected trees. After 3 years of nutritional treatments, fruit yield and HLB symptoms in field trees treated with ENPs have not differed significantly from the standard nutritional program. The only significant predictor of yield differences was the percent of declining trees, but there was no interaction with nutritional treatment, suggesting that nutritional programs have not affected yields of healthy or HLB affected trees in this study. Nutritional status of the trees after 3 years of ENP treatment have been assayed and preliminary analysis shows that although ENP treatments do have higher micronutrient concentrations compared to control treatments they are still deficient in these nutrients. This deficiency has increased over the course of the experiment as disease has progressed through the grove. Control and one of the ENP treatments are continuing for a fourth year to observe if a treatment effect will develop in year 4 as others have reported. Currently analysis is underway to determine if an interaction between nutrient status, disease severity, and yield can be observed for field trees. A similar analysis of with biomass measures in place of yield will be performed on greenhouse trees when final analysis of nutritional status is complete.
In this project Asian Citrus Psyllid is reared mainly for the development of the mass rearing of Tamarixia radiata parasitoids but also to supply other projects with ACP for testing the effectiveness of several insecticides. The mass reared T. radiata parasitoids were released in urban environment of the greater Los Angeles area for the biological control of ACP in backyards. Biological control of ACP appears to be the only economically viable method for reducing ACP populations in the urban areas. Methods were developed to optimize the host plant quality for the culturing of ACP on small plants suitable for rearing in small insectary rooms. Several plant species were tested, and under our conditions a combination of Curry Leaf plants (Murraya koenigii) and Volkameriana (Citrus volkameriana) performed well. ACP lays numerous eggs on Curry Leaf, while Volkameriana was very suitable for nymphal development. Both plants are capable of supporting the complete development of ACP on their own as well. For the rearing of T. radiata we have paid particular attention to maintaining genetic variation in our Tamarixia cultures by rearing a total of 16 different populations each initiated with a few individuals collected in from different locations in the native range of ACP in Pakistan. Within each of these small populations we avoid lab adaptation (= domestication) because there simply is no genetic variation that can be selected on. In order to restore the genetic variability of the overall population we mix individuals of the 16 subpopulations before we use them as parents in the mass rearing. The result of this approach is that we maximize the genetic variability of the released population and at the same time avoiding the negative impact of domestication. Post-release selection can then take place for genotypes that are adapted to the local circumstances in the released range. Over the 6 months covering this reporting period we have produced a total of approximately 23,000 T. radiata for release in the field. Many of the initial problems with the mass rearing have now been resolved and we estimate that we will be able to produce around 15,000 wasps per month for release over the next period. We are also testing using caged citrus trees in the field to determine if they can be used as field insectaries, thus vastly increasing the number of Tamarixia available for release. This work was inspired by the previous work done by Dan Flores (USDA-APHIS PPQ CPHST Mission Laboratory, Edinburg, Texas). Field cages have been designed and are in place. Experiments are underway to determine optimal release ratios of ACP, Tamarixia as a function of the number of leaf flushes present on the caged tree.
Studies show that predators (predaceous insects and spiders) form an important source of mortality for the Asian Citrus Psyllid (ACP) in Florida, much more important than the parasitoid Tamarixia radiata. In California we do not know if predators form an important mortality source as well, nor do we know which predators are involved. This project aims at determining the relative importance of different predator species as a mortality source for ACP by identifying which predators have fed on ACP. To determine if a predator has fed on ACP we will use PCR techniques to detect if ACP DNA is present in the gut of the predator. The complex of species that feed on ACP during the day may differ from that feeding on ACP at night, consequently we sample for predators both during the day and at night. This is the first reporting period for this project and during this period we have sequenced several genes of the ACP to determine their suitability for developing probes for ACP DNA detection in predator guts. Several DNA regions have been identified as potentially suitable for primer and probe development, and primers have been designed. They remain to be tested for specificity and ability to detect low DNA concentrations. Day time samples have been taken at two different dates, on one of these dates both an early night time sample and an early morning sample have been taken to collect night active predators. Ultimately, the goal of this project is to identify the relative importance of different predator species as a mortality source for ACP and to either develop cultural practices that may increase the populations of these predators in the field and/or to identify species that may be mass reared to help control ACP populations. To quantify the overall importance of predators for the control of ACP populations exclusion experiments are done. In these experiments we determine ACP mortality on terminal twigs that are completely protected from predation by enclosing the twig in a mesh bag, partially protected from predation by enclosing them in an open bag, or left exposed to predation by leaving the twig without enclosure. Different survival rates of ACP populations under these different experimental conditions will give an indication of the relative impact of predation on ACP population growth.
A method was developed and used to show that topical application of salivary sheath inhibitors to the surface of citrus leaves can block Asian citrus psyllids (ACP) from forming intact salivary sheaths and thus inhibit direct feeding on the citrus phloem. Because the phloem is the location of the Candidatus Liberibacter asiaticus (CLas) bacterium, it has been our hypothesis that deploying a ACP control strategy that prevents the ACP from reaching the phloem will prevent the ACP from either acquiring or transmitting the CLas. Also, manuscript was accepted in PLOS ONE describing methods used to produce pure salivary sheaths from a number of salivary sheath forming plant feeding hemipteran insects. The advantage of this method over previously used methods is that it allows analysis of specific sheath components (i.e. flang and primary shaft) without contamination of any other components typically present in diet and or plant tissues. This method is being used to provide structural confirmation to support the compositional data already obtained for ACP formed sheaths.
Our objective for this project has been to evaluate botanically derived compounds as potential repellents for practical application against Asian citrus psyllid (ACP) as part of potential integrated pest management strategies. In previous quarters, we evaluated five botanical oils as repellents for ACP. Of the five, only one, fir oil, was repellent in laboratory bioassays. We submitted a manuscript recently for scientific peer review, based on this research (to the Journal of Pest Science). In this manuscript, we reported data from: (1) olfactometer bioassays indicating that fir oil is repellent to ACP and that clove and camphor oils are attractive to ACP, and (2) laboratory settling experiments with fir oil treated plants, and (3) field trapping using baited yellow sticky cards. Based on these results, a new proprietary release device was developed by a commercial partner that we will be testing in the next quarter. This device releases fir oil at a controlled rate and we will be testing whether this rate has practical value in the field. We have already received these dispensers and we will initiate the experiment as soon as trees begin their next large flushing cycle to ensure there will be an adequate number of psyllids for a stringent test. In addition to what we have reported previously, we have continued experimentation towards identifying further botanical repellents. Since fir oil was repellent in laboratory bioassays, we decided to test turpentine as a less expensive alternative. Surprisingly, our settling bioassays with turpentine treated vs. control seedlings showed that the ACP may have a preference for settling on plants treated with turpentine odor (57% on turpentine treated seedlings and 43% on control seedlings). Since fir oil treatments were much more effective at repelling ACP than turpentine treatments, we will be focusing on this treatment in the subsequent field studies.
Key unknowns about Asian citrus psyllid biology in Florida: Overwintering sites and alternative hosts The goal of this project is to determine overwintering habits/sites 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. Recently, we submitted an article regarding our survey of Lake Kissimmee State Park to Florida Entomologist it has been accepted. From January to March, we performed extensive sampling with a high-powered insect vacuum system to determine ACP overwintering habits. Briefly, we sampled two areas of citrus production that were divided into two plots: one oriented north-south and the other east-west. For each plot, we sampled the citrus canopy at three different heights and four cardinals orientations. The purpose was to develop a two dimensional picture of ACP distribution within citrus groves during winter. Environmental data loggers were placed at these different locations to correlate temperature and relative humidity data with ACP distribution. Moreover, we collected and analyzed nutrient content of leaves at each location. During the next few months we will analyze the data and develop a scientific article to report this information. An additional aspect of this project is to assess how management of citrus groves has an impact on ACP population dynamics during winter months. During the 2012/13 winter, we sampled 40 citrus groves with various management programs (conventional, poorly managed, organic and abandoned) to understand the overwintering population dynamics of ACP over a broad range of grove types throughout Florida. Our results show that significantly more ACP are found in poorly managed groves than in groves under any other management. Our current objective is to correlate these discrete ACP distributions with certain biotic and abiotic data such as: citrus leaf nutrient analysis, landscape characteristics, soil data, meteorological data and ACP fitness analysis (survival and fecundity studies). Furthermore, we will compare these findings with ACP abundance data collected over 2 winter seasons from 40 more groves in Florida. These data will yield clues as to potential ‘winter reservoir groves’ that may require more intense management over winter months in order to reduce the population growth of ACP in the spring season.
The goal of this project is to identify insecticidal or antimicrobial peptides with efficacy against phloem-feeding citrus pests, including the Asian citrus psyllid (ACP). Previously, we reported that citrus plants expressing Peptide B showed promise in terms of deterring feeding by ACP, but that only four plants had been evaluated at the time of reporting. During this reporting cycle, we have completed additional replicates that corroborate our previous findings. Additionally, the final peptide to be tested, Peptide D, was evaluated in detached leaf assays and it was found to be the most efficacious out of the four peptides at significantly deterring feeding by adult ACP. Our results also showed that by 48 hours, the feeding behavior of ACP had returned to normal levels when insects fed on Peptide A, while Peptides B and D continued to deter feeding through 72 hours. Future experiments will examine protective effects beyond the 72 hour time point with the most promising peptides. In a preliminary study, we observed that when Asian citrus psyllids harboring Candidatus Liberibacter asiaticus (Las) at rates between 20-80% were held on plants expressing Peptides A, B or C, there was a reduction in the percentage of Las-positive insects after 15 and 30 days post-exposure to the plants, as well as, a reduction in the percentage of infected offspring. Plants necessary to set up a fully replicated experiment to investigate the potential phenomenon of peptide-conferred protection against Las have been grafted with CTV-peptide constructs, and once they have been screened and test positive for CTV expression, assays will be started to investigate these findings. Meanwhile, we have developed quantitative PCR primers for three of the four peptides under investigation in order to correlated psyllid responses to peptide expression in planta. This information will provide important information as to the potential for consistent management of Las transmission by ACP with the most promising peptides.
Biorational, non-neurotoxic insecticides play an important role in integrated pest management given that they typically target a narrower range of species as compared to conventional, broad-spectrum, neurotoxic insecticides. Also, non-neurotoxic insecticides often cause lower levels of toxicity to beneficial arthropods, including parasitic wasps and ladybird beetles, than conventional insecticides. During this report cycle, we have begun evaluation of three new compounds against Asian citrus psyllid (ACP) that fall under the non-neurotoxic category: novaluron, concanavalin A (jack bean lectin), and L-canavanine. Initial experiments with the chitin biosynthesis inhibitor, novaluron, indicate that this insecticide may hold potential as a control agent against ACP by inducing sub-lethal effects on the insect, including reduced egg hatch and reduced adult emergence. Based on these initial studies, Rimon’, the commercial blend of novaluron, is being evaluated at three field rates recommended by Chemtura for testing: 10, 15 and 20 fl oz. These rates are based on efficacy against other Hemipteran species in other fruit crops and on our initial experiments. This insecticide will be evaluated to determine the effect on egg hatch, adult emergence, and fecundity at these three rates. The other compounds under current evaluation, concanavalin A and L-canavanine, are being evaluated against adult ACP to determine feeding deterrence and effects on ACP mortality. Effects on feeding deterrence and mortality have been observed for other related insect species. Initial experiments suggest that L-canavanine, a non-proteinogenic amino acid, may deter feeding of ACP on citrus. However, concanavalin A had no effect on feeding or mortality of ACP in our initial experiment. However, the initial screens for both of these compounds were conducted over a brief time period (48H) and will be extended in subsequent experiments, where mortality effects may be more apparent for both compounds. Efforts to refine an artificial feeding assay to better evaluate these particular compounds are underway given that the current results caused higher than expected mortality in control treatments at 48 hours and longer.
The objective of this investigation is to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology, including host acceptance behaviors and population dynamics. We have have been conducting this investigation with complementary field and laboratory experiments. Regarding field experiments, we are still following the Keyplex’ program in the field. The most recent spray of nutritional supplements occurred on February 28th. The next application (post bloom application) will be performed mid-April. Since March, we have monitored the number of psyllids on each tree within our treatment plots weekly with the tap sampling method. We plan to continue this survey during the summer season with regular nymph counts and leaf nutrient analyses. We are also performing a qPCR analysis of all trees in the experiment, 6 months after the initial one, to investigate whether the infection rate has changed, depending on whether or not plots were treated with supplemental nutrients. Currently, our data indicate that infected trees are more attractive to ACP than non-infected trees. This has been already reported from our previous laboratory experiments, but we are observing this effect in the field for the first time. The effects of the supplemental nutrient regime on psyllid population densities are currently not yet statistically significant, but we have observed a trend of more psyllids found in plots treated with nutritional supplements as compared with untreated plots. However, in the laboratory we consistently observe that psyllids are more attracted to HLB-infected plants treated with a nutritional supplement as compared with non-supplemented controls. Interestingly, we have also observed that HLB-infected citrus plants were proportionally more infested with ACP adults in choice settling assays as Ct values (measured by quantitative real time PCR showing presumably higher bacterial concentration) were proportionally lower. We plan to perform a calibration curve for our qPCR method in order to determine the amount of bacterial DNA within these citrus plant treatment samples to confirm whether degree of bacterial infection impacts psyllid preference for choosing and feeding on plants. Our next objectives are to: (1) investigate the acquisition rate of the HLB-causing pathogen by psyllids from plants treated with supplemental nutrients as compared with controls, and (2) collect volatile chemicals from plants before and after the application of a nutrient spray cycle, in order to determine how this treatment affects the volatile emission pattens from plants. Our hypothesis is that changes in volatile emissions may be affecting behavior of ACP.
This is a cooperative research project between Co-PIs Joseph Morse, Jim Bethke, Frank Byrne, Beth Grafton-Cardwell, and Kris Godfrey. One objective is to coordinate with researchers working on chemical control of ACP in Florida, Texas, Arizona, and elsewhere. Towards that end, Grafton-Cardwell, Godfrey, and Morse met with Jawwad Quershi, Phil Stansly, and 2 CA PCAs in Orlando 2-5-13 at the Third International HLB conference to discuss cooperative trials focusing on organic products; the same group toured the UF Immokalee Research Center 2-6-13 to see ongoing field trials; and Byrne and Morse traveled to the UF Lake Alfred Research Center with 3 CA PCAs 2-8-13 to discuss ongoing research and collaboration with Michael Rogers and Lukasz Stelinski. The Orlando HLB conference is an excellent opportunity to network with other scientists working on ACP and HLB and much was learned and discussed. We are rearing ACP in a contained greenhouse at the Chula Vista Insectary (San Diego County; about 6 miles north of the Mexican border) under permit (#2847) from CDFA. This permit clearly notes experimental protocols and procedures so that the work is done as safely as possible to minimize the chance of ACP escape. At this site, Jim Bethke has initiated ACP tests on various organic and traditional pesticides of interest to California growers using 4 types of tests: adult topical adult drench adult residual and nymph topical tests. Data have been gathered to date on 14 traditional pesticides and 12 organic products, in many cases with multiple trials involving each material. A second location where we are working with ACP is at UC Riverside, working under permit inside the UCR Insectary Facility. Frank Byrne is conducting trials on various neonicotinoid insecticides and Morse is evaluating the baseline susceptibility of CA ACP to various pesticides (18 trials to date) in comparison with studies done in Florida. Two field trials have been run to date evaluating organic products useful in control of ACP; the first 10-4-12 evaluated the impact of 5 treatments on adult ACP; the second trial 3-5-13 evaluated 8 treatments against young nymphs and eggs. Kris Godfrey obtained a permit to rear ACP inside UC Davis’ Contained Research Facility and received 4 shipments of ACP (under CDFA permit) from Bethke’s colony to start the Davis colony (so that Chula Vista and Davis results might be similar). She has initiated testing of organic products and has several other lines of experimentation planned based on requests from the CRB. Beth Grafton-Cardwell has updated online ACP pest management guidelines and has given 8 presentations to various groups dealing with ACP since 10-1-12 (i.e. over the past 6 months). She has served as a focal point for communicating with ongoing ACP management programs in the SJV, Ventura, and elsewhere. In summary, we continue to expand our ability to conduct ACP research in California with a clear focus on management of HLB (rather than ACP).
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