During the past year, we have constructed four citrus tristeza virus (CTV) vectors containing four insecticidal peptides for management of the Asian citrus psyllid (ACP) and the brown citrus aphid (BCA). CTV expression vectors containing these peptides have been successfully bark flap-inoculated into plants to confirm systemic peptide expression and provide material for graft inoculation into citrus. To evaluate the effects of peptides on insect fitness, plants containing the CTV-expressed peptides have been used to graft-inoculated into citrus plants for psyllid bioassays. Since our last report, we have completed plant inoculations for each of the four CTV-peptide constructs originally proposed (henceforth A, B, C, and D). Most recently, fifteen plants have been grafted with peptide B and are being given latent period for the peptide to become established throughout the plants. The plants will be tested for the presence of CTV-peptide constructs using ELISA in a few days. Peptides C and D have been established in three and five plants, respectively, and additional plants will be grafted-inoculated to obtain a sufficient number of test plants for psyllid bioassays. Since the previous report, we have continued to conduct experiments to evaluate the lethal and sublethal effects of vector-expressed peptides A and B on development (egg and nymph), life history parameters (adult longevity, fecundity), and feeding of ACP and BCA. Bioassays with plants containing peptide A have been completed. Settling assays suggest that ACP adults prefer plants that do not contain either peptide A or B. Although adult survival was not significantly affected, development of eggs and nymphs were significantly longer when insects were exposed to plants containing peptide B compared with control plants. In addition, ACP feeding and fecundity were significantly reduced in the presence of peptide A compared with control plants. Similarly, the results of honeydew excretion assays suggest that peptide B reduces ACP feeding. In bioassays to evaluate the effect of peptides on BCA, survival of nymphs was significantly reduced when peptide A was present in citrus plants compared with control plants; however no other fitness parameters were significantly affected in response to peptide A. Longevity and fecundity of adult BCA were significantly reduced in response to peptide B plants compared with controls. The results from ACP and BCA bioassays with peptide B are based on four replicate plants only, due to limited availability of plants. Additional replications will be conducted as plants containing peptide B become available. Currently, artificial feeding bioassays are being conducted to evaluate the lethal concentration of a variety of insecticidal peptides, including the four originally proposed, against ACP. In addition, we have begun to conduct experiments to evaluate the effect of peptides on ACP endosymbionts, including Las. Insects from these ongoing bioassays are currently being collected and stored for subsequent analysis with qPCR analysis. In addition to the above ongoing experiments, our research plan for the next year is to conduct laboratory bioassays to evaluate the susceptibility of citrus leafminer, and T. radiata to citrus plants containing CTV-peptide constructs.
This project has 5 objectives: (1) evaluate efficiency of ACP control techniques in cooperation with growers, (2) develop efficient monitoring methods for ACP, (3) accelerate testing of new chemistries and techniques for ACP management, (4) evaluate the economic component of the comprehensive program, and (5) provide an information bridge between researchers, growers, and industry. The following is an update of ongoing field and laboratory experiments. The potential for ACP resistance is uppermost in many grower’s minds and management programs are based on rotation of modes of action. Nevertheless, many practical questions remain on how this is best accomplished. Experiments are underway to 1) compare selection rates for resistance using mixture (AgriFlex) versus rotation of its two components, and 2) evaluate stability of insecticide resistance and number of generations required to return tolerant populations to susceptibility. Following application of an LD80 dose (4 Jan), our colony experienced a crash. Because the untreated individuals were also affected, we are attributing this result to seasonal fluctuation of vigor. We are currently giving our murraya a 3-mo rest while we increase our laboratory psyllid populations. We continue to collaborate with the DPI CHRP program to evaluate results of the cooperative (area wide) ACP management program, in SW Florida (i.e. Gulf CHMA). We have presented this data for the last 10 cycles via interactive maps on our website (http://swfrec.ifas.ufl.edu/entomology/extension/chma/ for growers to use). These maps have allowed us to visualize ‘hot spots’ (i.e. those groves with high numbers of ACP for 3 consecutive cycles), and we are in the process of contacting growers to improve ACP management. Citrus leafminer (CLM) incidence and damage has been increasing, possibly due to increased use of broad spectrum insecticides to control ACP. Canker is also on the rise and growers are demanding assistance with management of CLM, thought to exacerbate the disease. We have been monitoring CLM using sticky card pheromone traps in citrus groves as well as a natural preserve (Okaloacoochee Slough State Forest) in an attempt to determine effective distances from source to trap. We have been using bucket pheromone traps dispensed in citrus groves and the slough to collect identifiable specimens for identification. Gracillariid leaf mining moths are very small, and often very difficult to identify solely based on morphology. We have used a combination of traditional morphological methods and COI barcoding, based on a mitochondrial DNA sequence. A sample of specimens that were collected with bucket traps was sent to the Lepidoptera sequencing lab of collaborator Kawahara at the McGuire Center for Lepidoptera and Biodiversity in Gainesville Florida for analysis. Thus far results show that there are many different Phyllocnistis species coming to the pheromone traps; P. citrella, P. insignis, P. vitegenella, and a newly discovered leafminer species to Florida. We have submitted grant applications for additional funding. We feel it would be beneficial to publish a paper that discusses genetic variation/species boundaries of Phyllocnistis found in our citrus groves and natural areas. Within the next few months, these pheromone will be used in conjunction with mark and recapture experiments to determine distance traveled by CLM, and development of a preliminary degree-day model. We have started a colony of CLM to rear the parasitoid Citrastichus phyllocnistoides and support a program for monitoring CLM susceptibility to key insecticides. This summer we will use a diagnostic dose to monitor resistance in field populations of CLM exposed to intensive versus modest insecticide use.
Low volume (LV) aerial and ground sprays have become an important method of application in Florida citrus. Our eval- uations of LV application of 435 horticultural mineral oil (HMO) have shown promising results the last 3 years. Here we document overall results from the 5th trial begun Feb 2011 in a 10.9 acre plot of ‘Valencia’ orange in Lee County com- paring LV spray of 435 HMO with the Grower Standard (GS) and an Untreated check (UC). The grower has been applying a foliar nutritional program throughout the block. Three treatments were set out in 3×3 Latin square design. A Proptec rotary atomizer P400D spray machine was used for all treatments. HMO was applied alone (no water) every 2 wks at 2 gpa. Between Feb-Apr, 3 sprays of oil were applied, and the GS was Carbaryl and Azasol. Mean ACP counts were below the 0.2 adults/stem tap sample threshold in all treatments with no significant differences. Due to high freeze- induced fruit drop, harvest was early (Feb) and juice content averaged less than 50% (0.41). Total brix was within normal range of 9-14 for all samples, but the acid content was below normal (1.0 – 0.5) in 5 of 8 samples tested. There was no significant treatment effect on yield, but highest production was seen from trees treated with the GS (12.6 lbs solids/tree followed by HMO (10.5) and UC (6.1). Between May-July, 7 sprays of HMO with Actara and Agri-mek for GS plots were applied. During May, significantly fewer psyllids were seen on GS trees than HMO trees, but during June these differences disappeared. We then added citrus leafminer (CLM) assessments to determine if treatments were effective at reducing CLM numbers. A damage assessment of leaves using a modified Horsfall-Barratt scale found significant differences between the GS and oil treatments, with the GS having less CLM damage. Between Aug-Oct, 5 sprays of HMO oil and 1 spray ea of Dimethoate and Delegate for GS were applied. Mean adult psyllid populations were lowest for GS, followed by Oil and UC. Thus, LV oil treatments were suppressing ACP, though not as effectively as the GS. From Nov 2011-Jan 2012, 3 sprays of HMO were applied, and all plots received a dormant spray of Danitol (Jan). Mean psyllid populations for GS and HMO treatments were significantly less than UC, with HMO treatments suppressing ACP as effectively as GS. Resets sampled in Oct for HLB and Ct titer showed no significant differences between treatments and low (< 3%) HLB incidence. CLM trap catches in Nov were significantly lower with HMO compared to GS and UC even though highest flush density was seen on HMO-treated trees. probably due to egg and larval mortality. Between Feb-Apr 2012, 4 HMO sprays and 1 GS of Micromite were applied. Mean ACP counts for GS and HMO were similar and lower than UC. Resets were sampled for % HLB, most samples were not usable due to frost damage. The 2nd harvest (27 Feb) showed HMO treatment with greatest lb solids followed by UC and GS. Acid/brix results are pending. CLM flight began early this year (2 Mar) with first peak at 16 Mar showing no significant differences between treatments. An experiment initiated spring 2011 on an 80ac block divided into 12 large plots in a RCBD with 4 treatments and 3 reps (Jul) evaluated aerial applications of Intrepid 2F, and Delegate. Significant control was obtained with the aerial application of Intrepid, but not as much or as enduring as with the ground application. An experiment initiated Feb 2011 compared LV (5 or 10 GPA - Proptec) vs. HV(40 or 120 GPA - Airblast) apps. of Movento SC along with Mustang Max 1.5 EC (5 gpa), Baythroid XL 1 EC (5 gpa) and Provado 1.6 F+Agrimek 0.15 EC (120 gpa). All treatments provided significant reduction in psyllid adults through 5 wks and nymphs through 4 wks except Mustang, low rate of Movento (5 gpa) and Provado + Agrimek.
we have completed the harvest for the fourth year of the Hamlin and Valencia trials with the Boyd Nutrient/SAR treatments. The ‘Hamlin’ orange block at SWFREC receiving the Boyd cocktail treatments was harvested in December before the freeze on January 4, 2011 with temperatures in Immokalee of 25 degrees F. Fruit yield on 8.2 foot tall trees from the better treatments averaged about 2+ boxes per tree. The better treatments contained Macro nutrients as DKP + KNO3, micro-nutrients Mg, Mn, Zn, Mo, B, and a Phosphite. Juice quality from the ‘Hamlin’ trees with HLB trial has not been affected. Citrus canker was a significant factor in our ‘Hamlin’ trial for the first time this year. Fruit loss due to canker varied from 40% in less vigorous trees to between 60% to 75% fruit loss in treatments stimulating more vigorous growth. Treatments with spray grade KNO3 + micro-nutrients had less fruit loss to canker. The less vigorous trees with less flush carried the larger fruit load with less fruit drop in the presence of citrus canker. Fruit yield from our 30 acre commercial ‘Valencia’ block experienced frozen fruit in the January freeze with some fruit loss, and still produced 1.6+ boxes per tree on 7 to 8 foot tall trees. This trial was harvested in March and juice quality data is not yet available. The highest yield per tree for ‘Hamlin’ and ‘Valencia’ continues to be treatments 2 (complete Boyd cocktail minus the SARs), treatment 9 (complete Boyd cocktail minus hydrogen peroxide, and treatment 1 (complete Boyd cocktail. These three treatments consistently are among the four highest yielding during the past three years. Treatment 3 (KNO3 + micro- nutrinets) has moved into the top three performers. Treatments 4, 5, 6, and 8 which do not contain the foliar applied nutrients Mg, Mn, Zn, Mo, and B are among the lower yielding trees. We are well into the 4th year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on Swingle to test 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. While we are continuing with the experiment, results from the first 3 years are being written up for publication. Psyllid populations for all treatments remained below the 0.20 threshold during each sampling period (13 Jan ‘ 21 Mar) except for 13 Jan when the nutritional only plots reached 0.23. Insecticide and Insecticide+nutritional had significantly (P< 0.05) fewer adult ACP than control or nutritional only on dates 13, 27 Jan; 10, 24 Feb; 7, 21 Mar. All three treatments had significantly (P < 0.05) fewer ACP than control on 21 Mar. Since our last report we applied a dormant spray of Danitol 4EC (12oz/ac) on 2 Feb in plots designated for insecticide treatment. Resets planted (June 2010) were tested for HLB detection (13 Jan) but showed no significant treatment effect on percent PCR positive for HLB (16% - 60% infection). All mature trees (10 years old) tested positive for HLB. Ct values were significantly less for Untreated (21.2) than the Insecticide only plots (22.5). However, there were no significant differences between the insecticide plus nutritional plots (21.6) and nutritional only plots (21.6). For the resets, there were no significant differences in Ct values (range 25.0 ' 33.4). All trees were harvested and all fruit weighed 8 Mar. Trees receiving the Insecticide + nutrition treatment produced greatest average yield (199.7 lbs/tree ' A) followed by nutritional only (182.3 - AB) Insecticide only (171.6 B) and Untreated (147.0 - C). We are waiting for results of brix/acid ratios.
This is the third and final year of a project on protecting newly planted citrus trees from Asian citrus psyllid (ACP) and huanglongbing (HLB). The project is collaborative between USDA-ARS (Hall) and University of Florida (Stansly). Update on USDA-ARS activities. Two experiments are being concluded by USDA-ARS in the Indian River citrus area at a grove just west of Fort Pierce. For research purposes, most of the grove is under a minimal ACP management program and HLB-infected trees are not removed. A detailed summary of the two experiments (as of late summer 2011) was presented in the September 2011 Annual Report. Since that time, notable is that regardless of the particular psyllid management program studied in each experiment, the percentage of trees infected by HLB increased to 90% within 42 months after planting in one experiment (infected trees removed as they were found and not replanted) and to nearly 100% within only 26 months after planting in the second experiment (infected trees not removed). This was based on qPCR of trees using the Wenbin Li primers and a cT threshold of 32. The results support that a grower will have difficulty protecting new plantings from HLB if concerted efforts are not made to control ACP not only in a young planting but also on an area-wide basis in the vicinity of a new planting. Simple calendar sprays were ineffective for completely preventing ACP infestations, even when hard pesticides were applied monthly. It may be more productive to use calendar sprays in conjunction with scouting to ensure infestations of eggs and nymphs are controlled, although such an approach may inflate the use of insecticides to more than once a month during some times of the year. In an experiment in which infected trees were not removed, the trees were ‘Valencia’ on ‘Carrizo’ rootstock and harvest data were collected during March 2012 (31 months after planting). An average of 10% trees per plot had died by March 2012. Across all three psyllid control treatments, an average of 72.5% of the trees produced fruit including fruit remaining on the trees plus fruit that had dropped to the ground. Harvestable fruit remained on 62.5% of the trees. Over all trees, we picked an average of 7.5 fruit per tree with an average of 137 grams per fruit (1,028 g of fruit per tree). Although yield of the 31 month old trees was low, yield was generally highest in plots of trees subjected to a complete insecticide program, intermediate in plots of trees subjected to a reduced insecticide program, and lowest in plots of trees subjected to oil sprays every three weeks. UF Update – A comparison of young trees treated four times annually with different rotational schedules of Admire, Platinum and Cyazypyr indicates that, regardless of the order that the insecticides were applied, significant control of ACP was achieved as compared to ACP levels in untreated trees. Cyazlypyr is a new insecticide very effective against ACP, and a label for citrus is anticipated. As the trees have grown the rate of this insecticide has had to be increased to maintain control of ACP but application frequency will be maintained at 3 times a year to adhere to expected label directions. This insecticide is of a novel MOA (diamide chemical class) and is effective against a number of important types of pests including fruit flies, leafminers, leaf-feeding beetles, whiteflies, hoppers, thrips and weevils. Lower percentages of trees tested HLB positive after almost two years in plots receiving insecticides (0 to 11% trees infected) than in plots of trees untreated (greater than 30 % of trees infected).
This project aims to 1) refine sampling methods for ACP, 2) test the influence of adult density and shoot infestation on precision of estimated means and distribution of population within blocks, and 3) evaluate methods for assessing psyllid density, shoot density, and infestation rates and their integration into a user friendly system accessible to consultants and managers. 1-2) An EDIS document describing the use and comparison of stem-tap sampling, sweet-net sampling and sticky traps for monitoring ACP adults in citrus groves was published (http://edis.ifas.ufl.edu/in867). Data showed that a little more than 100 tap samples would be necessary to detect with confidence 15 psyllids with 75% precision, a reasonable threshold during the growing season when trees are producing new growth. Young shoots are required for psyllid oviposition and immature development. Therefore, routine monitoring protocol developed for ACP integrates adult sampling, shoot density and infestation rates. For adults 100 tap samples per bock divided into 10 stops 5 along the perimeter to cover for edge effect and 5 inside the block are recommended. This scheme could lead to decision to spray only the block perimeter. At each stop 10 shoots containing feather flush are examined using hand lens to look for infestation with psyllid immature and search is terminated if 10 shoots are not found after 20 trees. Number of shoots examined, found infested and number of trees searched to find the examined shoots is recorded. Cost for materials and labor was almost same for stem-tap and sweep-net methods but 28 times more for sticky traps. Stem tap method is rapid, works under either dry or wet conditions and has proven to be reliable and consistent. Sweep net collect a lot of trash and incur a risk of canker spread. Whereas, sticky traps would cause delay in making management decisions and allowing psyllids more time to reproduce and acquire or transmit causal pathogen of HLB. Comparison of stem-tap- with vacuum sampling to detect ACP adults in two conventional blocks of citrus showed a strong positive relationship between the two methods explained by a linear equation y = 9.816x + 1.522 (r2 = 0.61) in ‘Earlygold’ and y = 14.79x + 0.044 (r2 = 0.74) in ‘Valencia’. Vacuum sampler detected psyllids in 53-55% samples compared to stem-tap which detected in 29-34% (n = 109-96). Additional data are being collected and analyzed. 3) Psyllid monitoring guidelines are provided at our website: http://www.imok.ufl.edu/entomology/extension/ including instructions and sampling sheets to conduct tap sampling to detect psylilds, other pests and beneficials http://www.imok.ufl.edu/docs/pdf/ento_ext_acp_sampling_english.pdf. This year approx. 3,000 tap sampling kits have been distributed to clientele through workshops conducted at SWFREC and IFAS extension. Two pest scouting workshops one each in 2011 and 2012 were conducted at SWFREC in which citrus growers were trained in monitoring psyllids and other pests and beneficials. In collaboration with the Protect US, Community Invasive Species Network a demonstration of tap sampling and shoot examination was posted at Protect US youtube channel. Stem-tap method has been widely adopted by the citrus industry and also by APHIS and DPI CHRP to monitor 6,000 blocks of citrus in Florida every 3 weeks in support of CHMA program. Tap sampling method was also used to evaluate several experimental and recommended insecticides at SWFREC and large scale studies in commercial citrus designed to control psyllids using insecticides, horticultural oils and nutritionals.
This project is focused on evaluating the impact of psyllid control programs on non-target pests, beneficial insects and mites. Four replicated studies comparing 43 foliar treatments of recommended and experimental insecticides against ACP and citrus leafminer were conducted at SWFREC and submitted for publication in the Journal of Arthropod Management Tests of the Entomological Society of America. Additionally, large replicated blocks of mature citrus in conventional groves untreated or treated with foliar sprays of insecticides, horticultural oils, and nutritionals were sampled for psyllids, 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 at two conventional groves in Hendry County and one each in Lee and Collier Counties. Studies conducted at SWFREC showed that most commonly used and experimental insecticides cause significant reduction in psyllid populations compared to the untreated control for three to four weeks. A few products, such as Delegate, Movento, Agri-flex and Voliam flexi, provided reduction in CLM populations which nevertheless, hardly lasted for two weeks. Most insecticidal sprays against ACP were seen impact beneficial insects and therefore reduce their effects on secondary pests. For example, arboreal ants thought to be critical for CLM were most affected in large plots treated with a calendar spray program for ACP control in Hendry county using selective and broad spectrum products compared to untreated control. Predation on CLM was reduced from 90% to 70% by calendar sprays and parasitism by the formally dominant introduced wasp Ageniaspis citricola was reduced to 3% of the total compared to other less specialized species. Calendar or grower standard sprays also impacted other non target pests and beneficial insects in large scale studies. For example, citrus red mite increased markedly in calendar sprays plots in Hendry county, presumably in response to suppression of predaceous mites. Similarly, more scales, particularly purple and chaff, were observed on fruits in the insecticide-treated plots in Hendry and Collier counties. CRM were also high on fruits in the treatments using insecticides in Collier county compared to treatments of only nutritionals and the untreated control. In contrast, only 2% of shoots on ‘Valencia’ orange were infested with woolly whitefly in plots treated with calendar sprays compared to 34% in control plots in Hendry county. However, shoot infestation with woolly whitefly averaged 36% and did not differ between plots treated with grower standard sprays, biweekly spray of 435 oil or untreated check in a block of ‘Valencia’ in Lee county. It seems that most calendar or grower standard treatments and experimental treatments suppressed psyllids but did not reduce non target pests in most situations, creating need for additional applications and negative impacts on predators and parasitoids. However, psyllid populations were very low during most of the reported period and fewer or no sprays were warranted based on thresholds. Publications: 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. Qureshi, J. A., B. C. Kostyk and P. A. Stansly. 2011. Effectiveness of selective insecticides to control Asian citrus psyllid and citrus leafminer during leaf flushing. Proc. Fla. State Hort. Soc. 124: (In press).
Objectives of this project include: 1) scale up production of Tamarixia radiata to levels that will allow better assessment of the potential impact an augmentation program may have on the ACP population 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. Funding for this project was released in August 2011. 1) This year 303,973, 300,627, 346,778 and 338,881 Tamarixia 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. About 4, 000 wasps from Pakistan colony were also sent to California for their colony and host specificity studies. An additional colony of Florida strain maintained at SWFREC produced 172,112 T. radiata. 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. We have observed parasitism rates of up to 60% or more at release sites in conventional citrus in southwest Florida during spring and summer compared to < 20% at sites with no releases, showing that augmentative release can potentially increase incidence of parasitism by T. radiata in the field. Similarly, parasitism rates averaged 50% in May and 80% in November in one of the organic groves in North Florida where parasitoids were released. However, in three other groves parasitism rates averaged below 25%. We also initiated two new colonies of the biparental colony of Diaphoencyrtus aligarhensis from Pakistan maintained at DPI and SWFREC and released a few in southwest Florida. 2) Collections are made at all release sites and recovered parasitoids were preserved in 95% ETOH and sent to Dr. Evan Braswell of USDA APHIS, Edinburg, TX, to characterize using molecular markers. 3) We have been assisting OrangeCo in their efforts to mass rear, release and evaluate T. radiata. Recently, we initiated with them collaborative experiments to determine the effects of release rates on psyllid populations and to look at the effects of soft and hard insecticide programs on incidence of T. radiata in citrus. Part of this research will be undertaken with the assistance of a self-financed master's student Ziyi Zhang AKA "Mike" with whom we had committee meeting at OrangeCo in Arcadia. chaired by Dr. Norm Leppla. Another master's student, Xulin Chen, whose assistantship is being paid for by this project, has been taking courses in Gainesville since August and has already undertaken a study of the use of CO2 for anesthetizing T. radiata to assist with manipulations during wasp rearing. Findings on the production, field release and evaluation of the parasitic wasp T. radiata against ACP in Florida were presented at two workshops at SWFREC and the annual meeting of the Entomological Society of America held in Reno, Nevada, and at the Subregional Workshop "Biological Control in Huanglongbing Recommendations for Central America" held in San Carlos, Costa Rica.
New outbreaks of invasive fruit flies (Diptera: Tephritidae) continue to threaten agriculture world-wide. Establishment of these pests often results in serious economic and environmental consequences associated with quarantine, control, and eradication programs. Early fruit fly detection and eradication in the United States requires deployment of large numbers of traps baited with the highly attractive male specific parapheromone lures trimedlure (TML), cue-lure (C-L), and methyl eugenol (ME) to detect such pests as Mediterranean fruit fly, Ceratitis capitata (Wiedemann), melon fly, Bactrocera cucurbitae (Coquillett), and oriental fruit fly, B. dorsalis (Hendel), respectively. The current study compared the performance of solid single lure cones and plugs in conjunction with DDVP insecticidal strips; liquid lure with naled formulations; and single, double, and triple solid lure wafers impregnated with insecticide. Treatments were placed in AWPM and Jackson traps under Hawaiian climatic conditions in habitats where B. dorsalis, C. capitata, and B. cucurbitae occur together. The overall goal of this study was to develop a more convenient, effective, and safer means to use male lures and insecticides for improved detection and male annihilation of invasive fruit flies. In survey trials near Kona, HI captures of C. capitata, B. cucurbitae, and B. dorsalis with Mallet TMR wafers were equal to those for the standard TML, ME, and C-L traps used in Florida and California. A solid Mallet TMR wafer is more convenient to handle, safer, and may be used in place of several individual lure and trap systems, potentially reducing costs of large survey and detection programs in Florida and California, and male annihilation programs in Hawaii. Trials of the new TMR wafers have now moved to evaluations in typical citrus growing areas of California. Mallet TMR dispensers are being weathered inside Jackson traps in citrus trees in Tulare, Ventura, Riverside, Kern, and Orange Counties of California. Two series of trials are being conducted in conditions representative of summer (August-September) and winter (January-March) conditions in California. Each week weathered wafers are sent to Kauai Island, HI or Hawaii Island, HI (USDA-ARS-PBARC) for fruit fly bioassays and to Farma Tech International, North Bend, Washington for chemical analysis using conventional gas liquid chromatography methods. Currently, approximately 30,000 sets of TML, ME, and C-L traps are maintained throughout the state. From a worker safety, convenience, and economic standpoint, Farma Tech TMR Mallet solid wafers with DDVP may be more cost effective, convenient, and safer to handle than current liquid lure and insecticide formulations (e.g. naled) used for detection programs for TML, ME and C-L responding flies in California. Cost/benefit analyses of Mallet TMR vs. standard trapping systems will be done.
Our research on use of double stranded RNA (dsRNA) targeting specific psyllid genes has previously shown that, when dsRNA targeting either a psyllid cathepsin or a psyllid vacuolar ATPase gene are fed in artificial diets to the Asian citrus psyllid, an increase in psyllid mortality is realized. Screening of psyllid performance on plants infected with a recombinant CTV vector containing psyllid DNA sequences from these genes that produce dsRNA within citrus phloem was continued. In leaf feeding assays, where psyllids are allowed to feed on excised leaves, leaves from plants containing the CTV-cathepsin construct showed greater mortality to adult psyllids than leaves from control plants. This was confirmed in triplicate studies and the effect of this feeding on psyllid gene regulation is now being characterized. Enough plants have also now been propagated to initiate whole plant challenge studies with psyllids to look at effect on psyllid nymph survival.
The strategy was to identify Asian citrus psyllid genes as targets for dsRNA-mediated RNA inhibition induced by feeding the insects dsRNAs that match coding regions of the selected genes. There was a bias placed on the selection of genes that would be necessary in the digestive tract epithelium lining since dsRNA molecules introduced by feeding would be presumed to be present at the highest concentrations in these cells. These cells are considered to be highly metabolically active in support of the production of oxidative enzymes used in digestion and the transport of nutrients, ions and numerous organic molecules from the digestive tract lumen into the endothelium cells, and from there, into the hemolymph (the insect circulatory system). Figure 1 shows a graph depicting the effect of feeding single gene sequence specific double stranded RNAs (dsRNAs) to the psyllids through the use of an artificial diet. Double-stranded RNA molecules matching eight gene targets were shown to induce significant mortality in adult Asian citrus psyllids when fed to the insects in artificial diets. Mortality (indicated as frequency: percent mortality divided by 100) is presented by normalizing to mortality seen in diets without any dsRNA. We define significant mortality as greater than 30% mortality ( a frequency of 0.3 on the x-axis). This is mortality above what is seen in diet without dsRNA after the insects have fed on the artificial diet for 6 days. A control dsRNA molecule used in this study targeted the sequence of a gene for a protein within another insect (the Diaprepes root weevil- DRW) and is listed in the graph as ‘DRW average’. We consistently saw mortality with the high concentrations of DRW control dsRNA (48 and 24 ng/uL) and this was also observed with dsRNA’s matching other presumed negative controls (i.e. dsRNAs cognate to the green fluorescent protein, GFP). The nature of this non-specific mortality is not yet understood but has been observed by other researchers working with the potato psyllid and the whitefly (Bemisia tabaci). Clearly, not all dsRNAs induced statistically significant mortality when added to diets, so at this time, the nonspecific mortality is not believed to be a general dsRNA toxicity. However, continued experimentation is needed to determine the nature of this toxicity. Interestingly, three of the eight mortality inducing dsRNAs target genes potentially involved in the electron transport pathway (ATP production from oxidative metabolism) and a forth, Ferritin, is involved in regulating iron availability which is a key cofactor in many electron transport pathway protein complexes. Therefore, of the dsRNA molecules that induced significant mortality, 50% could be influencing electron transport functions (ATP production). Of the other four, one is a membrane transporter of unknown substrate specificity, one is a regulatory of Ca2+ ion availability, one is involved in phenylalaline metabolism and the final one is a member of a unique family of Osiris proteins that are dose sensitive lethal and thought to play a role in development.
Two 3-year field experiments are being conducted in two commercial orange blocks in Hendry County (southwest Florida). One of the groves is planted with ‘Earlygold’ oranges and the other with ‘Valencia’ oranges. Average HLB incidence estimated in both groves at the beginning of the experiment based on PCR analysis of a random sample of 160 trees was 98% in ‘Earlygold’ and 42% in ‘Valencia’. Experimental design is randomized complete block with 4 replicates and 4 treatments: (1) No insecticide, (2) Calendar applications, in order to drive vector populations close to 0, (3) nominal threshold of 0.2 psyllids per tap, and (4) nominal threshold of 0.7 psyllids per tap. Calendar applications are being applied every month. In January, treatments (2) and (3) recieved a second dormant spary with zeta-cypermethrin (Mustang) at 4.3 fl oz/ac. In February, treatment (2) was sprayed with spirotetramat (Movento MPC) at 16 fl oz/ac and horticultural mineral oil 2%. All treatments received a foliar nutritional spray according to established protocols. Juice quality analyses from the “Earlygold” block fruit included estimates of Brix/acid ratio and the Lbs solids per box from samples take in each plot. In these analyses it was found significant differences for the Brix/acid ratio between treatments but not for the Lbs solids per box (F = 3.86; df = 3, 15; P = 0.0382 and F = 1.06; df = 3, 15; P = 0.4486 respectively). The highest Brix/acid ratio was obtained for treatment (2) (33.26 ‘ 0.25) and the lowest for treatments (1) and (3) (30.22 ‘ 0.23 and 30.25 ‘ 0.54 respectively). For all treatments, the Brix/acid ratio was higher during 2010 harvest (31.55 ‘ 0.49) than during 2011 harvest (26.05 ‘ 0.55). No differences were found between years for the Lbs solids per box. In the last week of November, the “Valencia” block was harvested and yields, yield increments with respect to the 2010 harvest as well as several fruit quality parameters were measured for each treatment. Yield values were correlated to the cumulative number of ACP adults obtained by stem tap sampling during the last season. A trend toward higher yields in the Calendar treatment (271.88 ‘ 57.75 boxes/acre) and lower yields in the treatment that did not received any sprays (232.03 ‘ 7.85 boxes per acre) was found but differences were not significant (F = 0.32; df = 3, 15; P =0.8086). Similarly, yields did not vary significantly with treatment compared to the previous harvest (F = 1.48; df = 3, 15; P = 0.285) although a trend for decreasing yields was found in the treatments where higher ACP populations was observed (-59.22 ‘ 39.78 and -20.92 ‘ 22.61 boxes/acre for treatments (1) and (4) respectively). On the other hand, a significant treatment effect on fruit diameter (F = 7.37; df = 3, 639; P < 0.0001), weight (F = 7.92; df = 3, 639; P = < 0.0001) and the fruit weight/volume ratio (F = 4.8 df = 3, 639; P < 0.0001) was seen, with higher values for the first two parameters in treatment (2), and for the last parameter in treatment (1). ACP cumulative numbers during 2011 were lower in treatment (2) than in treatment (1), but differences were only marginal (P = 0.063). In addition, no significant correlation was found between cumulative ACP numbers and yield (P = 0.1942). To evaluate the incidence that calendar sprays have on natural enemies an exclusion experiment on ACP infested flushes was conducted during the first flushing period of the year. Mortality in ACP cohorts due to predation was higher in treatment (1) than in (2) (38.29 ' 10.76% and 12.71 ' 10.11% respectively) (F = 3.94; df = 1, 21; P =0.0637). These results show that ACP natural enemies have been negatively affected by a year of calendar sprays.
At the beginning of this project growers were aggressively scouting for and removing HLB infected citrus trees with heavy duty front end loaders. This project had two objectives, the first was to evaluate chemicals that would kill individual citrus trees affected with HLB so they did not have to be removed and the second was to evaluate killing general areas of abandoned citrus trees that are present in Florida and present a large reservoir of the HLB bacteria for vector transmission to healthy commercial trees. For the first objective, canopy spray methods and trunk applications were used to test several chemicals. None of the usual citrus herbicides were effective by any application method, but the herbicide imazapyr effectively killed citrus trees by all application methods. It was consistent in effect at a reasonably low application rate (Albrigo et al, 200?). An alternative method of using the soil fumigant Midas (Iodomethane) was also tested and worked well on smaller trees, 8 or less inch diameter, but was very inconsistent on larger trees (Albrigo et al, 200?). The company manufacturing the fumigant choose not to continue evaluation. As part of this first objective, two machines were designed to apply the imazapyr to the tree trunk in machine cuts followed by the machine spraying the chemical. One machine was a hydraulically operated three saw blade system that moved around the trunk to cut and spray the cuts and was mounted on a small tractor. The second machine was a modified weed cutter with a slide guide to hold the cutter at a uniform distance from the ground and a backpack sprayer with nozzle mounted to the end of the cutter tube. A paper is in preparation describing the design and efficacy of these application machines. Because growers have mostly shifted to nutritional-SAR maintenance of HOLB infected trees no commercial development of the machines or clearance of the herbicide imazapyr for this use. Cooperating with the South Florida Water Management District we monitored aerial application of imazapyr to 560 acres of citrus in an area intended for water retention. The application was very successful in killing the citrus trees in most blocks and appeared as a good way to kill these abandoned stands. Material costs were about $50 per acre and would appear to be cost prohibitive.
In 2011, we started a new project on mandarins, which we conducted at a commercial grove in Bakersfield. The project is of interest for two main reasons. First, mandarins are often planted at a higher density than other varieties; and second, we were able to compare the uptake of imidacloprid into trees that were grown under different irrigation systems ‘ drip (average of 3 drippers per tree) versus sprinkler (placed centrally between trees) whereas other conditions (tree age and size, soil type, volume of water per tree) were similar in adjacent blocks with the two types of irrigation. The significance of tree density is important when the imidacloprid application rate is taken into account. The ‘per acre’ amount of insecticide allowed does not change when the tree density is increased. In short, changing the tree density will alter the amount of imidacloprid delivered to each tree. Our study on mandarins permitted us to determine how effective field rates of imidacloprid were on a high-density citrus planting. The impact of different irrigation systems on the efficacy of imidacloprid uptake is unclear. The manner in which trees are irrigated will likely affect the root pattern. With a drip system, the roots will be trained to a very precise area around each dripper, whereas with a sprinkler system the roots may be more dispersed. In our study, we did not evaluate the rooting patterns for the different systems, but followed the progress of imidacloprid uptake once the treatments had been applied. We also compared 3 application timings, beginning with the first application on June 9 soon after the honey bee netting had been removed from the trees. Additional treatments were applied on July 13 and Aug. 24. The trees used in our study were W. Murcott on ‘Carrizo’ rootstock, and were planted in blocks that were either drip-irrigated (11 years old) or sprinkler-irrigated (9 years old). As with all our trials, we strive to collect the youngest tissue available on the trees because this will be the target for the ACP adults to feed and oviposit on. Excellent uptake of imidacloprid occurred under both irrigation systems. In all but one of our 6 treatments, the 200 ppb threshold was achieved within 1 week of the application. The exception was the June treatment timing to the sprinkler-irrigated block, which took 2 weeks to reach the threshold. Overall, uptake was more rapid with the drip system although there was little to separate the two systems with the mid-summer timing. Peak imidacloprid levels were very high for all treatments, exceeding 1,800 ppb for the three drip applications, and at least 1,200 ppb for the sprinkler applications. In terms of persistence, the efficacy of the June application was 12 weeks; we are still processing data for timing 2 and timing 3 applications, but so far we can see that the persistence of the July applications (timing 2) has extended to 8 weeks, and the August applications (timing 3) to 6 weeks.
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. Unfortunately, the the boron and zinc nutrient deficiencies started to revert in the last quarter of 2011. We continue to truck distilled water to the greenhouse to water the trees. Since we have begun the distilled water treatment, the trees have slowly begun to revert to a nutrient deficient state. The results of the leaf analysis in February showed that the zinc levels have again become deficient but the boron levels had not dropped sufficiently. The rest of the project is waiting on nutrient symptom development. HLB symptom development continues in our plants with the different HLB strains. As soon as strong deficiency symptoms develop, then psyllid testing for objective 1 and 2 will commence and is expected to move rapidly. Tree maintenance has been continued but funds have not been received so all other activities have been curtailed.
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