December 2014 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: Tests with with the DMIs are completed with assays with a QoI/DMI on-going. Molecular characterization of CYP51 was completed and there were no unusual features. Manuscript preparation continues. Assays with SDHI’s have been initiated. Objective 2: We are waiting for symptoms to be expressed to collect the trial data. Objective 3: Preliminary analysis has been carried out on Groves I through III. Where Grove I shows total infectivity of the rows scouted, clustering analysis cannot be done. In Grove with emerging disease (Grove II) and intermediate grove (Grove III) both show clustering according to spatial analysis in R (Ripley’s K, Binomial distribution etc’). We are still waiting for color change in fruits to re-map Grove II and III for assessment of spread, and severity. Some initial scouting has begun but symptom expression is not yet at a high enough intensity to get a good estimate of disease intensity. Objective 4: Two sets of in vivo experiments were conducted using large numbers of naturally infected fruit. Fresh Valencia oranges from a block infected with citrus black spot were harvested and evaluated for presence/absence of CBS lesions and equal numbers of randomized fruit were distributed among the different treatments. Fruit were treated with one of seven fungicides at ambient or heated (10 or 13’C) temperatures. The fungicides include azoxystrobin, fludioxonil, pyrimethanil, phosphorous acid, Imazalil, thiabendazole, and sodium-o-phenylphenate. Each of these have formulations that are registered for postharvest use on citrus. Control fruit were either left dry or dipped in water alone. After treatments, the fruits were air dried and kept at 25’C, 70% relative humidity, 3 ppm ethylene, and continuous light condition to promote CBS lesion development. In further analysis of the data it was found that fungicide had a significant effect (P > 0.05) with TBZ having the lowest number of lesions. Neither the solution temperature nor the solution temperature and fungicide interaction had a significant effect on lesion number or percent incidence for asymptomatic fruit. There was a lot of variation among fruit that might have masked effects. There were no significant differences among treatments for symptomatic fruit as expected.
Growers apply insecticides to control ACP as a tactic to prevent or reduce the incidence of HLB but with little or no success. Plant resistance to colonization by ACP populations has been reported in the genotype Poncirus trifoliata L., which is cross compatible with citrus. The Citrus Research Center (CRC) in Riverside, California, maintains a large number of accessions of P. trifoliata. We conducted four free-choice experiments with a total of 29 CRC accessions of P. trifoliata and found that 19 were colonized by fewer eggs than a susceptible sweet-orange cultivar. Subsequent infestation densities of nymphs generally reflected reductions in oviposition but there were notable exceptions ‘ some accessions may be susceptible to oviposition but contain traits that confer antibiosis to nymphs. In no-choice experiments with seedling plants representing eight P. trifoliata accessions, oviposition rates were reduced as compared to sweet orange and it appeared that factors associated with mature leaves were involved. In no-choice experiments with adult ACP confined to flush cuttings, large reductions were observed in numbers of eggs and nymphs on each of five P. trifoliata accessions. Based on these results in conjunction with published reports, a number of accessions of P. trifoliata have exhibited resistance to ACP colonization. Accessions that have consistently shown resistance to oviposition include CRC 2554, 3206, 3207, 3209, 3218, 3338, 3412, 3486, 3547, 3548, 3588, and 3882, The level of resistance has been variable possibly due to environmental conditions, plant age, proximity of plants to susceptible germplasm, and other factors. We continue to study the behavior of ACP adults in choice and no-choice arenas on Citrus macrophylla and Poncirus trifoliata genotypes. Electronic penetration graph (EPG) studies suggest that ACP adults placed on resistant P. trifoliata genotypes spend significantly more time ingesting from xylem and less time ingesting from phloem vascular elements. Work by Dr. El-Desouky Ammar working in our lab and recently published (PLOS ONE 2014 9(10): e10919) suggests that differences in the internal distribution of sclerenchyma cells may inhibit stylet passage resulting in fewer successful feeding bouts from the phloem. Our EPG results support this hypothesis. Data from oviposition studies are being analyzed and will be presented in the next progress report.
We transiently expressed in plants artificial microRNAs (amiRNAs) targeting the V-ATPase mRNA of Bactericera cockerelli. We used agroinfection-compatible Tobacco mosaic virus (TMV) and Tomato mottle virus (ToMoV-A, TAV) expression vectors, and a virus-free 35S promoter driven binary vector and delivered amiRNAs through transient expression in plants and through artificial diets. For the in planta transient expression, the viral vectors, TMV (RNA viral vector) and TAV (DNA viral vector), and non-viral vector carrying amiRNA precursors were expressed via agro-infiltration in Nicotiana tabacum. We harvested small RNAs from tissues and re-sequenced products from these plants to ensure the specificity of amiRNA production. Bioinformatic analyses are underway. For artificial diet feeding experiments, we expressed amiRNAs via agro-infiltration in N. benthamiana plants and the processed amiRNAs were extracted from the infiltrated tissues and mixed with 20% sucrose for feeding tests. We evaluated the B. cockerelli from the feeding tests by mortality assays and reverse transcription (RT)-quantitative PCR (RT-qPCR) for target mRNA expression levels. Our results from the RT-qPCR were compared with the results of small RNA Northern blot and Hi-Seq illumina deep sequencing analysis. In single B. cockerelli-analysis of our initial plant feeding tests, the B. cockerelli fed on tissues expressing high amounts of the amiRNAs of target mRNA showed high variation of target gene expression levels versus more stable expression levels seen in controls. This variation could due to the distribution of amiRNAs in plant tissues and/or the areas where B. cockerelli fed. We are now developing transgenic plants expressing amiRNAs for further biological analysis.
The goal of this project was to test the effectiveness of the entomopathogenic fungus Isaria fumosorosea (Pfr 97) as a biological control tool for the Asian citrus psyllid (ACP) to complement ongoing biological control with the nymphal parasitoid Tamarixia radiata. adults can transmit the spores to existing populations of psyllids. The vertical transmission study was conducted in October-November 2013 at a residential trailer park in Weslaco. For this study, we used laboratory-reared psyllids that were regularly tested for Candidatus Liberibacter asiaticus (CLas) and confirmed CLas free. A group of 500 psyllids was collected from the lab-colony into a 25-ml tube and dusted with the fungus by adding 10 mg of Pfr 97 and gently shaking the tube for 10 seconds. Twenty adults ACP were aspirated in a glass vial and enclosed in a sleeve cage attached to a branch of mature grapefruit trees for a total of 10 replications per treatment. The four treatments tested were 1) flushing shoots infested with dusted ACP, 2) flushing shoots infested with healthy ACP, 3) mature shoots infested with dusted ACP and 4) mature shoots infested with healthy ACP. In the second study conducted in March-June 2014, ACP-infested flush shoots were selected from the same residential areas from lemon trees, and enclosed in sleeve cages. The initial number of adults and nymphs found feeding on these psyllids was counted in situ. Ten Pfr97-dusted psyllids were introduced in the sleeve cages of 10 previously infested flush shoots. Ten other flush shoots serving as control received 10 healthy psyllids. A sample of 20 adult psyllids per flush shoot was collected to determine their Isaria-infection status one week after introducing psyllids in the sleeve cages. One month later, the sleeve cages were removed and the total numbers of psyllid recovered tallied. In the vertical transmission study, mortality of healthy psyllids introduced after the Pfr97-infected psyllids ranged from 6 to 38%, and varied with flush shoot stage and prior contact of shoot with Isaria. Mortality was highest on mature shoots that were previously infested with Isaria-dusted psyllids (38%), while the lowest morality of 6% was recorded on control-young shoots. Prior exposure of flush shoots to Isaria-dusted psyllids increased adult ACP mortality by 17% on young flush shoots and by 26% on mature flush shoots. Three weeks after removal of adult psyllids, no additional psyllid was recovered from nature shoots and only few ACP adults and nymphs were recovered from shoots that were juvenile and expanding at the initiation of the trial. Only a subsample of 18 adults per treatment with young shoot were plated. About 39% (7 out of 18) of psyllids plated were infected with Isaria versus no infected psyllid in the control. These results suggest that Isaria-dusted adult psyllids that colonized a flush can transmit the entomopathogen to the next generation of psyllids and that vertical transmission occurs with Isaria. IIn the second study we observed that 72% of adult psyllids plated from flush shoots that were infested with Isaria-dusted psyllids were infected compared to only 3% in the control treatment. These results suggest that adding Isaria-dusted psyllids to established colonies of psyllids lead to infection of their congeners established on shoots. In fact, of the 20 adult ACP palted for Isaria infection, a maximum of 50% (10 dusted introduced) was expected to be infected, but there was an additional number of psyllids infected that could only come from lateral transmission of from infected individuals introduced in the sleeve cages. The total number of adult psyllids produced from flush shoots exposed to Pfr 97-inoculated ACP was reduced by 21% relative to the untreated control.
This project, in general, seeks to determine if there are any observable influences of foliar fertilizers on HLB-affected grapefruit in the Indian River marketing district. To-date, this project has established 3 separate research trials in commercial grapefruit groves and university research farms in St. Lucie county, Florida. Trial #1 is ~25 years old of ‘Flame’ grapefruit on Swingle rootstock. Trial #2 utilizes ~6 year old ‘Ruby Red’ on Sour orange trees. The first 2 trials are located in commercial groves. Trial #3 seeks to determine the effect(s) that foliar fertilizers have on young tree growth and their ability to protract HLB disease symptoms in 1 year old ‘Ray Ruby’ grapefruit on Kuharske rootstock. For trial #1, there are a total of 30 replicated experimental units; for trial #2, there are a total of 50 replicated experimental units; and, for the trial #3, there are a total of 24 replicated experimental units. Foliar fertilizer applications have been started since February 2014. Tree growth data, leaf nutrition data, and CLas titer measurements have been made. Fruit drop monitoring will begin in September 2014 in trials #1 and #2. In prosecution of these efforts, 2-part-time OPS employees have been hired to do our foliar sprays and collect data observations. As of January 2015, the first year of data collection on the commercial grove plots (trials #2 and #3) as well as the young tree trial at the IRREC (trial #1). First-year fruit yields were gathered in the commercial trials (trials #2 and #3) in December 2014. In addition, all of the foliar fertilizer applications were made (once per quarter) to all trials. Currently, data are being analyzed from the first growing season (2014).
The canker field trial of Zinkicide compared to industry standard management on grapefruit showed better control of canker on fruit than all other treatments. We also observed control of some fungal pathogens. A second year of this field trial used a half and quarter rate of Zinkicide compared to the previous year. The half rate provided equivalent canker control to copper, while the quarter rate started to show reduction in canker control. Analysis of the data from the nanoparticle and non-nanoparticle version of Zinkicide gives insight into possible systemic movement. Dr. Santra’s group has improved synthesis of Zinkicide to reduce the amount of some ingredients needed for synthesis while maintaining efficacy. This improvement is aimed at reducing cost of production. The research team continues working on commercial production and registration for canker although the time line for registration is still too preliminary to accurately predict.
This research seeks to determine whether young trees infected with CLas and displaying typical HLB symptoms can be brought to maturity and produce an economically viable yield. This will be achieved by managing a 58 acre grove of 3-year-old ‘Valencia’ / Kuharske Carrizo trees using a combination of three different foliar and three different ground applied nutritional programs. Factorial AxB treatments consist of A) ground-applied: 1) Liquid/dry+Ca (BHG standard), 2) Liquid+Ca, 3) Liquid/dry-Ca B) foliar-applied: 1) BHG standard-Ca, 2) BHG standard+Ca, 3) “Prescription”(+Ca). The prescription treatment was designed to be dynamic, customized for optimization, with feedback based on frequent leaf tissue analyses, visual symptoms, and the growth of the tree canopies and yield. There are six replications of treatments, with two being pure replications. The grove continues to grow well and also look normal, despite the nearly 100% HLB incidence. Foliation of canopies is dense, and leaf color in the summer and fall was a healthy green. There are no new measurements to report during this quarter. The next reported update will include the fruit yield and quality measurements of the 2014/15 season.
This is a project to develop novel approaches to controlling psyllids . Effective techniques to reduce the rate of Huanglongbing (HLB) spread are key to slowing its incidence, especially for new citrus plantings. RNA-interference (RNAi) is a natural regulatory and anti-viral response in eukaryotes and can be manipulated to target mRNAs/gene expression, including to control insects. Our on-going collaboration has found that RNAi inducers, expressed in citrus trees using the Citrus tristeza virus (CTV) vector, reduce the survival of adult Diaphorina citri moving onto the trees, and greatly reduce their reproduction and acquisition of Candidatus Liberibacter asiaticus by progeny. Our goal is to further improve RNAi activity such that it will help to manage D. citri and HLB, allow reduction in pesticide use and lower grower costs for U.S. citrus. Sequences of specific psyllid genes that are thought to be needed for the survival and reproduction of psyllids are cloned into the CTV vector. As the virus replicates in phloem cells, it produces large amounts of dsRNA intermediates that now also produces dsRNAs containing psyllid sequences. The normal plants RNAi defense mechanism processes the dsRNAs into small 21 nt siRNAs that target mRNA degradation. These siRNAs migrate from virus infected cells into the sieve element. As the psyllid feeds, it sucks up these siRNAs that now target the psyllid mRNAs and prevents the psyllid from making this protein. The lack of this protein has detrimental effects on the survival, reproduction, and CLas acquisition of psyllids. So far, we have seen reduction in survival of adult psyllids placed on RNAi expressing plants, but the effects on reproduction of the new generation of psyllids has been much greater. This is likely because the nymphs are rapidly growing an need lots of new protein synthesis and because they uptake large amounts of phloem sap. Although we have found sequences that can prevent normal expression of specific genes in psyllids, we are looking at several genes to identify sequences that work best. We recently have found that the RNAi target sequences that are expressed from near the 3′ terminus of the CTV vector appear to not be stable enough to be useful for controlling HLB spread in the field. We are recloning all of the target sequences in other positions within the CTV genome looking for an optimal compromise between efficacy and stability.
During this period we continued efforts for artificial micro RNA (amiRNA) delivery and efficacy assessment towards B. cockerelli, but also performed a series of new experiments targeting D. citri via artificial diet, dsRNA feeding. We chose 7 D. citri targets based on our analysis of which target RNAs may be susceptible to RNAi activity, and which could give detectable phenotypic effects. Our intent was to use dsRNAs to help identify mRNAs that we could later target by amiRNAs. We took two approaches. First we generated dsRNAs in vitro, and second, we purchased some in order to perform larger scale experiments. Like in previous studies by ourselves and others, we found that artificial diet feeding of dsRNAs to adult D. citri can induce mortality. All seven tested by us (AI, ATA, ATD, CD, CL, CS and TR) induced effects significantly greater than did the GFP dsRNA controls used at the same concentrations and over the same testing periods. The CS, CD and AI dsRNAs gave slightly greater effects in some experiments. Therefore, we next assessed quantitative effects of these dsRNAs on their specific mRNA targets by using RT-qPCR. All three gave significant target mRNA knockdown, even at very low concentrations used by us. Our efforts now will proceed to design amiRNAs for these targets by using bioinformatics approaches, but also NGS analysis of the D. citri degradome.
Canker disease on fruit and foliage was under evaluation in the following 2014 trials. 1) Soil-applied Actigard was evaluated for the second growing season an EPA-approved Experimental Use Program (EUP) in two east coast grapefruit groves in a collaboration with Syngenta. In general disease pressure was much higher this season and started earlier. Even though the1st applications were made at the same timing as last season, they were late. These factors, along with tree size (8 ft. tall) in one of the locations were reflected in about 20% greater disease control on fruit after four 3.2 oz/acre applications of Actigard compared to copper program for canker control. In a replicated plot trial in an adjacent block of 8 ft. trees, soil drenches of Actigard and season long rotations with Admire and Platinum were effective for suppressing canker and protecting fruit on young Ruby Red grapefruit trees. The control responses were comparable to those obtained with 21-day copper sprays. Registration of non-insecticidal Actigard will address concerns for use of neonicotinoids of larger size trees. 2) Foliar applications of soluble copper and novel bactericidal formulations were compared with standard film-forming copper formulations. Several results were obtained from 2014 trials of Ruby Red grapefruit in Vero Beach and Hamlin orange in south-central Florida: 1) Nordox 75WG, ChampION++ 30WDG and Kocide 2000 and 3000 with ‘1.0 lb of metallic copper per application significantly reduced canker incidence to ~ 20% on Ruby Red grapefruit 2) Nordox 30/30 with 0.5 lb of metallic copper and an equivalent amount of metallic Zn was less effective than Nordox 75WG with 1.0 lb of metallic copper. Nordox 30/30 with 1.0 lb metallic copper gave similar control to Nordox 75WG with 1.0 lb of metallic copper as would be expected if copper is the primary mode of action. 3) Two new Zinkicide formulations from Dr. Swadesh Santra a Nano-chemist at Univ. Central Florida with similar Zn content to Nordox 30/30 reduced canker incidence to 7.0-9.0% which is significantly lower than for all other copper standards. 4) Nordox 75 WG for 5 sprays followed by 5 sprays of Magna-Bon at the 100 ppm rate was nearly as effective as 1.33 lb of Nordox alone at 50% of the metallic copper per season. 5) Magna-Bon at 100 ppm and experimental nano-formulations of copper with or without quaternary ammonium: CS-CuSiNPs, MVCuSiNG pH 4.0, MV1-CuSiNG pH 4.0 plus Quat, or quaternary ammonium alone, Quat DDAC @ 2X or 1X gave nearly equivalent canker control to the copper standards at lower metallic rate. 6) For ‘Hamlin’ orange with average rainfall, canker incidence on fruit and drop was only 10 fruit per tree. Fruit drop from other causes (mainly HLB) was as high as the canker induced drop. 7) The effect of HLB on Hamlin tree vigor, susceptibility of foliage and fruit to infection, as fruit drop may have contributed to the lack of differences among copper formulations in incidence of fruit canker or fruit drop due to canker 3) The performance of two applications of FireWall (streptomycin) with and without the penetrant Nanocanopy was evaluated in a Ruby Red grapefruit trial. Canker incidence on fruit treated with Firewall + Nanocanopy was no different from FireWall alone. In greenhouse assay, the residual activity of FireWall was 10 weeks post foliar application. This confirms that the streptomycin a.i. is locally systemic, remains active and moves acropetally in citrus shoots. 4) Cu bioavailability and residual activity on grapefruit for copper oxide (Nordox 75 WG) and the copper-zinc formulations Nordox 30-30 was evaluated during two periods of summer season (July and August). Zinc may be enhancing the bioavailbility of Cu from the copper oxide in the 30/30 formulation compared to 75 WG copper oxide alone. These results will be presented to the product manufacturer to gain their perspective on this result. 5) Selected results will be presented at the Florida Citrus Show in Ft. Pierce on January 2015
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuable for monitoring CLM but need further evaluation to optimize use for management. The objectives of the proposed project include: (1) assess species composition of trap catches, (2) optimize spatial and temporal distribution of pheromone traps. Sampling from Silver Strand B9 block and the Okaloacoochee Slough State Forest continues. Species composition in trap captures continues to be evaluated by analysis for Cytochrome Oxidase 1 (CO1) sequence generation incorporating an anchored hybrid enrichment probe set from Illumina HiSeq data with the Lemmon Lab at FSU. The probe set is designed to capture a tiled, 250 bp region of the 658 bp CO1 ‘barcode region’. Blast-searches against the comprehensive BOLD sequence library have allowed a faster, bulk-sequencing approach for identification of leafminers. ‘Vince Ficarrotta continues to process samples from P. citrella and related specimens that were shipped to us in 2013. He has been extracting DNA and sequencing samples for COI barcoding. The Kawahara lab recently hired an additional assistant to help with extractions. Vincent is now fully competent in the Geneious program and other related for maximum likelihood phylogenetic analysis, and is planning to submit COI barcodes to Genbank for archiving. So far, 12% of the barcoded Phyllocnistis specimens were identified as species other than P. citrella, and increased the known species of Florida Phyllocnistis species to five. Photography of specimens prior to extraction, so that new species collected in OK Slough can be distinguished from P. citrella by external morphological characters, also continues. Photographs of the specimens will help elucidate these characters in order to provide more accurate identifications. David Plotkin continued work photographing moths through a microscope and generating COI sequence data. David presented leaf-miner work at the ESA meeting in Portland Oregon in November. Work by senior undergraduate student, Emma Roulette, continued dissecting specimens of the two unknown species of Phyllocnistis that are attracted to CLM lures. Examinations of genitalia and comparisons with type specimens in the National Museum of Natural History are being conducted to verify identities. A manuscript: “Placement Density and Longevity of Pheromone Traps for Monitoring of Citrus Leafminer, Phyllocnistis citrella (Lepidoptera: Gracillariidae)” by P. Vanaclocha, M. M. Jones, C. Monz’ and P. A. Stansly has been submitted for publication in a refereed journal. We have initiated two studies to evaluate Rb uptake and detection in CLM for mark-recapture applications. In the first, potted citrus plants have been spiked with RbCl (99% pure – Sigma-Aldrich, St. Louis, MO) at concentrations of 0, 5, 10, 50, 100, 500 and 1000 ppm by introducing 100ml solutions into the potting media. Spiked plants have demonstrated no signs of toxicity to date. For the second, two artificial diet recipes have been prepared. We are currently evaluating these recipes. Artificial diet will be spiked at concentrations of 0, 5, 10, 50, 100, 500 and 1000 ppm RbCl. Adults reared from these diets will be evaluated for Rb concentration by spectrophotometry and potential toxic effects of Rb (mortality, prolonged development time and mass). Once a calibration curve is established, releases will be made and captures compared to background Rb concentrations to establish recapture rates and spatial features of CLM populations.
The canker field trial of Zinkicide compared to industry standard management on grapefruit is ongoing with final disease assessment for the 2014-2015 crop complete. We are planning to start applications this spring with the hopes of collecting a second year of disease control data to confirm positive results observed in the first season. The HLB trial in young Valencia trees continues with regular treatment and disease assessment. The timing of first treatment and the trees flush cycle make it difficult to determine efficacy this soon after initial treatment. We are preparing to collect yield data in the next few months. We will also be closely monitoring new flush for vigor, symptoms and Las titer. HLB symptom ratings were also collected for infected trees in the canker trial because treatment began earlier. However, the data may be biased because the reduction in canker could reduce leaf drop and cause the trees to look healthier.
Current HLB management strategy involves protection of citrus plants from HLB transmitting vector, Asian Citrus Psyllid (ACP) and adaptation of a good nutrition program for preventing root loss as there is no cure available to date. The objective of this research project is to develop and study a new non-phytotoxic, environmentally friendly film-forming ACP repellent material, called hereafter Organo-Silica Composite Film (OSCF). This material form a film barrier on plant surface upon spray application. This barrier is expected to change ACP feeding behavior. Previously we reported synthesis of six new OSCF formulations (OS-SG6 through OS-SG10) using different polymer and cross-linker ingredients. Phytotoxicity testing on Vinca sp and Persian Lime showed no plant tissue damage from any of these formulations. We also looked into potential heat trapping by OSCF material. By using an infra-red (IR) temperature sensor, we monitored the temperature the OSCF coated plant surface (including leaves and stem) at different time points post application of the OSCF materials. As a control, Surround’, a recommended pest deterrent based on Kaolin Clay was included in our studies. Four of the six candidate OSCF materials showed plant temperature fluctuations comparable to that of Surround’ after application on our testing citrus plant, Persian Lime. From the four candidate OSCF formulations that did not exhibit phytotoxicity, OS-SG6 and OS-SG10 were chosen as the most promising candidates. This selection was based on formulation stability at room temperature and minimal change of plant surface temperature before and after spray application (i.e. minimal heat trapping when compared to the Surround’ control). These two OSCF formulations were delivered to UF-CREC collaborators for efficacy testing which will be conducted in a Psyllid house. Detailed materials characterization results on the OS-SG6 and OS-SG10 formulations will be reported in future reports.
Successful area-wide management of the Asian citrus psyllid (ACP) and Huanglongbing (HLB) requires effective control of the psyllid in abandoned citrus groves. Since ACP adults are highly mobile, they can disperse from abandoned to productive citrus groves and spread HLB. ACP is susceptible to Isaria fumosorosea (Ifr), a native entomopathogenic fungi. ‘Autodissemination’ is insect dispersal of a pathogen to members of its own population. Our project goals are to develop and field test an ‘autodissemination’ system for inoculating ACP with Ifr and use these infected psyllids to instigate epizootics that will decimate ACP populations in abandoned citrus groves and; thereby, reduce movement of both ACP and HLB to nearby managed groves. The Ifr formulation is being provided by Dr. Mark Jackson (USDA-ARS, Peoria, IL) and is based on a south Texas isolate (Ifr 3581). During the first year of our project, we field-tested the durability and efficacy of different cardboard and plastic autodisseminator components fabricated to our specifications by AlphaScent Inc. By September of 2014, we determined which components were most suitable for Texas conditions and ordered sufficient quantities from AlphaScent for fall and winter trials (September to December). Due to a delay in obtaining the raw plastic materials required, AlphaScent was not able to deliver the components until the second week of October 2014. During the fall and early winter (October-December) of 2014, we collaborated with Paramount Citrus to repeat field trials in Hildago County with one of their managed groves and an adjacent abandoned grove. The site was a managed Valencia Orange grove next to an abandoned Rio Red Grapefruit grove. The managed grove was treated with insecticides for ACP earlier in the fall and flood-irrigated before our field trial. The abandoned grove was not treated with insecticides or irrigated. At this site, we evaluated the impact of autodisseminators on ACP movement and infestation in plots of abandoned citrus trees and managed citrus trees. For treatment plots, we hung pairs of autodisseminators coated with Ifr-spore formulation and equipped with citrus-blend lures in trees on the edge rows of the abandoned groves. For control plots, we hung autodisseminators with only citrus-blend lures. One week before the start of our trial, we hung ACP sticky traps on the managed trees directly across from the abandoned trees to assess ACP movement from the abandoned grove to the managed grove prior to deployment of autodisseminators. To facilitate monitoring of ACP movement during our trial, we captured and released up to 4800 ACP adults marked with fluorescent powder on abandoned trees every week for three consecutive weeks. We used different fluorescent colors to distinguish psyllids released in treatment plots from psyllids released in control plots. Every seven days during the first to fourth weeks of the trials, we inspected and replaced dispensers and ACP traps. During the fifth week, we also ‘tap sampled’ managed trees and abandoned trees. Movement of ACP from abandoned trees to managed trees was greatly reduced by the deployment of autodisseminators coated with Ifr-spore formulation. Prior to autodisseminator deployment, the numbers of ACP adults trapped on managed trees in treatment plots and control plots were not significantly different. After autodisseminator deployment, weekly trap counts of ACP adults were 28-50 % less for treatment plots compared to control plots. Our findings showed that deployment of Ifr-autodisseminators at the end of the active growing season for commercial citrus in Texas can reduce the numbers of ACP adults moving from abandoned groves to managed groves. We plan to repeat this trial during the spring (March-May) of 2015 to determine if our autodisseminators can reduce movement of ACP from abandoned groves to managed groves at the start of the active growing season in Texas. Deployment of Ifr-autodisseminators at the end and / or start of the growing season should minimize intraguild interactions between Ifr and Tamarixa radiata.
Work continues with a susceptible CLM colony towards estimation of LD50s and LD80s for some commonly used insecticides. Larvae and adults from the susceptible colony are exposed for 48 h to different doses (0; 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100, 300, 600 and 1000 ppm) of: Actara 25WG, Agri-Mek 0.15EC, Cyazypyr, Danitol, Delegate WG, Dimethoate, Intrepid 2F and Micromite 80WGS. For CLM larvae: Actara (thiamethoxam) LD50=4.18 (CL95: 1.86-8.51) ppm, LD80=524.12 (CL95 209.70-1827) (n = 1047; .2 = 9.54; d.f = 10; Heterogeneity = 0.95); Agri-Mek (abamectin): LD50= 0.314 ppm (CL95: 0.13-0.69), LD80=16.68 ppm (CL95: 6.14-78.56) (n = 1249; .2 = 13.17; d.f = 8; Heterogeneity = 1.65); Cyazypyr (cyantraniliprole): LD50=43.36 ppm (CL95: 15.80-131.55), LD80=49,413 (CL95: 0.19E+07) ppm, (n = 1,196; .2 = 7.64; d.f = 8; Heterogeneity = 0.96); Danitol (zeta-cypermethrin): LD50=381.78 (CL95: 86.33-11,771), LD80= 37, 220 (CL95: 31.70-53761.1) ppm (n = 561; .2 = 5.29; d.f = 9; Heterogeneity = 0.59); Delegate (spinetoram): LD50=2.67 (CL95: 0.87-6.42), LD80= 93.75 (CL95: 33.89-489.58) ppm (n = 938; .2 = 10.69; d.f = 7; Heterogeneity = 1.53); Dimethoate (dimethoate) LD50=1.56 ppm (CL95: 0.15-56.31) and LD80=497.45 ppm (CL95: 22.27-0.37E+09) (n = 546; .2 = 13.07; d.f = 6; Heterogeneity = 2.18); and Micromite (diflubenzuron): LD50=74.30 (CL95: 13.63-285.15), LD80=5,723 ppm (CL95: 994-0.129E+07), (n = 816; .2 = 19.92; d.f = 9; Heterogeneity = 2.21). For adults: Micromite LD50=387.40 (CL95: 143-2817), LD80= 9392 (CL95: 0.192E+07) ppm have been estimated but further replicates are required. Contact toxicity for Intrepid LD50 and LD80 values remain inestimable at the concentrations tested (up to 10 ppm). We will continue tests of higher concentrations. Delegate (spinetoram) LD50=2.84 (CL95: 0.77-73.74), LD80= 183.52 (CL95: 16.47-0.44E+07) ppm (n = 430; .2 = 6.30; d.f = 4; Heterogeneity = 1.58). Additional tests of Actara 25WG, Agri-Mek 0.15EC, Cyazypyr, Danitol, and Dimethoate are slated for the coming weeks. Work also continues on the insect growth regulator, methoxyfenozide (Intrepid). This insecticide acts as an ecdysone agonist, interfering with molting, and exhibiting low contact toxicity as indicated above. Therefore, we have initiated larval bioassays on caged branches infested with CLM in the laboratory. Applications of doses of 0; 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100, 300, 600 and 1000 ppm are applied when infested leaves contain 3-4th instar larvae. Evaluations of mortality will be conducted 14d after application. We have also initiated work to develop a bioassay method using artificial diet and two diet recipes and are being tested. We are developing a method to collect large numbers of CLM eggs from the established laboratory colony. Eggs will be transferred to cups containing insecticide-spiked artificial diet to determine diagnostic doses for this compound against larval CLM. Field-collected larvae are currently being evaluated for their responses to these insecticides. Although cases of field resistance have thus far not been observed, populations of CLM are currently high in groves in SW Florida and subjected to frequent insecticide applications for control of ACP, CLM and other pests.
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