This program began in June 1, 2009 and ended in February 28, 2011. The goal of the program was to establish a monitoring program for Asian Citrus Psyllid to document an area wide spray psyllid suppression program. Participating agencies were: Douglas Bournique, Principal Investigator, Indian River Citrus League; Michael Rogers, PhD, UF, IFAS Lake Alfred; David Hall, PhD, USDA/ARS Ft. Pierce; Herbert N. Nigg, PhD, Professor Emeritus, UF; Calie Jenkins, FDACS; Jim Carter and Rusty Noah, FDACS, DPI, and Linda Minton, Southeastern Aerial Crop Service, Fort Pierce, Florida. The logistics of this program were as follows. An overall psyllid monitoring program for Indian River, St. Lucie and Martin counties fed data to Dr. Hall at USDA, Ft. Pierce. Data were correlated with the tracking system for area wide pesticide aerial applications through Southeastern Aerial Crop Service. Caribbean Fruit Fly monitoring was conducted in sync with aerial applications to determine if control of psyllid resulted in control of Caribbean Fruit Fly. Dr. Hall analyzed the psyllid data weekly and produced graphs and figures describing the results. The program was established with malathion based upon data provided by Dr. Rogers, UF. Mustang was added based upon psyllid data provided by Dr. Rogers and upon fly data provided by Dr. Nigg and Calie Jenkins. Asian Citrus psyllid was monitored from 6/1/2009 through 2/28/2011 in St. Lucie, Martin and Indian River counties. During the 21 month program 224,190 trees were monitored for psyllid; 29,449 psyllids were counted during this period. From June 2009 through May 2010 abandoned, fresh and juice groves were compared. Caribbean fruit fly was monitored with 400 McPhail traps distributed across a 45-square mile area within the spray program in Vero Beach/Indian River County, north and south of Highway 60 and west of I-95 (St. John’s Marsh area). Varieties included White Marsh, Pink Marsh, Star Ruby and Flame grapefruit and Navel, Hamlin, Valencia, Murcott and Temple oranges. Bait sprays were applied to maintain fly-free certification for export. Psyllid levels were higher in abandoned groves compared to commercial groves. The southeast corner of a block had the most psyllids; the center of a block had the lowest. Psyllids were widespread, but hotspots were common. Due to widespread and effective grower control of psyllid, this monitoring program provided little evidence that the area wide spray program was effective. Psyllid sprays in September 2010 and February 2011 were successful in specific blocks but overall area wide monitoring did not confirm this suppression. Data indicated that an area wide psyllid program also suppressed Caribbean Fruit Fly for the certification program. Grower participation and weather were unanticipated problems in the area wide program. This program established a monitoring program for psyllid populations that continues in 2011 through USDA and FDACS funding with regular reports from Dr. Hall, USDA. Through judicious use of funds, this project returned $20,188 to the Florida citrus growers for other programs.
We are attempting to develop RNAi-based strategies and reagents for the tomato/potato psyllid (Bactericerca cockerelli). We envision applications of our effort as a complementary means for controlling the Asian citrus psyllid, Diaphorina citri. Our hope is that by inducing effective RNAi in recipient psyllids, we can help to control psyllid-vectored plant pathogens with the ultimate target being Ca. Liberibacter asiaticus, the causal agent of citrus greening. We have cloned hundreds of B. cockerelli sequences and developed an artificial feeding system for their delivery and evaluation for RNAi effects in psyllids. Using this system, we screened the homologues of actin, ATPase, Hsp70 and CLIC for RNA interference effects against B. cockerelli. We found increased mortality of dsRNA-fed psyllids and the specific down-regulation of target gene expression. We also constructed an artificial chimeric sequence for actin, ATPase, Hsp70 and CLIC. Oral feeding of dsRNAs for this chimera sequence resulted in significantly higher mortality compared with the dsRNA of any of the individual sequence, demonstrating possible synergistic effects of these sequences on psyllid survival. Interestingly, combination of separately-synthesized dsRNA for these sequences did not increase psyllid mortality. The possible explanation is that some favorable sequence structures featured in the chimera sequence is missing in the combined dsRNAs. This is encouraging, and we will apply this sequence for in vivo expression system to test the efficiency for RNAi effects. Within a given mRNA transcript, the potency of different regions of the transcript sequences can be differentially susceptible for RNAi effects. There is accumulating evidence that the 3′-region of a mRNA is a better RNAi target than is the 5′-region. Based on this possibility, we have attempted to clone the full-length cDNAs for several selected psyllid target RNAs. We have cloned the complete coding sequence of the actin gene, and four dsRNA corresponding to different regions were synthesized and are under evaluation for qualitative and quantitative effects via artificial feeding experiments. The same approach is being used for additional psyllid genes. We are also attempting to develop means for more rapid evaluation of RNAi effects in plants by using plant virus-based expression systems. We have used a Tobacco mosaic virus (TMV)-based plant expression system to express B. cockerelli sequences in plants. However, TMV systemic expression of insert sequences requires approximately 3-4 weeks and resulting infections are non-uniform within tomatoes. We have now evaluated different virus vectors and host plants, including TMV, Tobacco rattle virus (TRV) and Potato virus X (PVX) in different host species such as tomatoes, tobacco, datura, tomatillo and Nicotiana clevelandii, all of which are good host plants for B. cockerelli. TMV systemic infections in tomatillo develop very rapidly, within one week and the tomato psyllid readily feeds on tomatillo. TRV and PVX both also offer opportunities in other plant species. The combination of viruses and plants will allow us to rapidly test RNAi activities in plants.
This project includes experiments being conducted by USDA-ARS and University of Florida on protecting newly planted citrus trees from Asian citrus psyllid and huanglongbing. This progress report reviews one experiment being conducted by USDA-ARS in east-central Florida. Three potential psyllid management programs are being investigated for protecting a new planting of citrus: (1) citrus subjected to an intensive insecticide program (8 treatments per year on a calendar schedule, annual chemical cost of $198/acre); (2) citrus interplanted with jasmine, with citrus subjected to a moderate insecticide program (4 treatments per year on a calendar schedule, annual chemical cost of $156/acre) and jasmine not treated with insecticides; and (3) citrus interplanted with jasmine, with citrus subjected to a moderate insecticide program (4 treatments per year on a calendar schedule, annual chemical cost of $156/acre) and jasmine regularly treated with insecticides (six applications of imidacloprid per year, annual chemical cost of $56/acre) (for treatment 3, a total annual chemical cost of $213/acre). The experiment was in the midst of high psyllid pressure and HLB inoculum. Numbers of psyllids captured on sticky traps indicated that relatively good psyllid control was achieved under the intensive insecticide program ‘ over all sample periods, there was a mean (SEM) number of 0.4 (0.1) psyllids per trap. Under the reduced insecticide program for citrus interplanted with jasmine, relatively poor control of psyllids was achieved in citrus when jasmine was treated with insecticides [1.4 (0.4) psyllids per trap] and even poorer control was achieved in in citrus with jasmine not treated with pesticide [4.7 (0.2) psyllids per trap]. Over all sample periods, psyllids were most abundant in jasmine not treated with insecticides [12.3 (2.9) adults per trap] and moderately abundant in jasmine treated with insecticides [2.4 (0.7) adults per trap]. Lady beetles were relatively abundant in jasmine plants whether jasmine was treated [0.11 (0.03) beetles per trap] or not treated with pesticide [0.13 (0.03) beetles per trap]. Compared to numbers of beetles trapped in jasmine, numbers trapped in citrus were similar ‘ 0.08 (0.02) per trap under treatment 1; 0.16 (0.04) per trap under treatment 2; and 0.11 (0.02) per trap under treatment 3. Although less than 1% of the trees under each treatment tested positive for HLB 12 months after planting, by August 2010 (27 months after planting), the percentage of trees infected by HLB under treatments 1, 2 and 3 averaged 28, 35, and 30%, respectively. There have been no significant differences among the three treatments in percentages of trees infected on any sample date. By February 2011 and across all three treatments, an average of 45.9% of the trees in each plot had become infected and removed. Based on these results, planting new citrus trees and getting them to a producing stage is difficult if HLB is endemic in the area surrounding the new planting and psyllids are not controlled area-wide. Under the circumstances of this small plot experiment, the incidence and spread of HLB have not been reduced under an intensive insecticide program consisting of eight calendar-based sprays annually. It remained possible that a reduction in the incidence and spread of HLB might have been seen in citrus planted with jasmine under either treatment 2 or 3 had these plants been larger in size when the citrus was planted. Also, basing psyllid sprays on scouting data may be superior to basing them on calendar dates, although more than eight sprays might have been needed.
This project started in May 2010 with the objective of evaluating impact of psyllid control programs on non target pests and beneficial insects. During first year we conducted six separate replicated experiments at SWFREC to evaluate impact of recommended and experimental insecticides using 54 treatments on psyllids, leafminers, ladybeetles, spiders, ants and lacewings in 15 yr old trees of ‘Valencia’ oranges. Highlights were reported in the quarterly reports. Two more replicated trials were initiated on oranges in commercial groves in Hendry county, one in a 35 acre block of ‘Early Gold’ and the other in a 16 acre block of ‘Valencia’. These experiments include 4 treatments: three spray programs for psyllid one receiving monthly calendar sprays, two intended to maintain ACP populations below thresholds of 0.2 or 0.7 psyllids per tap sample and an untreated check. Stem tap sampling, vacuum sampling, sticky traps and shoot examination are being used to monitor populations of psyllids and non target pests and beneficial insects. We are also ready to use exclusion techniques to evaluate pest mortality from predaceous and parasitic arthropods under different psyllid control programs and evaluate the relationship with intensity of insecticide use. About 100 cages to restrict predators and parasitoids from reaching the pests colonies on citrus trees were fabricated to make comparisons with unprotected colonies. We have also identified additional commercial groves to monitor populations of Florida red scale, lesser snow scale, and blackflies and whiteflies. Results from this year studies were presented at local and regional meetings and we are working on publications for Arthropod Management Tests, Proceedings of the Florida Horticultural Society, and peer reviewed journals. In addition to measuring the effects of insecticide use on psyllids and non targets we also surveyed grove managers in southwest FL on their insecticide use and incidence of non target pests. Thirty percent reported 6-11 sprays of insecticides to control psyllids during the past 12 months and 100%, 67%, and 33% of them reported seeing an increase in the populations of citrus leafminer, scales and mites, respectively. In contrast, 70% reported 5 or less sprays and only 14% and 29% of those reported increased citrus leafminer and mites, respectively. We also compared organic and conventional groves and found huge differences in the populations of ants and spiders between the two practices. An average of 22 ants and 11 spiders were observed per 140 tap samples conducted in the organic grove compared to 0.3 ants and 4 spiders observed in the conventional grove. Leafminer damage indicated a very serious infestation in the conventional grove compared to the organic grove, suggesting that increased insecticide use can lead to reduced populations of important natural enemies and increase in non target pests. P. A. Stansly, J. A. Qureshi and B. C. Kostyk. 2010. Efficacy of foliar applications of insecticides for control of Asian citrus psyllid and citrus leafminer in oranges during spring. Arthropod Management Tests (In press) P. A. Stansly, J. A. Qureshi and B. C. Kostyk. 2010. Foliar applications of BYI02960 compared to some commonly used insecticides for control of Asian citrus psyllid and citrus leafminer in oranges: summer. Arthropod Management Tests (In press) P. A. Stansly, J. A. Qureshi and B. C. Kostyk. 2010. Effect of spray volume and sprayer type on efficacy of insecticides for control of Asian citrus psyllid and citrus leafminer on oranges. Arthropod Management Tests (In press)
The goal of the present project is to establish economic thresholds under different juice price scenarios that optimize returns on investment when a nutrient/SAR package is being applied in groves with moderate to high incidence of HLB. Two 3-year field experiments were initiated 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 based on PCR analysis of a random sample of 160 trees is 98% in ‘Earlygold’ and 76% 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 will be applied approximately every 1-2 months and consist of a rotation of insecticides recommended for managing this pest. Adult psyllid populations are being monitored every two weeks by tap sampling. Flushing patterns and flush infestation are being estimated by assessing the number of new shoots per tree and evaluating the proportion infested with psyllids. Effects of treatments on natural enemy populations is being evaluated by counting beneficials in tap samples, suction samples and flush observations. Treatments (2), (3) and (4) received the dormant spray on January 20 in the ”Early Gold’ block and on January 21 in the “Valencia” block. Both blocks were sprayed with fenpropathrin (Danitol 2.4 EC) at 8 fl oz/ac. Treatment (2) received an additional spray in both blocks on March 16 using the IGR diflubenzuron (Micromite 80WGS) at 6.25 oz/ac. ACP numbers at the present, in all the treatments of the ‘Early Gold’ block are below the nominal thresholds established (0.01 ‘ 0.01, 0.03 ‘ 0.01, 0.02 ‘ 0.01 and 0.05 ‘ 0.02 ACP adults/tap in treatments (1), (2), (3) and (4) respectively). Greatest cumulative ACP values since the beginning of the experiment are being found in treatments (1) and (4) with significantly higher numbers than in treatment (4) (P = 0.0062; df = 3,15; F = 6.03). In the ‘Valencia’ block, ACP populations are increasing in treatments that have not yet been sprayed (0.53 ‘ 0.14 in treatment (1) and 0.23 ‘ 0.06 in treatment (4) at the beginning of April). Density of ACP adults remains close to zero in treatments (2) and (3). Despite of this, no statistical differences are being found yet in the cumulative ACP numbers between treatments (P = 0.9531; df = 3,15; F = 0.11). Block 1 and 2 were harvested on December 2010 and January 2011 respectively. As yet no significant treatment effect on yield has been observed (P = 0.3582; df = 3,15; F = 1.11 for the ‘Earlygold’ block and P = 0.7033; df = 3,15; F = 0.478 for the ‘Valencia’ block). Neither is the correlation between the yield loss and adult ACP cumulative numbers significant (r2 = 0.0026; P = 0.8516; df = 1,14; F = 0.03632 for the ‘Early Gold’ block and r2 = 0.1390; P = 0.1549; df = 1,14; F = 2,261). There were no significant differences among treatments in juice quality measured as the ratio Brix/acid and the Lbs of Solids per box. Predatory flies, spiders, parasitoids and arboreal ants are the beneficial groups that are being found in highest numbers. Calendar applications seem to be negatively affecting mainly spiders and arboreal ants. Further results will be needed to confirm this tendency.
In this project we are working on 1) refinement of sampling methods, 2) testing the influence of adult density and shoot infestation on precision of estimated means and distribution of population within blocks, and 3) evaluation of methods for assessing psyllid density, shoot density, and infestation rates and their integration into a user friendly system accessible to consultants and managers. Progress for the second year is summarized for each objective. 1) Comparisons of the stem tap sampling, sticky traps and sweep nets on data collected in the commercial groves showed that although all methods detect adult psyllids, the stem tap method is rapid, works under either dry or wet conditions, and has proven to be reliable and consistent. 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. Reliance on sticky traps would cause delay in making management decisions and allowing psyllids more time to reproduce and acquire or transmit causal pathogen of HLB. Sweep nets collect a lot of trash and incurs a risk of canker spread. An extension document (EDIS, ENY857/IN867) describing these methods and monitoring protocols for psyllids is in press. We are now comparing stem tap and vacuum sampling in commercial groves. The vacuum sampler is made from a leaf blower and has collected many psyllids where other methods fail because of low populations. 2) A peer review publication using regression and bootstrapping procedures to analyze data collected on comparisons of different sampling methods in commercial groves over a two year period is being prepared. We are using precision levels of 0.25 and 0.01 SEM:mean in order to determine the number of samples required using these methods for routine monitoring and analytical modeling respectively. 3) Detailed description of why, when, and how to monitor psyllids is provided at our website: swfrec.ifas.ufl.edu/entlab. Sampling sheets to conduct tap sampling and record data are available for download. Scouting workshops conducted in collaboration with Hendry county extension were attended by about 100 participants in February 2010 and 2011 before the start of the season to provide training in monitoring psyllids and other pests of citrus. This year training was recorded for posting on the Protect US, Community Invasive Species Network. A field demonstration of tap sampling method was part of the video. So far, more than 500 participants were trained through presentations and workshops on psyllid sampling and management. A survey of grove managers conducted by IFAS extension and SWFREC showed that to make management decisions 75% of SW Florida growers use tap sampling and 63% monitor psyllids more than 12 times a year. Scouts of Division of Plant Industry CHRP have also been trained to monitor psyllids using methods that we developed. They are using our methods to sample for psyllids in order to determine the effectiveness of area wide dormant sprays. The tap method has also been adopted by APHIS to monitor groves throughout the state in support of CHMA programs. Arevalo, A., J. Qureshi and P. Stansly. 2011. Sampling Asian citrus psyllid (ACP) in Florida citrus groves. EDIS (In press) Stansly, P., A. Arevalo and J. Qureshi. 2010. Monitoring methods for Asian citrus psyllid. Citrus Industry 91(4) 20-22.
Objectives were to: (1) import, release and evaluate new strains and species of parasitoid specific to D. citri. (2) identify genetic markers that can be used to track T. radiata in the environment, (3) develop efficient methods for mass rearing and release of T. radiata and possibly other species to increase biological control through augmentation of natural populations, and (4) transfer technology to industry clientele. A summary of accomplishments to date is given below. Objectives 1 and 3: Following initial evaluations in Florida citrus (Ref 1,2) we recommended augmentation of T. radiata to reduce psyllid populations. We brought in new strains from Vietnam, China, and Pakistan (Ref 3,5) established colonies at DPI- Gainesville as well as of the Florida strain originally brought in from Taiwan and South Vietnam both at DPI and SWFREC. So far, more than 250,000 parasitoids have been released and parasitism rates of 60% or more were observed during growing season at released sites compared to < 20% at non release sites or sites sampled in 2006-7. We also showed that parasitoid releases can be integrated with sprays of horticultural oils and nutritionals to reduce insecticide use during growing season to enhance biological control and reduce risk of pest resistance to insecticides. A Diaphorencyrtus aligarhensis colony from Pakistan was established at DPI ,Gainesville, and D. aligarhensis from China colony released with 6-19% parasitism observed in M. paniculata. We collaborated with the California Department of Food and Agriculture, USDA-ARS, USDA-APHIS, Orange Co. and FDACS-DPI to arrange and conduct a Tamaraxia rearing improvement Workshop in Florida, attended by researchers from Fl. TX. CA. Mexico, Costa Rica and Belize. Orange Co. started their production of T. radiata in 2010 and the DPI-Dundee is expected to come on line this year. Objective 2: A collaborative study with molecular biologist, USDA-ARS Mission TX using genetic markers to identify T. radiata from our colonies, Florida and Caribbean was published (Ref 3) and work aimed at identifying genetic origins of parasitoids recovered from field is in progress. A collaborative study on the searching behavior of T. radiata was also published (Ref 4). Objective 4: An extension document (Ref 5) on T. radiata was published at the Cornell Univeristy website "A Guide to Natural Enemies in North America" in addition to several talks given to clientele at local and regional meetings. References: (1) Qureshi, J. A., M. E. Rogers, D. G. Hall, and P. A. Stansly. 2009. Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology. 102: 247-256. (2) Qureshi, J.A., and Stansly P.A. 2009. Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129'136. (3) Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. J. Econ Entomol. 102: 2325-2332. (4) Mann, R.S., J. A. Qureshi, P. A. Stansly and L.L. Stelinski. 2010. Behavioral responses of Tamarixia radiata (Hymenoptera: Eulophidae) to volatiles emanating from Diaphorina citri Kuwayama (Hemiptera: Psyllidae) and citrus. Journal of Insect Behavior. 23: 447-458. (5) Qureshi, J. A., and P. A. Stansly. 2010. Tamarixia radiata Waterston [Hymenoptera: Eulophidae], an ectoparasitoid of Diaphorina citri Kuwayama [Hemiptera: Psyllidae]: http://www.nysaes.cornell.edu/ent/biocontrol/parasitoids/Tamarixia.html
The objectives of the 3-year projects were: 1) Developing a system for screening antimicrobial molecules against Las bacteria; 2) Screening and evaluating anti-microbial molecules for suppression of Las bacteria. The major accomplishments are summarized as follows: 1. Development of two screening systems (1) Regeneration system of Las-infected periwinkle cuttings: An optimized system using Las-infected periwinkle cuttings was developed to screen chemical compounds effective for controlling the bacterial population while simultaneously assessing their phytotoxicity. The optimal regeneration conditions were determined to be the use of vermiculite as a growth medium for the cuttings, and a fertilization routine using half-strength Murashige and Tucker medium supplemented with both naphthalene acetic acid (4 .g/ml) and indole-3- butyric acid (4 .g/ml). This system allowed a plant regeneration rate of 60.6% for Las-infected cuttings in contrast to the <1% regeneration rate with water alone. (2) Grafting system of HLB-affected citrus scions: The HLB-affected scions from different phenotypic types (yellow shoot and Blotchy mottle) and different cultivars (lemon and grape fruit) were grafted onto Las-free citrus rootstocks (sour orange and grapefruit). The results showed that more than 60% of HLB-affected lemon scions survived and 70% of the inoculated rootstocks were infected at 6 months after grafted using HLB-affected scions with high Las bacterial titers. The results also showed that plant growth regulators (PGRs) increased the survival rates of the grafted scions, but had no effects on the infection rates. 2. Screening antimicrobial compounds against Las bacteria Using the above two screening systems, several chemicals were evaluated for control of Las bacteria in the greenhouse, including antibiotics (Penicillin, Streptomycin, Oxytetracycline, Kasumycin, Metronidazole), a biocide (DBNPA), two peptides (D2A21 and D4E1), two fungicides (Zineb and captain) and Systemic Acquired Resistance (SAR) substances (SA, antiguard and ortho-phenylphenol). Some were effective, such as penicillin and streptomycin, both peptides and Oxytetracycline; some were partially effective, such as DBNPA. The SAR substances, Metronidazole and Zineb were not effective in this research. Kasumycin and Captain need to be tested further. 3. Monitoring the antibiotic effects in the field: Two kinds of antibiotic combinations, PS (Penicillin and Streptomycin) and KO (Kasumycin and Oxytetracycline), were tested in the field at the USHRL farm. The primary results showed that application of the PS via trunk injection eliminated or suppressed the Las bacterium in the HLB-affected citrus plants. The residues of antibiotics were also tested and were not detected in fruit after one months. The field trial is still in progress. Two papers has been or will be published in Phytopathology 2010 and 2011. Four abstracts or posters have been or will be presented at International Citrus meetings or the APS annual meeting. Future research: Continue to monitor treated, field trees; develop an improved method for treating trees with antibiotics; test additional antimicrobial compounds; test antimicrobial compounds in combination with growth enhancing molecules.
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. Objective 1 of our proposal intended to evaluate candidate genes for dsRNA-induced lethality of Diaphorina citri and our model organism, Myzus persicae, using artificial feeding assays. To date, we have cloned sequences at least 400 bp in length from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. While our results suggest that the M. persicae-specific dsRNA has a negative effect on both the lifespan of the insects and the number of offspring generated, additional independent assays are needed to enhance treatment replication to better resolve statistically significant differences among dsRNA treatments. Since the annual report, we have nearly completed our evaluation of the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, this approach may prove to be critical to the success of this study. This research requires the use of Gateway-based vectors that express the selected insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We have cloned and characterized two SUS1 promoter alleles (CsSUS1-1 and 2) from Citrus sinensis cv. valencia and have found them to drive phloem-specific expression in both Arabidopsis and tobacco when fused to a reporter gene. Deletion analyses have shown that two separate phloem-specific enhancer regions (nucleotides -1153 to -462, and -410 to -268) exist in CsSUS1-1. Conversely, only the latter enhancer region promotes phloem-specific expression in CsSUS1-2, while the former region drives constitutive expression. We have also successfully generated Gateway vectors that will result in the constitutive (35S promoter) or phloem-specific (CsSUS1 promoter) expression, respectively, of M. persicae-specific Coo2, GSTS1 and S4e dsRNA, as well as a control derived from GFP. In the fall, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. In the New Year, we will begin transforming citrus with the D. citri-specific gateway vectors for use in subsequent feeding assays with D. citri by Florida collaborators. Initial attempts at citrus transformation at Cornell suggest that Citrus sinensis cvs. Valencia and Hamlin can be transformed and regenerated. In summary, we have cloned a number of transcripts from both D. citri and our model organism, M. persicae, and have analyzed a subset of derived dsRNAs to test their effect on M. persicae using in vitro assays (objective 1). We have also cloned and characterized several novel phloem-specific promoters from C. sinensis, and have evaluated their expression patterns. We also created new Gateway-derived vectors bearing a native citrus phloem-specific promoter, for use in RNAi in our M. persicae model and evaluated them in planta (objective 2). Finally, we’ve now generated similar vectors specifically designed against D. citri (objective 3), and preparing to put them into citrus with our developing citrus transformation infrastructure.
This project is being terminated early due to departure of the post-doc working on the objectives. Certain objectives, including the work investigating the basis of resistance in Cleopatra mandarin will be continued as objectives of other grant proposals. Following this report, no more funds will be spent from this project and should be considered closed out. To summarize the results of the past two years work on this project: We examined the fitness of ACP on various citrus varieties to determine whether ACP populations might be higher on certain varieties. For example, previous reports in the literature suggested that grapefruit was a better host than most sweet orange varieties. In our studies, we found little meaningful difference in suitability of the commonly used citrus cultivars for ACP; in fact, grapefruit appeared to be less acceptable than sweet orange varieties. We also found that the potential for induced host preference did not appear to exist for ACP. Oviposition and survival rate of ACP reared on rutaceous plants in one genus and transferring the F1 generation to a rutaceous plant of an entirely different genus had no effect. One citrus root stock material that we examined, Cleopatra mandarin, was found to be resistant to ACP. Adult psyllids were found to lay eggs on Cleopatra mandarin plants, but upon eclosion, the nymphs failed to complete development to the adult stage. Evaluations of a series of Cleopatra mandarin hybrids identified a number of plants which varied in their susceptibility to ACP. Electrical penetration graph studies investigating the feeding behavior of psyllids on these plants has begun in attempts to help identify the mechanism for this host plant resistance. EPG recording and analysis of psyllid feeding on Cleopatra hybrids will continue in future planned grant proposals. The effect of host plant nutritional status was also examined. In particular, the effects of Nitrogen, Potassium and Boron were found to have the most influence on psyllid fitness. Similar to the results found in other piercing sucking insects such as aphids, increased levels of nitrogen resulted in higher reproductive rate, a shorter development time and overall larger body mass per individual. On the other hand, potassium significantly reduced both reproductive and growth rates. increased rates of Boron resulted in high rates of mortality of ACP. EPG studies of ACP on Boron treated plants altered certain aspects of psyllid feeding behavior.
Progress on a new special regulatory trap for capturing and preserving citrus psyllids in situ has been advanced enough to develop some prototypes for further field testing and refinement. Laboratory bioassays that examined psyllid behavior in response to physical details on the surface of the traps is ongoing. Our premise is that eventually some types of attractants will be identified and deployed with trap visual cues optimized in yellow to enhance the numbers responding to the vicinity of traps. However, the trap capture efficiency will depend on the psyllid behavior on the trap relative to their willingness to orient, land and enter the trap. We are concentrating on testing and improving trap components to optimize these behaviors.
In a previous report, we showed that acquisition efficiency of Ca. Liberibacter asiaticus (Las) by Diaphorina citri was related to the duration of acquisition access periods (AAP), increasing with time available for feeding on infected plants. In this report, we describe the relationship between duration of AAP and transmission efficiency by nymphs and adults. Groups of 30 psyllid adults (1-wk old) or 3rd-4th instar nymphs were confined on leaves of a young shoot of a symptomatic infected plant, with recently expanded leaves, inside sleeve cages, for AAPs of 1.5, 6, 12, 24, 48 or 96 h. After the AAP, the insects of each group were first transferred to healthy citrus seedlings in order to complete a latent period of 14 days at 25C, and then transferred to healthy test seedlings (5 insects/plant) for two consecutive 7-day inoculation access periods (IAP). After the second IAP, total DNA of each insect (sample of 10-20 insects per AAP treatment) was extracted and submitted to qPCR for detection of Ca. L. asiaticus. The experiment was repeated three times, using different source plants of the pathogen for the AAPs. Transmission efficiency of Las by D. citri nymphs and adults, as measured by the proportion of test plants that became infected after exposure to the psyllids during the two consecutive 7-day IAPs, also increased with AAP duration. Las transmission rates increased from 14 to 33% when nymphs were submitted to AAPs ranging from 1.5 to 48 h. In the case of Las acquisition by adults, transmission to test plants was first detected when the insects were allowed a 6-h AAP, reaching a maximum of 19% for an AAP duration of 48 h. Considering that the test plants were inoculated by groups of five psyllids, we used the method proposed by Swallow (1985) to estimate the transmission probabilities by single vectors (P), by using the formula, P = 1-(1-p)1/k , in which p is the proportion of infected test plants and k is the number of individuals used to inoculate each test plant. The transmission probabilities by single psyllids after an AAP of 48 h by nymphs and adults were estimated in 0,077 (or 7.7%) and 0,042 (or 4.2%), respectively. These transmission probabilities are rather low, considering that mean acquisition rates after a 48-h AAP by nymphs and adults were around 90% and 60%, respectively. The occurrence of transmission rates >10 times lower that the acquisition rates strongly suggest the existence of a selective barrier within the psyllid body that may be affecting the passage of the bacterial cells to internal organs involved in inoculation of the pathogen. According to Ammar et al. (2011), a lower proportion of salivary glands were colonized by Las cells in relation to other organs in infective psyllids, suggesting that the passage to the salivary gland may be an important barrier involved in Las transmission. Because significant proportions of nymphs and adults were able to acquire and transmit Las after relatively short AAPs (1.5 and 6 h, respectively), we conduct an additional experiment to evaluate the possibility of bacterium acquisition with AAPs shorter than 1.5 h. Groups of 30 psyllid adults (1-wk old) or third-instar nymphs were confined on leaves of a young shoot of a symptomatic infected plant, with recently expanded leaves, inside sleeve cages, for AAPs of 15, 30, 45, 60 and 90 min. After the AAP, the insects of each group were first transferred to healthy citrus seedlings in order to complete a latent period of 14 days at 25C, and then transferred to healthy test seedlings (5 insects/plant) for two consecutive 7-day inoculation access periods (IAP). After the second IAP, total DNA of each insect (sample of 15 insects per AAP treatment) was extracted and submitted to qPCR for detection of Ca. L. asiaticus. The qPCR results showed that nymphs and adults successfully acquired the pathogen after AAPs ’30 min. No acquisition was observed during the shortest PAA tested (15 min), suggesting that >15 min of psyllid exposure to infected plants is necessary for stylet penetration and contact with infected tissues for successful acquisition of Las.
Report: ACP dwelling in rutaceous plants outside of commercial citrus orchards are a source of HLB. If not controlled, these ACP will stymie the effectiveness of area-wide management programs aimed at containing the spread of HLB in commercial citrus. The proposed research is to develop an autodisseminator (‘dispenser’) of pathogen spores for controlling ACP in residential and organic citrus. The working hypothesis is that ACP will visit the dispenser, pick-up spores, and infect other psyllids when they return to the foliage. The dispenser will be coated with spores of the fungus Isaria fumosorosea (Ifr), a known pathogen of ACP, and will be designed to attract and retain psyllids and to efficiently infect them with spores. Greenhouse tests will determine if infected psyllids can transmit the pathogen to immatures and adult cohorts. Tests will be conducted in local communities to determine if the dispenser can effectively infect ACP in residential trees and to assess spore viability over time. Information from these tests will be used to optimize ACP attraction to the dispenser and spore viability and transfer. The ultimate goal is to design a dispenser that is effective and safe, acceptable to regulatory agencies, and readily adopted by homeowners and organic growers. Exposure of free-fly ACP in a greenhouse resulted in an average of 55% of the ACP becoming infected with pathogen spores (n = 3 trials), demonstrating that the proposed concept is very plausible. The dispenser used for greenhouse tests is constructed from a 21.25 cm x 27.5 cm (8.5 in. x 11 in.) sheet of bright yellow stock paper rolled into a tube with eight pleated ridges running lengthwise across its surface. ACP prefers to crawl along edges and the ridges increase ACP retention on the device, in turn increasing the likelihood of spore infection. The inner portion of each pleat is coated with a thin line of SPLAT (ISCA Technologies, Inc.), an emulsified wax used to dispense scent compounds. The SPLAT contains a standardize aliquot (10’l scent mixture per 10 ml SPLAT) of a terpene mixture based on the emission of volatiles from growing terminal shoots of ACP host plants. Ongoing tests are showing that scented SPLAT induces significantly more probing by ACP than unscented SPLAT. Greenhouse tests will be soon be initiated to determine whether nymphs can be infected from adults carrying Ifr blastospores.
Field trials were conducted in fall 2010 to determine how best to apply systemic insecticides to young trees to gain the longest lasting duration of psyllid control possible. Three neonicotinoid insecticides were evaluated in replicated field plots on trees approximately two years of age. These three products were imidacloprid (Admire), thiamethoxam (Platinum) and clothianidin (a product expected to be registered for use in citrus in the coming year). Each of these products were evaluated using three different application methods; soil drench, soil band spray and trunk application with multiple application rates for each method tested. For the trunk applications with imidacloprid, in addition to evaluating Admire, trunk applications were also made using Confidor 200SL which is the formulation reportedly used in the past for trunk applications in South Africa. The results from our first trial showed that soil-band applications (at the rates and volumes applied) were the least effective method for applying neonics for systemic protection of young trees. The traditional soil-drench application and trunk application appeared to provide the most consistent results in terms of psyllid control. Because of the low psyllid populations present, we were not able to determine whether trunk applications made using different rates provided better control than soil drench applications in this trial. We did however show that our available imidacloprid formulations were equal to, if not more effective than the product (Confidor) used in South Africa that has been touted as providing months of residual control of African psyllids. Work is continuing this spring to further investigate the most effective rate and application method of these products on trees of varying sizes. Work was also initiated in fall 2010 to determine the effect of combining psyllid repellents with soil-applied neonicotinoids to further reduce the likelihood that young trees will become infected with the HLB pathogen before reaching bearing age. These studies have been successful in determining the minimum dose required to deter psyllid feeding and we are currently doing residue studies to determine the effect of rainfall on duration of efficacy.
Imidacloprid is a systemic insecticide that is applied to the soil through irrigation systems. Imidacloprid follows the water-conducting channels (xylem) within the tree and is thus easily distributed to all tissues. Many insects, including the Asian citrus psyllid (ACP), feed from the vascular system. Systemic pesticides like imidacloprid are, therefore, ideal against such pests since they directly target the insects during feeding. One of the attractive features of imidacloprid is its persistence. A good application can result in several months of protection. And, because of its systemic properties, imidacloprid will move from older tissues into developing leaf tissues during flushes. In our research we are studying the conditions within citrus groves that are conducive to better imidacloprid uptake. At a commercial grapefruit grove in Riverside County, we evaluated 2 application rates (1X label rate and an experimental 2X label rate) of imidacloprid in 50-year old trees growing on sandy soil. Late Summer/early Fall applications of imidacloprid were very effective with rapid uptake and persistence through the winter months to the following Spring. At the higher experimental 2X rate, the imidacloprid concentrations in young flush recovered to levels that would be toxic to ACP. With early Spring applications, there was a long delay before the appearance of imidacloprid within the leaf tissue at ACP target threshold levels. The experimental 2X rate was noticeably more effective than the 1X rate, providing good protection to trees at a time that is likely to be critical for ACP control should the insect establish on commercial citrus in California. The delay in uptake observed with the Spring applications may be linked to poor root development at that time of year. Applications later in the year occur at a time when root activity is well established, thereby allowing for a more rapid rate of uptake. At a second site in Ventura County, 24-year old lemon trees growing on a heavy clay soil were treated with the label rate of imidacloprid. Treatments between June and August resulted in very poor uptake and on no occasion during the season did imidacloprid concentrations reach the ACP threshold level. Interestingly, imidacloprid levels within the trees appeared to spike at several times during the season, albeit at low levels. This pattern is likely due to the irrigation schedule used at this site, which is on a 3-week cycle. However, with long periods between irrigations, imidacloprid uptake was less effective. The impact of irrigation frequency on imidacloprid uptake was compounded by the heavy soil conditions at the lemon grove. Imidacloprid can become bound up in heavy clay soils, particularly when water is not available to keep the imidacloprid solubilized. With no water to release it from the binding sites on soil particles, imidacloprid uptake may be compromised. At the grapefruit grove, irrigations were more frequent (weekly) and, combined with the sandier soil conditions, there was less opportunity for imidacloprid to become bound up and unavailable for uptake. Comparisons of imidacloprid behavior in citrus groves are critical to our understanding of the role that imidacloprid will play in the management of ACP.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
