Objective 1 is to conduct a field evaluation nutritional sprays for control of HLB and HLB symptom expression and yield. The field study was set up May 2010 in Southern Grove, Hendry Co., FL. Six treatments were set in 4 plots of 150 trees per treatment (interior 10 trees in each block were identified for PCR, leaf nutrition sampling, tree health and yield evaluation). Treatments were 1) non-treated check; 2) Nutri-Phite sprayed 3times bimonthly; 3) N-Sure sprayed bimonthly; 4) Agra Sol Mn/Zn/Fe plus Nutri-Phite plus triazone urea sprayed bimonthly; 5) Keyplex 1400 DP plus Nutriphite plus triazone urea sprayed bimonthly; 6) Wettable powder nutrients (Diamond R #2) plus Nutri-Phite P+K sprayed bimonthly. The materials were applied to both sides of the tree in 125 gallons per acre with an airblast sprayer driven at 2 mph to obtain thorough coverage. Three applications have been completed as of October 2010. Ratings of tree health, leaf nutritional status and PCR status will commence in November 2010. Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application. Hamlin sweet orange trees have been inoculated and are being treated bimonthly with the nutritional sprays. Infection rate and progress are being monitored by qPCR monthly. One month after inoculation, the new flush on a few of graft inoculated trees was weakly positive for Las. Two months after inoculation, new flush on some trees is strongly positive for Las; however, too few of the trees are positive to determine any treatment effects. Three month post inoculation samples are currently being processed. Sampling and fixing of plant material for microscopy of phloem and leaf blade tissue began at two months and continues monthly. Fixed samples will be observed by microscopy once strongly symptomatic tissue is available for comparison.
Transmission of Candidatus Liberibacter asiaticus by the Asian citrus psyllid (ACP), Diaphorina citri, probably involves complex psyllid-pathogen interactions, which should be understood in order to improve management strategies to control HLB. The goal of this research is to determine factors that influence the risks of acquisition or inoculation of the pathogen (Candidatus Liberibacter asiaticus – CLas) by D. citri, e.g vector developmental stage, feeding periods, leaf age and symptom expression/bacterial population in disease plants, in order to optimize strategies to avoid or reduce disease spread within and between citrus groves. We already set up the first four of five proposed studies and the project is progressing as planned originally. Partial results of previous studies showed that bacterial acquisition can occur when ACP adults feed on asymptomatic infected plants, although acquisition efficiency is higher on citrus plants with higher bacterial titers, usually symptomatic. We also have reported that acquisition efficiency by psyllid adults is markedly affected by citrus leaf age and duration of acquisition access periods (AAP). Efficient acquisition depends on the availability of young leaves in infected plants and long AAPs (>2 days). In this quarterly report, we present details of the first study, concluded in September/2010, which was carried out to determine how acquisition efficiency of CLas varies in relation to ACP developmental stages. Health insects of known age were obtained from a lab colony maintained on Murraya paniculata. In each experiment trial, groups of 40 adults with 3-5 days post-emergence and newly-molted nymphs of all development stadia (1st, 2nd, 3rd, 4th and 5th instars) were confined on young leaves of a same symptomatic branch of an infected plant, during a 48-h AAP. Psyllids of each treatment (developmental stage) were confined on a separate leaf. After the AAP, the insects were maintained on healthy sweet orange seedlings for 21 days (healthy seedlings were replaced after the first 14 days). Surviving psyllids were then fixed in 100% ethanol and individually tested for the presence of CLas by real-time quantitative PCR (qPCR). As a negative control, a group of 40 psyllids of the same rearing batch was transferred directly to the healthy seedlings (without previous AAP on infected plants). The experiment was repeated three times using distinct pathogen source plants. Acquisition efficiencies by 1st, 2nd, 3rd, 4th and 5th instars, as measured by the proportion of infective psyllids by qPCR, were 68.4, 60, 66.6, 84 and 65.3%, respectively. In contrast, only 42.9% individuals were infective when acquisition took place during the adult stage. Interestingly, a higher concentration of CLas was observed in psyllids that acquired the bacterium during the 4th or 5th instar. These results show that all developmental stages of ACP are able to acquire the bacterium with moderate to high efficiencies during a 48-h AAP on infected plants, but nymphs are more efficient than adults. We are now concluding PCR assays for detection of CLas in the test seedlings exposed to the psyllids of each treatment, to check if the higher acquisition rates by nymphs result in higher transmission rates of this pathogen.
Imidacloprid (Confidor 700 GrDA), 0.35 g AI/plant and thiamethoxam (Actara 250 WG) 0.25 g AI/plant, applied in the nursery tree bags, before planting, was efficient to control ACP until 60 days after application. The time to cause 100% of ACP mortality was between 5 to 7 days after the confinement of adults in treated plants. However, researches using electrical penetration graph (EPG) showed that in plants treated with imidacloprid and thiamethoxam, after the first feeding on phloem, the adults do not do more probing. We carried out the first PCR of the plants in this experiment and the results were negative, no plants have been detected the presence of the bacterium L. Ca asiaticus. No transmission results yet. We finish the second experiment that was performed to determine if the systemic insecticides are effective until 90 days after application and its effect on transmission of the bacteria. In this experiment, the time to reach 100% of mortality ranged from 3 to 7 days for both systemic insecticides tested (imidacloprid and thiamethoxam). The insecticides were effective up to 90 days after application. The results of PCR carried out for the ACP, in some periods, were positive for 100% of the samples, consisting of 10 insects tested, but in the confinement held at 46 days after application, in any sample was detected the presence of the bacteria. No acquisition in this period. In bioassays performed at 75 and 90 days after application, the percentage of positive samples was 50 to 70% and 10 to 40%, respectively. We finish the experiment 2, the difference from the experiment 1 is the application of varying doses of the systemic insecticides and confinement of the ACP in plants treated only 7 days after application. To thiamethoxam (Actara 250 WG), the doses tested were: 1, 0.5, 0.1 and 0.05 g/nursery tree and imidacloprid (Provado 200 SC) were: 1.75, 0.9, 0.2 and 0.08 mL/nursery tree. We also finish the experiment 3, using different insecticide spraying to determine if they prevent the transmission and for how long. In both experiments the systemic insecticides were effective in control of ACP, but have no result of transmission. Plants treated with insecticide, the proportion of insects reaching the phloem was similar between plants treated with imidacloprid (0.35 g AI/tree), thiamethoxam (0.25 g AI/tree) and control (untreated plants), being respectively 74, 72 and 76%. The time to perform the first ACP salivation was also similar between treatments, 118.4, 103.2, and 112.6 minutes, respectively. However, the time of phloem ingestion is drastically reduced compared to untreated plants. Apparently, ACP can only distinguish between plants with and without treatment from the moment that start ingesting the phloem sap. In this case, it was observed that after ingestion of sap with insecticide, the ACP removes the stylet from the plant and rarely returns to start a new probe on the same plant. With systemic insecticides, the main interference on probing behavior occurred during the phloem phase; phloem sap ingestion, as measured by duration of waveform E2, is significantly reduced (approximately 91%), and the insect subsequently withdrawals the stylets from the plant and rarely restarts a new probe. We finish the experiment to evaluate the effect of oil on the feeding behavior of ACP and its effect on repellency of the vector. The results showed that up to 21 days after application, mineral oil, 1.5% and 1.0%, shows repellency to adults of D. citri. Using electrical penetration graphs (EPG) techniques, we are studying the probing behavior of ACP in plants that were applied mineral oil. Plants sprayed with insecticides and M.O. affect mainly the initial phase of probing (pathway). In the case of insecticides, D. citri was able to perform probes 1 DAA, but it was able to reach the phloem only in the evaluation of 15 DAA. However, on plants treated with M.O. around 32% of insects were able to reach the phloem 1 DAA. We started the EPG experiment using pymetrozine.
Work on Objective 1 was initiated this season. The purpose of this objective was 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. Using the same material and rate as used in South Africa where several months of control are touted, we will be able to determine if we can obtain the same level of control reported from South Africa and also determine if by altering application method can we use our currently labeled products to obtain a level of control similar to that reported from S. Africa under Florida growing conditions. Currently, this trial is still ongoing with weekly evaluations of psyllid adults and nymphs being made. We are also collecting data on control of citrus leafminer to ensure that changing application methods does not have any negative effects on control of this pest as well. Results should be reported in the next quarterly progress report.
Previously we reported on results from this project indicating that Cleopatra mandarin is an unsuitable host for the Asian citrus psyllid with little or no reproduction occurring on this citrus species (Citrus reshni Hort. ex Tan.). These results were recently published: Tsagkarakis, A.E. and M.E. Rogers. 2010. Suitability of ‘Cleopatra’ mandarin as a host plant for Diaphorina citri (Hemiptera: Psyllidae). Florida Entomologist 93 (3): 451-453. As a followup to this work, we conducted additional studies with Cleopatra mandarin hybrids to determine if there would be differences among these hybrids in susceptibility to psyllids that might be used in future psyllid management approaches. For these experiments, 30 field grown mature Cleopatra mandarin hybrids were selected for evaluation. For each of these hybrids selected, young flush was available for psyllid oviposition at the time experiments were conducted. The initial evaluation was conducted by caging adult psyllids on the new flushes of each of the 30 hybrids which were selected at random. The flushes remained caged for the next 21 days to allow for psyllid oviposition, development of nymphs and subsequent emergence to the adult stage to ensure that psyllids were able to complete their lifecycle on these hybrids. In this initial evaluation, there was successful psyllid reproduction on 9 of the 30 hybrids. It appeared that there was some gradient of suitability between these plants however with some plants having only 1 or 2 surviving offspring whereas other hybrids produced up to 30 offspring per flushing terminal. This experiment was repeated a second time using all the plants for which some psyllid reproduction was found and an equal number of plants for which no psyllid reproduction was previously found. In this second round of evaluations, we again found psyllids were able to reproduce and complete development successfully on all of the Cleopatra hybrids for which there previously had been relatively high levels of reproductive success (10-30 offspring per branch). However, we did not see successful development on some of the hybrids which previously had low levels of psyllid development. We also did not see any reproduction on the hybrids for which there previously was no reproduction recorded. Thus, based on the outcome of these two evaluations, we have identified three groups of Cleopatra mandarin hybrids with varying levels of susceptibility to psyllids; no reproduction, little or no reproduction, and moderate to high reproductive success. The next step in this work will be to evaluate plants from these three susceptibility groups to determine if there are physical or chemical differences that are responsible for the suitability of the host plant for psyllid development.
A paper resulting from this funded project was recently published: Pelz-Stelinski, K.S., Brlansky, R.H., Ebert, T.A. and M.E. Rogers. 2010. Transmission parameters for Candidatus Liberibacter asiaticus by Asian Citrus Psyllid (Hemiptera: Psyllidae). Journal of Economic Entomology 103 (5): 1531-1541. ABSTRACT The purpose of this investigation was to evaluate acquisition and inoculation efficiency of Candidatus Liberibacter asiaticus (Las), the pathogen associated with huanglongbing (HLB) or citrus greening disease, by the Asian citrus psyllid, Diaphorina citri (Kuwayama). In laboratory studies, nymphs reared on Las-infected plants were more likely to acquire the bacterium than adults. Acquisition by nymphs ranged from 60-100%, whereas acquisition by adults only reached 40% after 5 wk of feeding on Ca. Las infected plants. Similar rates of pathogen acquisition by psyllids following nymphal and adult feeding were observed in the field. Transmission of Ca. Las from parent to offspring (transovarial) occurred at a rate of 2-6%. One year after psyllid inoculations, successful transmission by individual D. citri ranged from 4-10%, while groups of 100 or more D. citri transmitted the pathogen at a rate of approximately 88%. In addition, the proportion of Ca. Las-positive adult psyllids, determined using quantitative real-time PCR, decreased over time when held on healthy plants. Due to the low rate of pathogen acquisition and long period required for successful inoculation by adult D. citri, experiments designed to determine the latent period required for replication and successful inoculation of Ca. Las by D. citri did not result in Las infected plants after more than one year of incubation following inoculation. Collectively, these results indicate that adult D. citri which acquire the HLB pathogen as adults are poor vectors of the pathogen compared to adults which acquired the pathogen as nymphs. END ABSTRACT. In other ongoing related work…experiments to evaluate the effect of temperature on acquisition are nearly finished. Preliminary data indicate that peak acquisition occurs between 25-30C, with approximately 50% of D. citri becoming infected after two weeks of feeding on infected plants. Only 4% of psyllids acquired Las at 20C. In addition, Las acquisition decreased at temperatures above 30C, although acquisition was not as low as that observed at 20C. Collectively, these data suggest that little acquisition of Las occurs during winter months when temperatures are low. Currently, experiments are underway to evaluate the effect of temperature on transmission. Studies to determine whether there are fluctuations in the seasonality of pathogen transmission by psyllids continues. Since this study began in 2008, by analyzing more than 80,000 psyllids collected from 6 studies sites across the state we have documented significant grove-to-grove, year-to-year and month-to-month variability in the number of Las+ psyllids. However, there has been no consistent pattern in the month-to-month change in number of Las+ psyllids within each grove. The changes in abundance of Las+ psyllids is most likely a cyclical pattern associated more with plant related factors than weather per se. The one pattern most evident from this work however is the overall abundance of Las+ psyllids from year-to-year. In 2008, a low level of Las+ psyllids were collected from most sites followed by a dramatic increase in the abundance of Las+ psyllids at all sites in 2009. In 2010, to date, the overall abundance of Las+ psyllids has been much lower, possibly as a result of enhanced psyllid control programs being conducted.
All experiments are already established and we are now waiting for symptoms appearance. Exp.1 – It was observed the predominance of diseased plants with positive conventional PCR for CLas in the two compartments where citrus plants served as primary source of inoculum of CLas and CLam. In compartment 1, CLas infected 9.6% of test plants (15 of 156) and in compartment 2, CLas infected 13.5% (21 of 156) and Clam infected 1.9% of plants (3 of 156). The first CLas and CLam citrus infected plant were detected respectively 6 and 27 months after the beginning of the experiment. From the three Clam citrus infected plants, two had also CLas in a mix infection. There were 12 infected citrus plants with CLam in compartment 4, where Murraya exotica served as source of inoculum and no plant with positive for CLam in compartment 3, where citrus served as inoculum source. Since the ACP population was much higher in the last three months of 2009, all plants were moved to other insect-proof screenhouse and will be there for symptoms observation and leaf sampling for PCR analysis at least until December 2011. A new leaf sampling was done at the beginning of October to check the presence of both liberibacters in new test plants. A replication of this experiment was started in October 2009 to repeat this experiment. Exp. 2- A factorial designed experiment with two HLB bacterium species (CLam and CLas) and two sources of inoculum (symptomatic and asymptomatic) were established. Five to ten adults of Diaphorina citri had the access to infected sweet orange plants (symptomatic and asymptomatic) during 2-3 weeks. Both those adults and the adults emerged from nymphs developed in those infected sweet orange plants were transferred to young flushes of health sweet orange plants (test plants) to evaluate the proportion of transmission of the bacterium species. The adults used in the tentatively transmission of liberibacters were processed by PCR. Also, during 12-18 months all test plants used to HLB-insect transmission will be evaluated about the symptom expression and the presence/concentration of the HLB bacterium by qPCR. We are waiting for the PCR results. Exp. 3 – No HLB-symptoms and infection were observed yet on inoculated trees at different sweet orange varieties and ages that were encaged under insect proof screenhouses. As an alternative experiment, even without control of number and time of infections, 30 trees with initial symptoms of HLB were selected in different citrus blocks (B1 – Hamlin/Swingle planted in 2007; B2 – Valencia/Volkamer planted in 2007; B3 – Valencia/Swingle planted in 2007; B4 -Valencia/Swingle planted in 1999, and B5 – Valencia/Swingle planted in 1995) in a farm without removal of symptomatic trees but with strong ACP control program. In that farm, last inspection and eradication of HLB-symptomatic trees was done in April 2010. The symptomatic tree selection was done in the end of June 2010, so the symptoms on selected trees were quite new (less than 2 months). The average initial severity was 10.8%, 8.6%, 7.9%, 4.6%, and 6.2% respectively for B1 to B5. The disease severity (% of symptomatic canopy) of each tree is being periodically assessed. A new group of new symptomatic trees in the same blocks will be selected in December/10.
We continued the assessments of two experiments under high and low external inoculum pressure (E1 and E2, respectively). At the end of September 2010, HLB incidence in plots without vector control was 76.8% and 6.2% in E1 and E2, respectively. In plots with vector control program, the disease incidence was 53.1% for E1 (insecticide sprays every 14 days) and 4.6% and 6.3% for E2 (every 14 days and 28 days, respectively). This results indicates that with regional management of HLB, with less intensive vector control program (insecticide sprays every 28 days, or even no insecticide sprays) it is possible to be more efficient than a high intensive vector control program (insecticide sprays every 14 days) applied locally within a region without regional management. It has been observed that cumulative ACP adult population caught per yellow stick trap in plots without vector control program in E1 was high and quite stable year after year (5.0 in 2007, 30.5 in 2008, 10.8 in 2009 and 13.9 in 2010), while the cumulative population in plots without vector control program in E2 has decreased year after year (11.3 in 2007, 2.4 in 2008, 2.2 in 2009 and 0.1 in 2010). It is an indication that the local population of ACP do not stay in the plots without vector control during all the year (probably after some period or year season all adults move to other place), and the yearly population with be dependent of the influx of ACP from outside citrus blocks or alternative hosts. With the implementation of regional HLB management around E2, the influx of migrant ACP population has decreased yearly causing the reduction of the initial ACP population on plots without vector control. No significant differences on HLB progress rate and HLB incidence among different frequencies of local inoculum reduction in each experiment was observed yet. However in a regional scale, the 3-4 inspection/eradication of symptomatic HLB trees around E2, were enough to reduce the average incidence of HLB in E2 compared with E1 surrounded by citrus without any eradication of HLB infected trees. PCR-positive ACPs have been observed both in plots with and without vector control program for E1 and no psyllid sample was positive for Candidatus Liberibacter spp. in Exp.2. This result could explain why even with similar population of adults ACP in plots with vector control, the incidence of HLB-symptomatic trees were much lower in vector controlled plots of E2. Spatial analysis of annual maps of HLB-symptomatic trees were done at USDA lab in Fort Pierce by Gottwald’s team by stochastic models (MCMC) to verify the effects of each treatment on primary and secondary spread of HLB. Now we are interpreting the outcome results. The assessments on both experiments will continue to allow more detailed temporal and spatial analysis, as well economical analysis, and get final conclusions. These and others preliminary results were recently presented in two workshops in Mexico (2nd International Workshop on Huanglongbing and Asian Citrus Psyllid – July/10, and the 2nd International Citrus Research Workshop, July/10), and two meetings in Brazil (the Annual Meeting of Brazilian Society of Phytopathology, Aug/10, and in the Brazilian Workshop of Plant Disease Epidemiology, Oct/10). The results of this project are also being presented to growers to help the establishment of growers’ groups for regional management of HLB in S’o Paulo, Brazil.
July, 2010 ‘ Met with Dr. Steve Rogers in Winter Haven to discuss and submit raw data and reports developed from the project. August, 2010 – No Activity September 2, 2010 ‘ Submitted a no-cost extension to FCPRAC due to the volume and complexity of the data resulting from the project.
No progress in the field trials has been made since the October update. Currently we are planning multiple field trials to begin in spring 2011 that will validate the greenhouse studies on the effects of altering host plant nutrition on psyllid population dynamics. These trials will evaluate use of differing nutrient strategies to help manage psyllid populations using certain nutrients applied at rates that would be feasible for commercial citrus production without undue harm to the overall grove production or tree health. We are also in the process of propagating more ACP resistant and susceptible Cleo hybrids for more in depth studies on the mechanism of resistance to ACP observed in some Cleo plants in the greenhouse and field trials.
The results so far indicate that with regional management of HLB: – The disease epidemics starts later (299 days) and is slower (disease progress rate reduced in 75%); – Increases the efficiency of local control measures because reduces the migrant psyllid population (ACP population is decreased year after year even in plots without insecticide application), reduces the regional inoculum sources, and finally reduces the migrant infective psyllid population that reach the local plots. – Reduces the control costs because with less intensive vector control program (insecticide sprays every 28 days, or even no insecticide sprays) it is possible to be more efficient than a high intensive vector control program (insecticide sprays every 14 days) applied locally within a region without regional management. – Allows resets and solid block replanting with the guarantee of yield. In plots of E2 without and with vector control the yield still increasing (2 ton/ha in the first, 16 ton/ha in the second and 38 ton/ha in the third harvest) while in plots of E1 the yield decreased year after year (2 ton/ha in the first, 9 ton/ha in the second and 10 ton/ha in the third harvest for plots with insecticide application, and 2 ton/ha in the first, 4 ton/ha in the second and 3 ton/ha in the third harvest for plots without insecticide application). Now we are interpreting the outcome results from spatial analysis of annual maps of HLB-symptomatic trees to verify the effects of each treatment on primary and secondary spread of HLB. Experiment 1 (E1) under high external inoculum pressure was finished at the end of November 2010 with 76.7% of infected trees in plots without vector control and 53.0% in plots with vector control. Experiment 2 (E2) under low external inoculum pressure is with 4-6% of eradicated trees and will be assessed for more time for better understand about the temporal longevity of a grove under regional management of HLB. These preliminary results will be presented in the 2nd International Research Conference on Huanglongbing in Orlando ‘ January/11. A paper with the temporal analysis of both experiments is on preparation and would be submitted for publication until April/11. The results of this project are also being presented to growers to help the establishment of growers’ groups for regional management of HLB in other countries.
Research on low volume spray application technology has been continued with complementing field and laboratory investigations. Field investigations were conducted to gain a better understanding of how efficacy of various chemistries may differ when applied at different times of the year. In addition, the effect of various adjuvants on droplet size and efficacy of low volume sprays has been investigated. Laboratory investigations have focused on the impact of spray droplet size on mortality of Asian citrus psyllid (ACP) as well as how adjuvants impact droplet size range. Results from the field investigations suggest that applications of the pyrethroid, Danitol, are not equally effective against ACP at all times of the year and under all environmental conditions. The results suggest that effectiveness of this treatment applied by low volume may be lower later in the summer as compared with winter and early spring applications. This may be due to increased humidity and wetness of foliage in the summer as compared with the fall and early spring. Our laboratory evaluations with several pesticides to date (Danitol, Lorsban, Dibrom) consistently show that mortality of psyllid eggs, nymphs, and adults increases as droplet size is decreases. With all products tested in the laboratory to date optimal psyllid kill appears to be with about 100 micron droplets. However, droplet size measurement of Danitol, when sprayed with a commonly used low volume applicator, revealed larger than the optimum and more variable droplet size range. To remedy this problem, we have investigated whether addition of various adjuvants to Danitol could reduce average droplet size. We found that addition of the organosilicone adjuvant (Sylgard) at 5 % by volume could reduce average droplet size of the low volume Danitol spray for optimal psyllid kill and compliance with the label requirement. Addition of this adjuvant also makes droplets much more uniform in size. The effect of additional organosilicone adjuvants on droplet size of low volume application is currently being investigated. We are currently investigating whether those adjuvants could improve efficacy of low volume sprays with insecticides such as Danitol and whether their addition could improve efficacy when applied to wet foliage.
This project started in May and the previous report was submitted in August. The main objective of the project is to evaluate impact of psyllid control programs on non target pests and beneficial insects in citrus groves. Increased use of insecticides to control psyllid continues to show negative impact on non target pests of citrus. In a recent survey of grove managers in southwest FL, 30% 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. Studies conducted before the advent of HLB in Florida in 2005 showed huge impact of predation, particularly by ants, on citrus leafminer populations. It appears that increased use of insecticides in conventional groves is significantly impacting populations of ants and/or other natural enemies. A recent comparison of organic and conventional groves in Charlotte county showed huge difference 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. Trees in both groves were not flushing at the time of sampling, but old damage indicated a very serious infestation of citrus leafminer in the conventional grove compared to the organic grove. Ants and spiders were also the only common predators in the citrus block in Glades county which was also sampled in September and is being treated with nutritional mix and 435 Oil @ 2%. Ants and spiders averaged 0.5 and 0.3 per 10 tap samples in the treated blocks respectively, compared to 1.4 and 0.6, respectively, in the untreated plots. Only a few young shoots were available and 38% and 75% were infested in treated and untreated plots, respectively. A trial comparing low vs. high volume applications of Movento along with Mustang Max, Baythroid, Provado, and Agrimek was initiated to look at impact on psyllids and non target pests and beneficial insects. Data is being collected and will be presented in the next report. Two more replicated trials are being initiated on oranges in Hendry county, one in a 35 acre block of ‘Early Gold’ and the other in a 16 acre block of ‘Valencia’. There are 4 treatments: three spray programs 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. The plan is to use exclusion cages to evaluate mortality from predaceous and parasitic arthropods in each plot and evaluate the relationship with intensity of insecticide use. Additionally, we are monitoring infestations of Florida red scale, lesser snow scale and black fly in commercial groves.
We are attempting to develop an RNAi-based approach for controlling psyllids. Our goal is to identify and then use specific psyllid RNA sequences to induce RNA interference activity in recipient psyllids. To make rapid progress we are using the tomato psyllid (Bactericerca cockerelli), which colonizes herbaceous plants and is the vector of Liberibacter spp. (Ca. L. psyllaurous), which is believed to cause psyllid yellows in tomato. Currently, sequence information of B. cockerelli is very limited in available databases. However, we were able to clone 28 gene fragments from B. cockerelli by using D. citri homologous primers from the D. citri midgut cDNA library database. We also generated a B. cockerelli normalized cDNA library and so far have sequenced less than 100 targets. We have developed three approaches for delivering candidate RNAs to psyllids including a Tobacco mosaic virus (TMV)-based expression system and tomato plants; micro-injection of dsRNAs directly into the psyllid hemocoel; and artificial diet membrane feeding in vitro. We have used our artificial diet system for screening candidate dsRNAs for B .cockerelli. Psyllid acquisition of the artificial diet was confirmed by food dye and Cy3-labelled dsRNA visualization. Both are obviously apparent in the psyllid gut tissues. When the dsRNAs for psyllid sequences were added into the food, we saw increased mortality of B. cockerelli for some sequences at higher concentrations of dsRNA. Four of ten sequences caused substantial psyllid mortality as compared with GFP dsRNA controls. Consistent and specific down-regulation of endogenous mRNA also was revealed by qPCR, and corresponding siRNAs were detected by Northern blot analysis. These latter two analyses demonstrate RNAi activity in psyllids. Second, we are using a TMV-based plant expression system which is efficient for producing dsRNAs/siRNAs for insert psyllid sequences in tomato plants. So far, decreased survival of B .cockerelli was detected for 2 from 6 sequences after 10 day feeding on plants. We are in the process of analyzing psyllids for RNA hallmarks of RNAi activity. Direct intrathoracic injection of dsRNAs into the psyllid hemocoel is also being used to test candidate sequences. Survival for injected psyllids ranges from 50 ‘ 60% three days post injection. We are using injection only as a positive control for comparison with the other two approaches, and as an alternative approach to identify candidate dsRNAs for subsequent analysis by oral feeding. In conclusion, we developed an applicable system for screening of psyllid sequences for RNAi effects, and efforts for transgenic plants are also underway. We have cloned the promising sequences into plant expression vector to produce dsRNA/SiRNA by transgenic approaches, and we will use these vectors to get transgenic tomato.
Objective 1. Localization of Liberibacter asiaticus (LAS) in the hemolymph and other tissues and organs of the Asian citrus psyllid (ACP). [A] Using Fluorescence In situ hybridization (FISH): We continued our studies to improve the sensitivity and specificity of several FISH protocols to localize LAS in hemolymph smears and dissected organs of ACP, and in leaf sections from HLB-infected plants as positive controls. So far, LAS has been detected in the hemolymph, filter chamber and midgut of LAS-infected ACP both from laboratory colonies and infected field trees, but not in healthy controls. It has been also detected in leaf sections from HLB-infected citrus plants but not in those from healthy ones. [B] Using quantitative RT-PCR on dissected insect organs: We tested different RT-PCR protocols for detection and quantification of LAS in dissected salivary glands, alimentary canals and other parts of individual ACP adults either from our laboratory colony or collected from field HLB-infected citrus trees. In field collected psyllids, the proportion of infected salivary glands (47%) was significantly lower than those of the alimentary canal (72%) or other body parts (79%). A similar trend was found with ACP adults reared as nymphs in our lab. on HLB-infected citrus. Interestingly, the relative titer of LAS, compared to psyllid genomic DNA in each ACP sample, was significantly higher in both the salivary gland and alimentary canal compared to that in the rest of the insect body. These results strongly suggest that the salivary glands constitute a major transmission barrier to LAS in the psyllid vector, and that LAS may replicate or accumulate in both the alimentary canal and salivary gland of ACP. [C] We continue to use a combination of transmission electron microscopy (TEM) and RT-PCR to compare the ultrastructure of ACP adults that have never been exposed to infected plants with those collected from HLB-infected citrus and are PCR-positive for LAS. This is providing a very useful library on the ultrastructure of the alimentary canal, salivary glands and other organs of ACP as well as its bacterial symbiotes, and will form the basis for future studies on LAS pathogen-vector interactions in ACP at the cellular and tissue levels. Objective 2. Clarification of various acquisition and transmission parameters between ACP and LAS. [A] We have started two large experiments to compare between young (2nd-3rd-instar) nymphs and adults having various acquisition access periods (AAP) on HLB-infected citrus plants. Previous reports have indicated that ACP nymphs are more efficient in LAS acquisition than adults, and that LAS probably multiplies in ACP nymphs but not adults, but the effects of various AAPs on nymphal and adult stages in this regard have not been carefully studied so far. Our experiments are thus designed to study the effects of various AAPs on both acquisition and transmission of LAS in ACP, as well as on LAS replication in both nymphs and adults. [B] Percentages of LAS-infected psyllids tended to be lower during late spring or early summer with no other obvious seasonal trends during 2008-2009 at a USDA grove in Fort Pierce, FL. A general decline in percentages of infected psyllids appears to have taken place during 2009-2010. No seasonal differences in percentages of infected psyllids or titers of LaAS in psyllids have been found with respect to psyllid sex or color morph. Among infected (CT<36) psyllids from the grove, an average of 18% (range 0 - 64%) psyllids transmitted LAS to healthy seedlings in monthly experiments, with no seasonal trends identified. Transmission rates are being investigated for each sex and for each of the three color morphs.
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