This project’s goal is to determine factors influencing transmission efficiency of Candidatus Liberibacter asiaticus by the Asian citrus psyllid (ACP), Diaphorina citri, which have implications on current HLB management strategies, particularly rouging and vector control. We are currently studying how acquisition efficiency varies depending on vector developmental stage (study 1), citrus phenology (young leaves x mature leaves) (study 2) and pathogen titer and symptom expression in source plants (study 3). We will also study the time period required for psyllid adults to inoculate the pathogen in healthy citrus (study 4) and if a systemic insecticide can affect this process (study 5). So far we have started experiments related to studies 1-4, and have partial results for studies 1 and 2, which are described below. The project is progressing as planned in the original proposal. In study 1, we are comparing different ACP nymphal stadia (1st, 2nd, 3rd, 4th and 5th instars) and adults (1 wk old) with respect to acquisition efficiency of Ca. L. Liberibacter on citrus. Around 10-20 psyllid adults and nymphs of each instar were confined on distinct leaves of a young shoot of a symptomatic infected plant, with recently expanded leaves, inside leaf cages. After an acquisition access period (AAP) of 48 h, the insects of each age treatment were first transferred to healthy citrus seedlings for a latent period of 15 days, and then transferred to healthy test seedlings (5 insects/plant) for a 7-day inoculation access period (IAP). After the IAP, total DNA of each insect was extracted and the sample was submitted to real-time PCR. In a first trial of this experiment (set up in May/09), the results suggested that nymphs acquire the bacterium more efficiently than the adults. Two recent trials set up in July/09 and Sept/09 confirmed this trend; mean psyllid infectivity rates of 100, 79, 80, 75, 83 and 60% were obtained when acquisition occurred during the 1st, 2nd, 3rd, 4th, 5th instars and adults, respectively. We are still waiting the data of a fourth trial (Oct/09) to run a statistical analysis, but these partial results indicate that the pathogen is efficiently acquired by all nymphal instars of ACP. In study 2, we observed the effect of leaf age (young and asymptomatic x symptomatic mature leaves) in source plants infected with Ca. L. asiaticus on acquisition efficiency and probing behavior of ACP adults. In a first experiment, groups of healthy adults were confined separately on a fully-expanded young leaf and on a mature (symptomatic) leaf of source plants of Ca. L. asiaticus. After a 4-day AAP, psyllids from each treatment were kept on healthy plants for 24-day latent period and then tested for infectivity by qPCR. ACP adults acquired Ca. L. asiaticus with higher efficiency on young (asymptomatic) (20.4% infective individuals) than on mature (0%) leaves from infected plants. We now repeated this experiment with inclusion of a third leaf age treatment (not fully-expanded young leaves), and observed that acquisition efficiency on mature leaves was lower (6.0% infective individuals) than on fully-expanded (25.1%) or not fully-expanded (9.1%) young leaves, as reported previously. By analyzing the probing behavior of adult females of ACP on mature x young (fully-expanded) leaves of infected citrus by the Electrical Penetration Graph (EPG) technique, we noted that phloem ingestion was longer and observed more often on young leaves. Within 5 h, around 50% individuals on young leaves started sustained phloem ingestion (E2), whereas less than 15% individuals on mature leaves did so. On mature leaves, the insects spent most of the time with the stylets in the parenchyma (pathway phase) or non-probing. The higher frequency and longer duration of phloem ingestion appears to explain at least in part the higher acquisition efficiency of Ca. L. asiaticus when ACP is confined on young asymptomatic leaves.
The objectives of this project are establishing baseline toxicity data of various insecticides to field populations of the Asian citrus psyllid (ACP). Determining the resistance and cross-resistance development potential in field populations of the ACP and determining the mechanisms of insecticide resistance as part of insecticide resistance management program. Using a topical bioassay method, the baseline susceptibility data (LD50) have been generated for 14 insecticides that are labeled or in the process of labeling (chlorpyrifos, dimethoate, malathion, aldicarb, carbaryl, abamectin, bifenthrin, cypermethrin, fenpropathrin, lambda-cyhalothrin, acetamiprid, imidacloprid, thiamethoxam and spinetoram) for psyllid control in Florida citrus. The toxicity data of a laboratory strain which was established in 2005 and has not been exposed to insecticides were used for comparison. Bioassays were conducted on five psyllid populations collected from one grove each in five counties (Hendry, Indian River, Lake, Polk, St. Lucie) for determining the baseline susceptibility levels and to compare them with laboratory strain. In general, all five psyllid populations collected from five Counties showed decreased susceptibility to all the tested compounds compared with laboratory strain with few exceptions. The decrease in susceptibility to different insecticides was ranged from 1 to 34-fold (Hendry County); 1 to 18-fold (Indian River County); 1 to 14-fold (Lake County); 1 to 12-fold (Polk County); and 1 to 14-fold (St. Lucie County). For Hendry County psyllid population, the highest decrease in susceptibility was observed for imidacloprid (34-fold) followed by thiamethoxam (10-fold). For Indian River County psyllid population, the highest decrease in susceptibility was observed for chlorpyrifos (18-fold) followed by imidacloprid (10-fold). For Lake County psyllid population, the highest decrease in susceptibility was observed for imidacloprid (14-fold) followed by thiamethoxam (12-fold). For Polk County psyllid population, the highest decrease in susceptibility was observed for chlorpyrifos (12-fold) followed by imidacloprid (7-fold). For St. Lucie County psyllid population, the highest decrease in susceptibility was observed for chlorpyrifos (14-fold) followed by imidacloprid (9-fold). Psyllid populations from five Counties are still highly susceptible to most of the synthetic pyrethroids tested when compared to laboratory strain. Further work to screen psyllid populations collected from 4-5 different locations in Florida is under way for determining the baseline toxicity and monitoring resistance levels. In another study, two field colonies have been established in a greenhouse and are being subjected to imidacloprid or chlorpyrifos selection pressure in every generation for developing resistant colonies. This is because field collected psyllid populations from five Counties showed decreased susceptibility to imidacloprid and chlorpyrifos. These colonies will be used for further studies on determining the resistance and cross-resistance development potential in psyllids and mechanisms of resistance. Thus far selection of five successive generations has been completed for imidacloprid and a resistance level of up to 150-fold has been observed. Further selection of future generations and determining the mechanisms of resistance by toxicological and biochemical studies are under progress. The information from these studies should help monitor the onset and progress of resistance in psyllids to different insecticides which in turn will enable us to take remedial measures as part of insecticide resistance management program.
Effects of host plant nutritional status on Diaphorina citri Kuwayama fitness: In recent experiments we focused on the two most important essentials, Nitrogen and Potassium in several combinations. ‘Valencia’ orange plants were potted individually in containers using sand. Five different fertilization levels were applied consisting of high and low nitrogen and potassium levels in all their combinations. We also included a nutrition deficient treatment. Psyllid fitness was checked in each of these treatments. Results shown that the adult weight of the psyllids fed on deficient plants was significantly lower than in all the other treatments. Additionally, the average number of eggs per female laid by psyllids reared on deficient plants was significantly lower than in all the other treatments. The effect of boron on psyllid fitness was tested. Sour orange seedlings were sprayed with 275ppm boron (0.25% Borax). Two days after treatment, 10 psyllids were caged in each seedling for a week afterwhich time mortality was assessed. IN these experiments, there was a significant effect of boron on psyllid survival with higher rates of psyllid mortality occurring in the boron treated plants. Effect of host plant species on psyllid fitness: Of the various citrus species tested thus far for effects on psyllid fitness, we have shown that Cleopatra mandarin is not a suitable host for the Asian citrus psyllid. Specifically, the survivorship of psyllid immature stages on Cleopatra mandarin did not exceed 5%, whereas survivorship on sour orange was 62.21%. Furthermore, significantly fewer eggs found to be laid on Cleopatra mandarin than sour orange. The potential for induced host preference of the psyllids was evaluated. Psyllids reared on host plants of two different genera (Murraya and Citrus) were transferred to different Citrus species and check for survivorship. Specifically, psyllids form colonies reared on Murraya koenigii (curry leaf tree) and Citrus aurantium (sour orange) were caged in Sour Orange ‘Valencia’ Sweet Orange, ‘Flame’ Grape Fruit ‘Sunburst’ tangerine and Volkameriana lemon. Results shown that no significant difference occurred in the survivorship of psyllids that were previously reared on the two hosts. Effect of the Plant Growth Regulators (PGR’s) on psyllids fitness: citrus seedlings were sprayed with 6 different PGR’s (Embark, Sumagic, Atrimmec, Profile, Cycocel). Psyllid fecundity and survivorship on those plants was studied. The initial results shown that in all PGRs tested, except Cycocel, psyllids laid fewer eggs per female compared to the untreated plants. Regarding the survivorship, significant differences occurred only between Cycocel and Embark, which was higher than in Profile.
Activities (2009) of Pasco B. Avery, postdoctoral associate who is supported by these funds to provide assistance to citrus growers on the east coast. 1) Packers of Indian River, Ltd. – Ft. Pierce : Continue monitoring psyllids along the border using yellow sticky cards. Data collected is being used to produce a distribution graphic for assessing the psyllid population dynamics throughout the year for the grower. Populations peaked in March and August. 2) IMG Citrus ‘ Fellsmere: Assessing and comparing the abundance of psyllids in orange and grapefruit trees managed by the same chemical spray program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. Results thus far indicate that there appear to be more psyllids found on orange than grapefruit trees with similar flushing patterns; but have only assessed for 2 months since August. 3) Pine Ranch, Inc. ‘ Lorida: Continue assessing the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps, tap and flush samples. Generally the population is low, but the numbers of psyllids may be influenced by the abandoned grove near the monitoring site. 4) River Country Citrus, Inc. ‘ Okeechobee: Continue assessing the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps, tap and flush samples on young trees. Very low numbers present on young (2 yrs old) trees using imidacloprid. Also assessing and comparing the abundance of psyllids in orange and grapefruit trees managed by the same oil spray program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. In general, results indicate that there appear to be more psyllids found on orange than grapefruit trees with similar flushing patterns; but have only assessed for 2 months since August. 5) Lindsey Groves ‘ Vero Beach: Assessing and comparing the abundance of psyllids in orange and grapefruit trees managed by the same organic approved program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. In general, results indicate that there appear to be more psyllids found on oranges than grapefruit with similar flushing patterns; but have only assessed for 2 months since August. 6) Premier Citrus ‘ Vero Beach: Assessing and comparing the abundance of psyllids in orange and grapefruit trees managed by the same chemical spray program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. In general, results indicate that there appear to be no differences in psyllid abundance between orange and grapefruit trees with similar flushing patterns; but have only assessed for 2 months since August.
Acquisition of pathogen: For assessment of adult acquisition, groups of 10-50 healthy adult ACP were confined in mesh enclosures on psyllid-free branches of infected citrus plants for acquisition access periods (AAPs) ranging from 1 to 52 days. To test nymphal acquisition of Las, flush containing first instar nymphs from our healthy psyllid colony was excised and placed on mature leaves of Las-infected citrus plants. Branches containing nymphs were enclosed in mesh bags to prevent dispersal of nymphs. Adults from eight replicate groups were collected upon emergence and preserved for Las-detection as described above. The acquisition of the HLB pathogen by adult ACP, determined by real-time PCR, ranged from 0% (< 1 week of acquisition feeding) to 38.8% (> 5 weeks of feeding). When ACP were reared on infected plants for acquisition as nymphs, the percentage of positive psyllids rose to 62.2%. Transovarial transmission: Sexually mature, Las-infected D. citri obtained from a laboratory colony were caged on uninfected sweet orange seedlings for oviposition. Eggs laid by single females were collected in pools of 20-30 eggs or transferred to healthy ACP host plants. Nymphs arising from the eggs of single, Las-positive females were collected as 1st-2nd instars in pools of 20-30 nymphs. Of the pools tested, 2.1% of eggs and 6.8% of nymphs were positive for Las. In addition, 2.4% of adults emerging from the eggs of infected females also tested positive for Las. Cumulatively, these results suggest that bacteria are transmitted to offspring at a low rate. Transmission of Las by individual ACP: Psyllids from an infected colony were held individually on healthy sweet orange seedlings and allowed to feed for inoculation access periods (IAPs) of 1, 4, 7, 15, and 28 d. Plants used in transmissions were held in an insect-proof greenhouse and tested bimonthly via real-time PCR for the presence of Las. To date, Las detection in plants ranged from 0% (28d IAP) to 10% 4 day IAP). Results of experiments to determine the rate of transmission by healthy ACP will be included in the next report pending plant testing. In determining the effects of host plants on the acquisition and transmission of Ca. Liberibacter asiaticus (Ca. Las) two hosts were studied this quarter. Chinese box orange (Severinia buxifolia) previously listed as a host for the Asian psyllid and a host of HLB was utilized as well as rough lemon (C. jambhii). Healthy Asian citrus psyllids readily fed on Las infected box oranges and rough lemons and after the appropriate acquisition periods were PCR tested. Between 30-40% of the psyllids from the both hosts were PCR positive for Ca. Las. To determine transmission rates from Chinese orange boxwood psyllids reared on infected plants were transferred in groups to Valencia orange seedlings. Infection results are pending. HLB infected rough lemon stems were PCR assayed at various locations to determine if symptomatic or asymptomatic differed in bacterial concentrations. Psyllid acquisition experiments from rough lemon are in progress. Seasonality of HLB infected psyllids: During 2009, psyllid collections continued at the same 5 locations sampled during 2008. During 2009, a noticeable increase in the overall percentage of HLB infected psyllids was found. While there was still a trend for periodic increases and decreases in the percentage of HLB+ psyllids, the periods when infection rates were highest occurred in January, April and July. While the average psyllid infection rate was less than 5% across all study sites, at one of these locations (Lake Alfred) the percentage of HLB+ psyllids was above 15% on each of these three months. Data collected thus far in 2009 from a 6th new location (Homestead) have shown that there does appear to be fluctuations in the number of HLB+ psyllids even where 100% of the citrus host plants are HLB+. However, the numbers of infected psyllids is much greater ranging from 20-100% based on data analyzed thus far.
This research project is directed towards controlling psyllids using RNAi technologies, as well as, protease inhibitors and peptide hormones that, if expressed in transgenic citrus, would kill adult and nymph psyllids. We tested this approach by feeding TMOF, a mosquito decapeptide hormone that stops trypsin biosynthesis in adults and larval gut, and a citrus weevil cysteine protease inhibitor (CPI). Each was added separately to an artificial diet on which psyllids were allowed to feed through a membrane. After 10 days of feeding TMOF (10 µg/µL) and CPI (3 µg/µL), all the psyllids that fed on the diet died (100% mortality). In control diets without TMOF or CPI only 40% died. The earliest significant effect on psyllids survival was observed at day 4 after feeding TMOF. The mortality of Psyllids that fed TMOF for 4 days was 15% whereas the mortality of the control group was less than 5%. Psyllids that were fed CPI did not show a significant higher mortality than controls until day 7. However, CPI concentration in the diet is 3.3-fold lower than the concentration that was used for TMOF. Because CPI is produced by genetic engineering of E. coli, its availability is limited, whereas TMOF was commercially synthesized. To continue these studies we are now fermenting large amounts of E. coli in order to purify high amounts of CPI to study the effect of different doses and optimal concentrations of this inhibitor on psyllids. We are cognizant to the possibility that both proteins when fed together may show synergism and will be looking for such phenomenon that was recently discovered in our lab when TMOF and cry toxins were fed together to mosquito larvae. The ultimate purpose of this project is to develop transgenic citrus plants expressing these proteins. Our results show that this will be feasible after dose response curves will be developed to calculate the minimal amounts of the recombinant proteins that are needed to be circulated in the citrus phloem to effectively control adult and nymph psyllids. Currently we are synthesizing double-stranded(ds) RNA targeting 11 different psyllid genes encoding three different classes of essential proteins that control cell division, digestion, water and ion balance in psyllids cells. The DNA that encodes fragments of each of the 11 genes was synthesized and cloned into appropriate vectors. Primers for the dsRNA synthesis using the RNA replicator kit (Finnzyme) were synthesized and used to produce dsRNA. After optimizing the procedure, dsRNA of individual genes will be fed to the psyllids using artificial diet and the effect of the dsRNAs on psyllid-mortality will be monitored for 10 days. At different times during the feeding, psyllids will assayed by qPCR or Northern blot analyses for RNA transcripts that are being targeted by the dsRNAs. To enhance psyllids survival on the artificial diet, we optimized the diet composition by incorporating antimicrobial agent to reduce fungal growth in the diet, and identified suitable buffers to support optimal diet and pH balance during the feeding period. In summary we: 1. Prepared 11 DNA clones that will be converted to dsRNA for feeding experiments 2. Optimized the feeding diets with antimicrobial and fungal agent and balanced the pH. 3. Showed that feeding TMOF or CPI for 10 days causes 100% mortality to psyllids.
We are attempting to identify and then deliver to psyllids, RNAs capable of inducing RNA interference (RNAi) activity in recipient psyllids. Our goal is to use RNAi to confer a negative phenotype (even death) in psyllids, such that they cannot colonize and/or reproduce on selected plants. We believe that by controlling the psyllid vector this will aid other efforts to control HLB/citrus greening. We don’t know which RNA sequences will prove to be the best for our effort, and we are using both directional cloning of specific sequences and a random cDNA library to identify effective sequences. In order to test and identify effective interfering RNAs, we are attempting to develop an efficient, high throughput screening approach that can be used with random and/or specific potential interfering RNA sequences. We are using the tomato psyllid (Bactericerca cockerelli), which colonizes herbaceous plants and is the vector of another Liberibacter spp. (C. L. psyllaurous) in our studies. B. cockerelli readily feeds on and colonizes most tomato cultivars tested by us so far, and on potatoes. It also appears to transmit C. L. psyllaurous to these plants based on our PCR-based detection analyses. We also showed that pJL36 Tobacco mosaic virus-based expression vector can be used to deliver specific RNAs into the plant phloem, and that psyllids can acquire some of these RNAs by feeding on corresponding TMV-infected tomato plants. Moreover, by using recombinant TMV we can induce production of specific siRNAs corresponding to the recombinant sequence in tomatoes. Thus, we will use this approach to induce production of siRNAs corresponding to psyllid genes in plants and to evaluate candidate sequences for RNAi activity against the tomato psyllid. We have already cloned sequences for eight psyllid genes. These are highly conserved insect gene sequences such as for actin, and we will use these in our initial experiments. Our initial sequences were obtained using primer sequences based on the Asian citrus psyllid (D.citri), but using B. cockerelli RNA as the template. We cloned these sequences into pGEM-T easy, and the dsRNAs for these sequences were synthesized. We are attempting to use microinjection to deliver these dsRNAs into psyllid nymphs to assess their abilities to induce RNAi effects. Initial efforts showed that the survival of psyllid nymphs after injection is low but acceptable. Further experiments are needed to improve injection and increase psyllid survival. Also, we are attempting to orally deliver dsRNAs to psyllids. We also have cloned eight gene sequences into pJL36 and have infiltrated tomato plants using Agrobacterium tumefaciens. We will place B. cockerelli psyllids on these plants and to determine we can induce specific RNAi effects in recipient psyllids. We have also identified 1904 contig sequences from a D. citri EST database and identified 179 midgut sequences among these. Primers for 80 midgut contigs, which are immediate genes of interest for us, were synthesized and are being used to clone homologous sequences from B. cockerelli. Our hypothesis is that the midgut genes will be important to be targeted by orally-acquired siRNA or dsRNA. We have also taken efforts for an unbiased, high-throughput approach to evaluate and identify additional B. cockerelli target sequences. The normalized cDNA library construction is underway by Bio S&T inc. In this library, the random sequences from B. cockerelli will be directly cloned ino the pJL36 plasmid, and transformed into Agrobacterium tumefaciens GV3101. This will allow us to test them directly by tomato inoculation and psyllid feeding experiments.
This is a cooperative research project between Co-PIs Joseph Morse, Jim Bethke, Frank Byrne, Beth Grafton-Cardwell, and Kris Godfrey. One objective is to coordinate with researchers working on chemical control of ACP in Florida, Texas, Arizona, and elsewhere. Towards that end Grafton-Cardwell and Morse met with Michael Rogers (UF) in San Diego in July to discuss current research and Godfrey and Grafton-Cardwell participated in the Third Citrus Health Research Forum in Denver recently. We are rearing ACP in a contained greenhouse at the Chula Vista Insectary (San Diego County; about 6 miles north of the Mexican border) under permit (#2847) from CDFA. This permit clearly notes experimental protocols and procedures so that the work is done as safely as possible to minimize the chance of ACP escape. At this site, Jim Bethke has initiated ACP tests on various organic and traditional pesticides of interest to California growers. A second location where we are working with ACP is at UC Riverside, working under permit inside the UCR Insectary Facility. Frank Byrne is conducting trials on various neonicotinoid insecticides, Morse is evaluating the baseline susceptibility of CA ACP to various pesticides in comparison to studies done in Florida, and Morse is collaborating with Mark Hoddle in evaluating the impact of registered organic pesticides on both ACP and Tamarixia. Kris Godfrey recently obtained a permit to rear ACP inside UC Davis’ Contained Research Facility and she is presently growing host plant material in order to start a colony this month or next. In collaboration with Jim Bethke, she will focus her efforts on evaluating experimental organic pesticides and microbials. In summary, we continue to expand our ability to conduct ACP research in California. To date, we have been mostly dependent on research done elsewhere but we are beginning to build the infrastructure and capability for conducting trials of specific interest to California here.
Transmission of Candidatus Liberibacter asiaticus by citrus psyllids requires acquisition of the bacteria by the psyllid, passage through the psyllids body, and release of the bacteria in feeding. The causal agent is a fastidious prokaryote that is extremely difficult to grow in quantity in pure culture. We have been attempting to augment a medium acceptable to psyllid feeding on a membrane which contains living bacterial cells. We have been able to demonstrate release of the bacteria into the membrane by feeding psyllids but have not been routinely successful in psyllid acquisition of the cultured bacteria from the membrane sachet. This work is continuing while we are developing a good micro-injection method for introducing the bacterial culture into the psyllid hemolymph. Upon successful establishment of infective psyllids, the plant inoculation process will proceed immediately to obtain biological proof of PCR results.
We have made good research progress this quarter. Need for trap in regulatory efforts to reduce spread of greening bacteria – 2009 samples. Of all samples from citrus or kumquat about 19% were positive for citrus greening with 21% of discount garden centers and 16 % from miscellaneous retailers. If both nymphs and adults were collected, about 2/3 of the time, nymphs were positive, suggesting infected plants. Field testing of traps: We conducted two field tests of about 25 different trap configurations as prototypes in a replicated experiment in two field locations in southern Florida: Immokalee and Fort Pierce. Results from these two tests helped us to focus further experiments on prototypes with configurations that trap adult psyllids with some degree of efficacy. These results also enabled us to better focus some laboratory experiments to gain a better understanding of psyllid behavior. Laboratory testing of traps: We have conducted several and continue to conduct other laboratory bioassays to determine the behaviors used by psyllids during approach, landing and post landing on key parts of traps. We are characterizing the physical properties of traps relative to their impact on psyllid walking, jumping and flight behavior. These experiments will enable us to optimize trap configurations and to develop an estimate of the importance and the relative trade-offs in efficacy among trap components. Once we have a better understanding of how psyllids respond to individual trap physical properties, we can change the configuration of various components in a focused effort to improve overall capture efficacy. We expect to develop an improved trap for Asian citrus psyllids that can be used both in groves and for regulatory purposes in places where citrus plants are sold. This trap would collect the insects and preserve them using methodology that will enable testing for the presence of HLB pathogens at some later date.
As of September, 2009, we have over 16 actively growing Asian citrus psyllid derived insect cell lines, These cell lines remain heterogeneous in composition and one of our current goals is to adapt and select for more homogenous cell populations. We now routinely grow our cells in media without any antibiotics, a needed step in order to use these cells to attempt to culture the citrus greening agent (see below). In the broadest sense, we have two distinct cell culture types, (a) suspension cells, and (b) cells that attach and spread along the tissue culture flask substrata. These two cell types have different potential uses, e.g. the attached cells may be better suited for culturing of bacteria, whereas the cells in suspension may facilitate isolation of psyllid specific viruses. Images of the cells show elongated diverse cell types in the attached cell cultures, including epithelial-like cells attached to the tissue flask surface and growing in a monolayer from clumps of globular floating cells to round cells growing in small patches. The suspension cell cultures appear more uniform as small and large clumps of round shaped cells. Growth and maintenance of the cell cultures has become routine. We have established collaborations with Drs. Wang and Davis at the Lake Alfred Citrus Research Center in order to attempt to cultivate Liberibacter asiaticus with the cell lines.
USDA test 1. A new block of young, HLB’free citrus (Valencia on Carrizo) was planted on May 1, 2008. Three psyllid control treatments (programs) are being compared in this planting: 1) a monoculture of citrus receiving monthly insecticide applications; 2) citrus interplanted with orange jasmine with a relaxed insecticide program for the citrus and orange jasmine not treated with insecticides; and 3) citrus interplanted with orange jasmine with a relaxed insecticide program for the citrus and regular applications of imidacloprid to orange jasmine. For plots with jasmine, a jasmine plant was planted between each citrus tree along some rows in each plot. Treatment 2 is being studied because psyllids may be strongly attracted to jasmine and killed, reducing numbers of psyllids that go to citrus. Treatment 3 is being studied because psyllids may be strongly attracted to jasmine thus reducing numbers of psyllids that go to citrus, and population levels natural enemies of the psyllid may be enhanced by having jasmine in the vicinity of citrus. None of the trees tested HLB positive just before planting in May 2008. None of the trees tested positive during August 2008. During November, a single tree tested positive. This tree, which was removed, was located in one of the Treatment 2 plots. During February 2009, a second tree in the same treatment’2 plot tested positive and was removed. During May 2009, 3 trees tested positive in one of the treatment-3 plots, and 2 trees tested positive in one of the treatment-1 plots. A mean of 0.9, 0.6, and 0.6% of the trees became infected within a year in plots under treatments 1, 2 and 3, respectively. From May 2008 to May 2009, a total of $163, $259, and $93 per acre worth of insecticides (materials only) was applied to treatments 1, 2 and 3. The large cost of treatment 2 was due to two applications of Bayer Feed and Protect to jasmine. USDA test 2. A new block of young, HLB-free citrus (Valencia on Carrizo) was planted during December 2008. Three treatments (psyllid control programs) are being compared in this planting: 1) citrus receiving monthly insecticide applications; 2) citrus under a relaxed insecticide program; and 3) citrus treated once every three weeks with spray oil. There are three replications of each treatment. Coincidently, the trees are being grown under an open hydroponic planting system. None of the trees tested positive prior to planting. All of the trees were treated just after planting with imidacloprid. A hard freeze on Jan 22 damaged almost every tree, and a number of the trees died. These were replanted during February. All trees were treated with imidacloprid during February, April and May giving time for the trees to recover. The experiment was to officially began in June, but five trees tested positive in June. We removed these and replaced them with healthy trees. As of today, we have no results to present for this planting. UF. An experiment is being conducted to evaluate the ability of systemic insecticides to protect a new planting of citrus from psyllids and consequently HLB infection. We planted 160 Hamlins in two rows at 6 ft spacing on 03 Mar 2009. Half of each row was considered a replicate and divided into two main plots, treated and untreated. Treated plots were split into two subplots on 13 March, one receiving a liquid formulation of imidacloprid (Nuprid 2f @ 32 oz/acre ‘ 0.5 lbs a.i./acre) and the other receiving a solid formulation of the same (Suscon 13 @ 10 lbs/acre ‘ 0.5 lbs a.i./acre). Evaluations for psyllids were conducted on 3/25, 4/3, 4/30, 6/5, 6/29 and 8/3. No psyllid infestations have been observed. The trees have grown slowly, thus we upped our fertilization program to get the trees more established. Problems have diminished with deer feeding on flush. We have not yet tested any trees for HLB.
The purpose of this investigation has been to develop, evaluate, and optimize biorational management tools for Asian citrus psyllid (ACP) including insect growth regulators (IGRs) and antifeedants. In the second series of laboratory studies with insect growth regulators, we investigated the activity of both Buprofezin (Applaud) and Diflubenzuron (Micromite) on ACP eggs, nymphs and adults to evaluate their potential usefulness as biorational insecticides for inclusion into an integrated pest management (IPM) strategy for ACP control. Both chemicals exhibited strong ovicidal and larvicidal activity against ACP eggs and nymphs, respectively, in age- and concentration-dependent manners. Fewer eggs hatched into nymphs at the higher concentrations tested (80-160 µg mL-1). A significantly lower percentage of early instar nymphs (first, second and third) survived and emerged into adults at the higher concentrations tested (80-160 µg mL-1) compared with late instar nymphs (fourth and fifth). Furthermore, both chemicals exhibited transovarial activity by significantly reducing the fecundity of females and viability of eggs deposited by females that emerged from treated fifth instar nymphs. Topical application of each chemical to adults also significantly reduced fecundity and egg viability. Application of each chemical at 160 µg mL-1 resulted in the highest inhibition of egg hatch in younger eggs (0-48 h old) laid before or after treatment and strongest suppression of adult emergence from early instar nymphs compared with other rates tested. Both Buprofezin and Diflubenzuron also markedly reduced female fecundity and egg viability for adults that were exposed either directly or indirectly. The direct (ovicidal and larvicidal) and indirect (transovarial) effects of Buprofezin and Diflubenzuron against immature and adult ACP, respectively, suggest that integration of these insecticides as part of an IPM strategy should negatively impact ACP populations over time. Current studies are underway to determine the effects of field aged residues. Also, further field scale testing is underway to determine how to best incorporate each chemical into an integrated management program for ACP. Depending on dosage, both of these insect growth regulators reduced or eliminated psyllid egg hatch and reduced or eliminated nymph to adult survival (the effect was greater when younger nymphs were treated). Furthermore, adult female psyllids exposed to both of these IGRs produced fewer eggs and viability of eggs from treated females was reduced. In a separate investigation, we have studied a psyllid antifeedant, named Pymetrozine. If psyllid feeding can be prevented or greatly reduced, perhaps HLB transmission can be reduced or prevented. Pymetrozine is a chemical that is known to paralyze the muscles involved in plant probing in plant-sap sucking insects such as aphids. Pymetrozine is also known to prevent transmission of aphid and whitefly transmitted viruses. In our initial investigations, we found that at a 100 ppm dosage applied to citrus plants, Pymetrozine inhibits acquisition of Liberibacter by psyllids on Valencia, Clepatra Mandarin, and Persian lime by approximately 50%. However, acquisition is not completed prevented and occurs in approximately 20% of psyllids. Our ongoing research is focussing on optimizing the dosage of Pymetrozine to see if we can further reduce acquisition of Liberibacter by psyllids. Also, our initial investigation has shown that only 25% of psyllid nymphs feeding on Pymetrozine-treated plants survive. This is because the majority of psyllids feeding on Pymetrozine-treated plants are starved and do not develop into adults. Thus this chemical both reduces acquisition of the greening pathogen and kills psyllid nymphs by starvation. Our next steps in addition to optimizing dosage are to determine if pymetrozine affects transmission of HLB and to investigate the effects of this chemical applied to citrus in the field on both psyllid mortality and transmission of HLB.
The goal of these investigations has been to develop an effective attractant for Tamarixia radiata, the main parasitic wasp attacking Asian citrus psyllid (ACP) in Florida. Development of an effective attractant for this insect will allow for accurate monitoring of this beneficial insect and it will allow us to recruit and establish high populations of this beneficial insect to improve biological control of ACP. We partnered with an industry collaborator (Alpha Scents, West Linn, OR) to develop an appropriate dispenser for releasing .-Butyrolactone. We have developed a polyethylene-tube dispenser for releasing this chemical. In previous studies, we found that .-Butyrolactone serves as an attractant for Tamarixia radiata wasps in laboratory experiments. We have conducted initial field trapping tests, which have been inconsistent. In the early summer, traps baited with .-Butyrolactone appeared to catch more Tamarixia radiata than unbaited traps, but this trend did not hold up in the late summer months. Current research is aimed at determining whether more consistent captures of Tamarixia radiata can be achieved by optimizing the dosage of the attractant. Also, we are investigating whether these inconsistent captures are due to the seasonal phenology of the wasps in terms of population densities, natural behavior, and commercial citrus management practices. We have also investigated a second chemical, Methyl Salicylate (MeSA) and associated dispenser from a second collaborator (AgBio, Corporation). This chemical is known to recruit beneficial insects and improve biological control. We have conducted our initial field testing of MeSA to determine whether we can improve biological control of ACP. There is abundant research showing that Methyl Salicilate (MeSA) is an effective chemical for simultaneously repelling pests and attracting natural enemies. Deployment of MeSA from controlled release devices has been shown to keep pests including aphids, thrips, and mites below economically damaging levels. In addition, this chemical has been shown to attract predatory mites, predatory bugs, coccinellids, lacewings, parasitic wasps and flies, and predatory flies. This chemical is readily synthesized at low cost. The company AgBio, the US sales rep of Chem-Tica, (who produces and distributes MeSA dispensers to researchers) was contacted and we received MeSA dispensers from them. In our initial investigation, we confirmed that deployment of MeSA reduces ACP populations and increases populations of psyllid natural enemies in small scale, non-commercial plots. Currently, we are identifying and cataloging the types of natural enemies that were recruited by deployment of MeSA as many more types of natural enemies were recruited by this chemical in addition to Tamarixia radiata. Our future goal is to optimize MeSA dispensers for larger scale use and to determine if similar results can be achieved in commercially managed groves. We plan on continuing work with AgBio to determine if MeSA dispensers will improve biological control of the psyllid. If large scale field trials prove effective, then this company will be in position to commercialize the product for use in Florida citrus.
The purpose of this proposal is to identify and develop attractants, both pheromone and host-plant based, for the Asian citrus psyllid (ACP). The intent is to develop a highly effective attract-and-kill control system for ACP with such attractants, as well as to develop highly effective monitoring traps to effectively evaluate ACP population densities to better determine the need for spraying. Most recently, we have moved forward with developing an attract-and-kill formulation for ACP with our industry partner and Co-PI Darek Czokajlo. In the past decade, a gel matrix with UV-protective properties was developed as an attracticide for lepidopteran pests. This formulation was registered in Switzerland under the trade name Sirene and subsequently as LastCall in the U.S.A., Europe, and South Africa. The original target pest of this formulation was the codling moth in apples; however, LastCall has been adopted for multiple other pests since then. One of the main goals of this proposal is to develop effective attract-and-kill for ACP by adopting the controlled release gel, termed MalEx, which is the currently licensed name for a formulation similar to LastCall. There is a large precedent for effective attracticides targeting insect pests of agricultural crops, which combine the use of a very low dose of both synthetic attractant (plant volatile or sex pheromone) and insecticide. Such formulations are typically applied as small droplets, which release pheromone at a rate highly attractive to males. Responsive males are thought to follow the plumes from attracticide droplets and obtain a lethal dose of toxicant upon final contact with the source of attractant. Thus, the MaleEx formulation is well suited for this purpose. In our initial research, the MaleEx formulation was loaded with permethrin as the insecticide. However, we learned that it would be difficult to register the formulation for use in Florida citrus with this active ingredient. Thus, our recent investigations have focussed on developing a formulation that is laced with the neonicotinoid insecticide imidacloprid instead of permethrin. We compared formulations containing 6, 14, and 22% imidalcloprid against Asian citrus psyllids in the laboratory. We found the the 14% imidacloprid formulation is superior to the 6% formulation, but that there was no added benefit of the 22% formulation. Thus, we have optimized the dosage of toxicant in the MalEx attract-and-kill formulation for the psyllid using imidacloprid as the insecticide. In seperate trials working on a different attract-and-kill formulation consisting of an emulsified wax formulation (SPLAT, ISCA Technologies), we compared the insecticides Spinosad, Methoxyfenozide and Tebufenozide against the psyllid. We found that Methoxyfenozide and Tebufenozide are not effective with this formulation and that Spinosad is only marginally effective resulting in about 50% mortality. Our ongoing studies are focusing on testing more insecticides with the wax formulation so that more effective psyllid kill can be achieved. We have continued our work on developing a psyllid attractant in collaboration with both USDA colleagues and our industry partner, Alpha Scents. Most recently, we have conducted four field trapping experiments of synthetic citrus plant volatiles that have been shown to attract psyllids in the laboratory. Our tests to date have been inconsistent. In some cases, the proprietary blend of plant volatiles increase captures of psyllids by 2-3 fold, but in other cases there is no apparent increase in psyllid capture as compared with blank control traps. We are continuing our work on optimizing blends and dosages of currently identified chemicals. We also continue our efforts at identifying further, perhaps more potent attractant chemicals. We are also continuing our research on optimizing trap placement for best psyllid monitoring within groves. This also includes further research on optimizing trap color and design.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
