We have initiated two new collaborations in various efforts for the application of the D. citri insect cell lines developed. (1) Dr. W. Hunter, Objective: provide cultured cells for genomic characterization of D. citri. We have scaled-up the growth and culturing of several lines in order to have enough material for extraction and characterization of the genomic DNA derived from the cultured. The requested amount of cells ~0.1-1 g of material. We have gathered approximately 0.2 g of material and will forward the frozen cells to Dr. Hunter’s lab presently. It is anticipated that an additional ~ 0.2 g of cells will be ready within the next month. (2) Dr. Lisa Fontaine-Bodin,CIRAD-BIOS, Montpellier, France, Objective: to use the cell lines in attempts to culture the various European and African Liberibacter strains. In the part quarter, we have sent Dr. Fontaine our cells and they are apparently viable and the cell lines should be established in the lab. It is anticipated that attempts to cultivate the various Liberibacter strains available in Dr. Fontaine’s lab will proceed in the coming year. The laboratory continues in attempts to increase the robustness of the cells lines. A periodicity in the growth has been noted which current efforts are aimed at mitigating. Suspension cells lines appear more uniform, however, contain large clumps and doubling times remain in 2-3 weeks. Attached cells remain heterogeneous with doubling times near 2-4 weeks. Antibiotic free cell lines have also been established and are now in routine growth in the lab.
The purpose of this multi-faceted research project has been to develop or uncover biorational alternatives to conventional pesticides for management of Asian citrus psyllid (ACP). One tactic that we have been recently exploring is the use of systemic induced resistance (SAR) against ACP. .-aminobutyric acid (BABA) is known to induce resistance against several microbial pathogens, nematodes and insects in several host plants. The current investigation was undertaken to determine whether BABA would induce resistance against ACP in citrus under greenhouse conditions. We examined the effect of five different concentrations of BABA applied as a root drench to citrus plants, on the performance of ACP. Observations were made on the number of eggs, nymphs and adults produced per plant. In addition, leaf-dip bioassays were performed using similar concentrations of BABA to rule out the possibility of any direct toxic effect of BABA on early nymphal instar (2nd), later nymphal instar (4th) and adults ACP. The results revealed that BABA-induced resistance in citrus plants can suppress the growth and development of ACP at various growth stages. The total mean (‘ SEM) number of eggs produced per plant was significantly higher in control plants (97.9 ‘ 8.8) than in plants treated with 25 (43.5 ‘ 12.8) and 100 (44.8 ‘ 14.9) mM of BABA. The total mean number of nymphs produced per plant was significantly higher in control plants (74.0 ‘ 7.3) than in plants treated with 25 (47.9 ‘ 10.2), 50 (37.7 ‘ 7.9) and 100 (364.2 ‘ 10.4) mM of BABA. Likewise, the mean number of males and females was significantly higher on control plants (male: 4.7 ‘ 1.3; female: 5.6 ‘ 1.1) than on plants treated with 25 (male: 3.2 ‘ 1.0; female: 2.7 ‘ 0.9), 50 (male: 1.5 ‘ 0.4; female: 1.6 ‘ 0.5) and 100 (male: 1.2 ‘ 0.3; female: 1.4 ‘ 0.7) mM of BABA. The percent mortality of early nymphal instar (2nd), later nymphal instar (4th) and adults of ACP as a result of different BABA concentrations was not significantly different from the percent mortality observed in the control treatment. The above results suggest that the reduced growth and development of ACP on BABA-treated plants is a function of induced resistance in citrus plants, rather than direct toxicity by BABA on various developmental stages of ACP. The results of the current study suggest that BABA has potential as a SAR treatment for management of ACP that may supplement conventional insecticides. However, the effects of BABA under field conditions and its effect on no-target organisms still require further investigation. In addition, since BABA acts by potentiating a normally under-expressed defense pathway; therefore, genetic tools could be used to trigger such pathways by genetic alteration and possible development of transgenic cultivars.
Asian citrus psyllids (ACP) generally rely on olfaction and vision for detection of host cues. Plant volatiles from Allium spp. (Alliaceae) are known to repel several arthropod species. Recently, we examined the effect of garlic chive, (A. tuberosum Rottl.) and wild onion (A. canadense L.) volatiles on behavior of ACP in a laboratory two-port divided T-olfactometer. Citrus leaf volatiles attracted significantly more ACP adults than clean air. Volatiles from crushed garlic chive leaves, garlic chive essential oil, garlic chive plants, wild onion plants and crushed wild onion leaves all repelled ACP adults when compared with clean air, with the first two being significantly more repellent than the others. However, when tested with citrus volatiles only crushed garlic chive leaves and garlic chive essential oil were repellent and crushed wild onions leaves were not. Chemical analysis of the headspace components of crushed garlic chive leaves and garlic chive essential oil by gas chromatography-mass spectrometry revealed that monosulfides, disulfides and trisulfides were the primary sulfur volatiles present. In general, trisulfides (dimethyl trisulfide) inhibited the response of ACP to citrus volatiles more than disulfides (dimethyl disulfide, allyl methyl disulfide, allyl disulfide). Monosulfides did not affect the behaviour of ACP adults. A blend of dimethyl trisulfide and dimethyl disulfide in 1:1 ratio showed an additive effect on inhibition of ACP response to citrus volatiles. The plant volatiles from Allium spp. did not affect the behavior of the ACP ecto-parasitoid Tamarixia radiata (Waterston). Thus Allium spp. or the tri- and di-sulphides could be integrated into management programmes for ACP without affecting natural enemies. The current results provide evidence that volatiles from crushed garlic chive leaves inhibited the response of ACP to its normally attractive host plant volatiles. These volatiles also appeared to have inhibited the psyllid’s normal geotactic and phototactic responses. Furthermore, our results suggest that the sulfur volatiles released by wounded A. tuberosum leaves affected the behavior of ACP. Our current efforts are focusing on formulating these sulfur compounds into controlled release devices for deployment in the field. In field investigations, we continue to evaluate the effect of DMDS on psyllid populations when deployed in the field in the SPLAT release device from ISCA. In one field experiment we eliminated psyllid populations with a standard Danitol spray and subsequently applied DMDS in SPLAT to half the plots, while leaving the other half untreated. We found that it took psyllids significantly longer to re-colonize citrus plots that were first treated with Danitol and then by DMDS than those that were treated with Danitol only. We initiated another large field experiment evaluating DMDS in SPLAT in a commercially managed grove. All plots receive psyllid management sprays, but half also received additional DMDS treatments. Under this low psyllid population density situation, we have observed no additional benefit of adding DMDS. Thus, our results with applying DMDS in the field have been inconsistent to date, with certain experiments showing promise, while others showing little to no effect of the DMDS treatment. We have also discovered that DMDS is highly phyto-toxic. Application of SPLAT containing DMDS was found to kill large tree branches. We are looking into alternate formulations and application procedures to overcome this hurdle. We also have three new SPLAT-DMDS formulations for extended longevity that we are evaluating; however, those tests are ongoing.
The objective of this project is to elucidate mechanisms of resistance in ACP to insecticides and to develop appropriate resistance management strategies. Microorganisms are known to alter insect host physiology, which may benefit or harm the host. Most recently, we determined the effect of Candidatus Liberibacter asiaticus (Las), the bacterium presumably responsible for causing huanglongbing (HLB) disease, on the physiology of its vector, the Asian citrus psyllid (ACP). Specifically, we determined the effects of Las infection on susceptibility of ACP to selected insecticides. Furthermore, total protein content, general esterase, glutathione S-transferase (GST) and cytochrome P450 activities were quantified in Las-infected and uninfected ACP to gain insight into the possible mechanism(s) responsible for altered susceptibility to insecticides due to Las infection. LC50 values were significantly lower for Las-infected than uninfected ACP adults for chlorpyriphos and spinetoram. Furthermore, there was a general trend for lower LC50 values for three other insecticides for Las-infected ACP; however, the differences were not statistically significant. Total protein content (‘g ml-1) was significantly lower in Las-infected (23.5 ‘ 1.3 in head + thorax; 27.7 ‘ 1.9 in abdomen) than uninfected (29.7 ‘ 2.1 in head + thorax; 35.0 ‘ 2.3 in abdomen) ACP. Likewise, mean (‘ SEM) general esterase enzyme activity (nmol min-1 mg-1 protein) was significantly lower in Las-infected (111.6 ‘ 4.5 in head + thorax; 109.5 ‘ 3.7 in abdomen) than uninfected (135.9 ‘ 7.5 in head + thorax; 206.1 ‘ 23.7 in abdomen) ACP. GST activity (‘mol/min/mg protein) was found to be significantly lower in Las-infected ACP (468.23 ‘ 26.87) when compared to the corresponding uninfected adults (757.63 ‘ 59.46). Likewise, mean P450 activity (EU of cytochrome P450/mg of protein) was significantly higher among the uninfected (0.49 ‘ 0.05) than in Las-infected (0.23 ‘ 0.02) ACP. Susceptibility of ACP to selected insecticides from five major chemistries was greater in Las-infected than uninfected ACP. The lower total protein content, and reduced general esterase, GST and P450 activities in Las-infected than uninfected ACP may partly explain the observed higher insecticide susceptibility of Las-infected ACP. Therefore, the results of our study indicate that Las infection may be detrimental to ACP suggesting a non-symbiotic relationship. Higher mortality of Las-infected than uninfected ACP suggests that Las-infected psyllids may be selected against under commercial ACP management practices relying on insecticides. Selection against Las-infected ACP may limit spread of HLB. This hypothesis is consistent with the notion that insecticide resistance contributes to the spread of vector-borne disease. Our subsequent investigations should help elucidate the mechanisms of altered host physiology with respect to insecticide resistance management programs for ACP. In other concurrent investigations, we have recently determined baseline toxicity of various insecticides to late nymphal instar (4th) ACP to determine if resistance levels differ between ACP adults and nymphs. Baseline data were collected using an ACP culture maintained at a CREC greenhouse for 5 commonly used insecticides. LC50 (mg ai/l) values of the susceptible greenhouse population were compared with those of field collected populations from 2 sites. LC50 of carbaryl (50.71) and spinetoram (3.88) was significantly higher for the population collected from one of the field sites when compared to the greenhouse population (carbaryl: 17.59; spinetoram: 0.66). Immature psyllids collected from two field sites were significantly less susceptible to imidacloprid (with LC50 values of 0.84 and 0.50) than the susceptible lab population (0.22). Our results show that immature psyllid nymphs exhibit resistance levels similar to those previously documented for adults.
The goal of this project is to determine if infection by Candidatus Liberibacter affects the response of Asian citrus psyllid (ACP) to its citrus host plants to understand a critical component of disease spread. In this project we evaluated if healthy psyllids are attracted more to HLB infected or healthy trees. Also, we determined whether this behavior changes when the ACP vector becomes infected with the pathogen. We conducted a series of behavioral experiments to investigate whether HLB-infected citrus plants are differentially attractive to ACP as compared with healthy citrus plants. We also examined if psyllids known to be infected with the pathogen behaved differently from uninfected controls in response to both healthy and HLB-infected plants. Our preliminary results indicate that HLB-infected citrus plants are more attractive to ACP adults than healthy plants in two-choice olfactometer experiments. More ACP were attracted to HLB-infected plants than to healthy plants in open-air cage experiments. However, subsequent dispersal of ACP adults to healthy plants following their initial choice indicated that final settling preference was for healthy rather than diseased plants. Initial movement of ACP to infected plants and further dispersal to healthy plants may be explained by production of deceptive volatile compounds by HLB-infected plants to attract ACP adults in the field to facilitate the spread of bacteria as occurs with apple phytoplasma Candidatus Phytoplasma mali, responsible for apple proliferation disease. Ca.P. mali hijacks the apple trees to produce specific chemical that attracts the plant-sap sucking psyllid vector to infected trees. This is a major factor for facilitating disease spread in apple. Alternatively, the yellow color of HLB-diseased plants due to chlorosis and yellowing of shoots may attract the ACP initially but psyllids move to healthy plants after sampling the phloem of diseased trees. The movement to new plants could be due to poor nutritional status of HLB infected plants. It is known that ACP adults are attracted to yellow color; therefore, initial attraction of ACP adults to diseased plants may be due to chlorosis of leaves caused by HLB. Also HLB-infected plants are deficient in zinc, iron, manganese, calcium, sulfur and/or boron and hence the subsequent movement of psyllids to healthy plants could be due the poor host suitability of HLB-infected plants. To answer these questions, we are analyzing the head space volatiles produced by HLB infected plants versus healthy plants to evaluate if the differential response of ACP to HLB infected and healthy citrus plants is due to differential chemical production. In addition we are continuing to conduct more experiments in light and dark conditions to evaluate the role of visual cues in the differential response of ACP to HLB infected and healthy citrus plants.
The objective of this project is to evaluate methyl salicylate dispensers to determine whether their deployment in citrus can enhance biological control of Asian citrus psyllid. Locations were selected for the field trials with the commercially available methyl salicylate (MeSA) lure, Predalure (AgBio Inc.; Denver, CO). The first site was an unmanaged grove of ‘Valencia’ oranges near Clermont, FL and plots were established on 4/13/10. Four plots were designated to have 1 lure hung per tree and 4 plots were designated as control plots. The plots were 0.4 ha in area with a 0.4 ha buffer between the plots. Twelve unbaited Pherocon AM yellow sticky traps were placed in all plots in rows 2, 5, 7 and 9 in trees 3, 7 and 12 to determine pretreatment populations of Asian citrus psyllid (ACP) adults and natural enemies. The trees were in late bloom stage. On 4/20/10, MeSA lures were attached to the trees with twist ties at mid-canopy height and sticky traps were replaced. The plots were then checked every 2 weeks. Sampling included replacement of sticky traps, sweep net samples of trees 6 and 11 in rows 2, 7 and 9 of all plots and 5 flush samples/plot, when suitable flush was available. The flush samples were examined in the laboratory and the number of ACP eggs and nymphs were counted. The flush were then placed in a jelly cup with water and a mylar cylinder with a mesh top placed around the flush to capture emerging ACP and parasitoid adults. They were placed in an incubator maintained at 25oC and egg, nymphs and adult ACP as well as any parasitoids were counted at 5-10 day intervals. In the field, MeSA lures were replaced on 6/24/10. The second location was established on 4/22/10 in a minimally managed grove of mixed citrus cultivars in Lake Alfred, FL. In this grove, only 3 replications of MeSA treated plots and controls were possible. Traps were placed as before and lures were hung 1/tree on 4/29/10 (post-bloom) with biweekly sampling as above. Lures were replaced in this grove on 6/30/10. The third site was a heavily managed grove of ‘Hamlin’ oranges in Auburndale, FL at the beginning of fruit set. Replicated plots (4) of MeSA treated and controls were established as above on 5/5/10 with the lures placed in the treated plots on 5/11/10. Biweekly sampling was conducted with traps, sweeps and flush (when available). Lures will be replaced in this grove on 5/15/10. To date, very few ACP and beneficial insects have been collected in sweep samples in any of the 3 groves. Flush has only been available for 3 sampling dates at the Clermont grove and no parasitoids have been found although there was successful adult ACP development from the eggs and nymphs present. Traps are being held in refrigeration until the ACP and beneficial species can be counted. The unusually cold winter most likely reduced overwintering ACP population levels and also had an effect on the flush cycle of the citrus trees, but the most recent samples indicate an increase in ACP populations. An experiment to investigate the effects of MeSA lures on the behavior of ACP is being conducted. When feather flush suitable for oviposition by ACP appeared all but 5 flush were removed and the plants placed in 58 X 58 X 90 cm screen cages. Five plants were designated as treatment and 5 as control. In the cages with treatment plants, 1 MeSA lure was suspended from the top of the cage centrally. The control cages had no lures. All cages were placed under natural conditions outside the laboratory and treatment cages were separated from the control cages by a distance of 50 m. On July 8, 2010, 50 adult ACP from a greenhouse colony were released in each cage and the number of psyllids settling on the plants was observed at 1 hour, 24 hours, 5 days and will be continued at 7 days and 10 days. At 10 days the adult ACP will be removed from the plants and examined to determine the number of males and females. The flush will be examined and the number of ACP eggs and nymphs will be counted.
We are beginning the second year of our project. During this year we are focused on evaluating psyllid-derived sequences which can be used as double-stranded RNAs to yield RNA interference-induced negative effects (even death) in recipient psyllids. We are using the tomato/potato psyllid, Bactericerca cockerelli, and its solanaceous plant hosts as an herbaceous plant model system. This herbaceous system is much easier and faster to manipulate than is the perennial citrus system, but we believe our efforts will lead to subsequent application toward the asian citrus psyllid, Diaphorina citri. We have well over 100 candidate sequences identified from last years work, and several thousand more cloned and stored. We are now comparing specific sequences for RNAi activity via three delivery methods: direct injection into the psyllid hemocoel, in vitro acquisition via feeding through parafilm membranes, and by feeding on tomato plants infected with recombinant Tobacco mosaic virus (TMV) constructs containing the candidate sequences. B. cockerelli midgut cDNAs, and those generated last year from the B. cockerelli normalized cDNA library, were engineered for use as templates to generate dsRNAs in vitro (via T7 RNA polymerase transcription) and for insertion into TMV. In vitro-generated dsRNAs were adjusted to specific concentrations and used for subsequent direct delivery experiments while TMV was used to express the sequences in whole plants. The in vitro-generated dsRNAs were tested by feeding psyllids through stretched parafilm membranes containing the candidate dsRNAs in a solution of 15% sucrose. B. cockerelli psyllids readily feed on this solution for up to 7 days, thus allowing adequate time for acquiring the test dsRNAs and assessing potential RNAi effects. We have demonstrated that fluorescent, Cy3-labelled dsRNAs were acquired by membrane feeding. We were able to visualize these in psyllid guts by using fluorescence microscopy after feeding. We also used qPCR to identify acquired dsRNAs and to quantify target mRNAs in psyllids. In some experiments dsRNAs evaluated in initial membrane feeding experiments caused mortality in psyllids. However, we are investigating now the quantitative effects of these treatments to ensure that phenotypic effects, including psyllid mortality, are due to the specific sequence and not due to overly abundant dsRNAs. The dsRNAs also were evaluated via micro-manipulator driven intra-thoracic injection (200 nL/psyllid). Mortality is fairly high, ~50%, due to injection even when injecting buffer controls. Therefore, we are injecting large numbers of insects (at least 15 per treatment) and including buffer controls so as to have sufficient numbers for statistical analysis. We are using injection here only as a comparator, intra-thoracic injection is a standard means to induce RNAi effects in insects so it is a very good positive control for comparison against oral delivery via membrane feeding and TMV-infected plant acquisition. The same psyllid sequences are being evaluated via psyllid feeding on recombinant TMV-infected tomato plants. Our data show that psyllids feeding on these plants acquire the specific test RNAs, as determined by RT-PCR and qRT-PCR. However, this approach is still a little problematic as TMV sometimes loses the inserted test RNA sequence during tomato plant infection, therefore all plants used for feeding experiments are carefully evaluated for inserted sequence retention. We are also assessing how much and what forms of RNAs are acquired during whole plant feeding.
The first year of a 2-year research proposal (FDACS Contract Number 58-1920-9-925 40) was successfully completed when the second year funds were terminated and a no cost extension was granted to complete loose ends. Our research objectives were: (1) Devise and perform alternative methods (microinjection and membrane uptake) to complete Koch’s postulates using a pure culture of bacteria isolated and cultured in our laboratory and healthy psyllids as a transmission tool; and, (2) following successful inoculation or loading of the psyllids, we would complete Koch’s postulates. We have been able to cultivate a strain of ‘Candidatus Liberibacter asiaticus’ (Las) Taiwan (B239) in our laboratory using different media components and it has been shown to be Las by PCR and sequencing. We have followed the procedure previous described in our last quarterly report to transmit the bacteria produced in culture media or from infected dodder using stretched parafilm membrane sachets containing 10% sucrose solutions in 1X TE buffer in which the cultured bacteria were suspended. The titer of the bacteria in sucrose was roughly assessed using real time PCR (Ct values ranged from 20 to 38). Seedlings were grown at 25 C with natural daylight supplemented with HID lamps to extend the daylength to 18 hr. No symptoms were observed at 3 months, however, those positive by PCR at 3 months developed typical blotchy mottle symptoms by 6 months. We have repeated membrane inoculations with cultivated bacteria from infected dodder and citrus plants but will not be able to complete the final steps of Koch’s postulates because of lack of funding. A second approach used direct microinjection of the culture fluid into the hemolymph of adult psyllids. Psyllids were immobilized with a low velocity stream of C02 and approximately 0.01’l of bacterial culture was injected into the abdomen of each adult psyllid as described in the last quarterly. After 6 months, we have observed 2 of 7 sweet orange seedlings inoculated with injected bacteria exhibiting HLB symptoms. This has been repeated and we are waiting for results. While this does not serve as additional confirmation of Koch’s postulates for Las, it does provide important insights into the interaction of Las with the vector. We have been continuing to refine our membrane and micro-injection techniques to obtain additional confirmatory data on the ability of D. citri to transmit cultivatable (HLB causing) bacteria obtained from HLB infected sweet orange, dodder, or infectious psyllids.
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 started 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 started the experiment 3, using different insecticide spraying to determine if they prevent the transmission and for how long. 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: imidacloprid 6.1, 9.9 and 6.9 min, respectively for 15, 35 and 95 days after application (DAA); thiamethoxam 9.6, 14.5 and 17.5 min, respectively for 15, 35 and 95 DAA; Control 142.0, 80.3 and 129.0 minutes, respectively for 15, 35 and 95 DAA. 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. In plants sprayed with mineral oil decreased the percentage of psyllids that can reach the phloem when compared with plants not sprayed, 20 and 70% respectively. However, the few insects that reach the phloem of treated plants carry out long periods of ingestion in this vascular tissue (‘ 1 h). We began 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%, 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. The results showed that the number of insects reaching the phloem is lower. The next step is the study of lower doses of mineral oil.
The objective of this project is to screen citrus germplasm for resistance to the Asian citrus psyllid (ACP). Although citrus huanglongbing (HLB) is a century-old disease and the control of ACP is the key factor for HLB management, there is little information regarding citrus host resistance to ACP. Our preliminary results indicate there is ACP resistance in citrus germplasm. Historically, most (if not all) citrus resistance to HLB has been evaluated using graft inoculations, which sometimes resulted in plants becoming infected that appeared to have resistance in the field. What is needed is research to find varieties with resistance to the psyllid. Greenhouse and field evaluations of the USDA germplasm collection will be conducted. There already exists in China individual citrus plants that are thought to be HLB-resistant,but likely some of these are ACP resistant. The identification of a psyllid-resistant variety (or individual mutant) could revolutionize HLB management strategies. If we can identify ACP resistant germplasm, we can identify resistance genes for use in traditional and molecular breeding . Our intentions are to screen hundreds of sources of USDA and Chinese germplasm for resistance to ACP and to field test these to see if resistance to the psyllid negates HLB or greatly facilitates control. We expect to identify psyllid resistant citrus genotypes (or individual mutants from field-resistant collections) and/or citrus relatives within the Rutaceae that have psyllid resistance. We expect to determine traits that confer resistance and to identify traits that might be transferred to citrus varieties currently grown in order to make them resistant to the psyllid and thus less prone to contracting HLB. The ultimate return from this project would be an effective management strategy to control ACP and HLB that is less costly and friendlier to the environment and non-target organisms than the repetitive use of broad spectrum insecticides. A post doc was found and hired to conduct this research. A trip was made to China to firm up research plans with the Dr. Liu Bo and the Fujian Academy of Agricultural Sciences. Seeds representing the entire citrus/citrus relative collection at USDA-ARS-NCGR were obtained and have been planted. A field planting of many citrus genotypes and relatives was screen for psyllid infestations during July and August. Some of these genotypes were obviously highly susceptible to infestations by the psyllid while others appeared to be avoided.
Overall goal of the project was to develop and/or optimize low volume spray application technologies for controlling psyllids in Florida citrus production. Our research included both laboratory and field studies. In the laboratory, first we tested several fluorescent tracers to develop a reliable methodology for qualitative and quantitative assessment of spray deposition in low and ultralow volume applications. We used a controlled droplet applicator to generate different spray droplet sizes to investigate the effect of droplet size on the mortality of ACP. The tests included various commercially available pesticide formulations that are labeled for citrus applications. The technique involved spraying different droplet sizes on psyllid infested potted trees (Swingle) and counting the number of ACP eggs, nymphs, and adults before spraying and at 3 days and 7 days post-treatment. We tested ‘Lorsban Advanced’ ‘Danitol 2.4EC’ (synthetic pyrethroid), ‘Lorsban Advanced’ (organophosphate), and ‘Dibrom 8E’ (organophosphate), each at six droplet sizes. Our results showed that the smaller the droplet size, the greater is the percent mortality of all life stages of the ACP. For all chemistries, the results suggested up to 80 percent control of nymphs using sprays with droplet VMD of about 40 ‘ 100 ‘m. Danitol and Dibrom produced this effect by day 3, with Lorsban taking until day 7. The egg and adult stages showed similar trends, but with greater levels of control. Three other formulations screened were EverGreen (natural pyrethroid), AgriMek (abamectin), and Knack (pyriproxifen). Nonetheless, chemistries involving suspended particles could not be applied readily with our laboratory apparatus. In their present formulations, Movento (spirotetramat) and Provado (imidacloprid) are not compatible with the laboratory droplet applicator. In the field, we continued comparing various available application technologies. In one experiment, we compared high volume (dilute) spraying with low volume applications. The test involved evaluating two cold foggers and a mist blower. We also conducted several field trials with our LV-8 low volume applicator, which is becoming commonly used in the Florida citrus industry. We have applied over 4o treatments in Florida citrus groves to optimize chemistries that are labeled for the psyllid control, such as Danitol, as well as testing of unlabelled compounds. For labeled compounds, we have investigated the effect of product rate and application timing. For unlabelled compounds, we have conducted efficacy testing at standard rates to generate the needed data for label changes. Thus far, we have found no difference in efficacy between the available technologies. However, we may find differences with some of the more selective chemistries, so we are continuing this research by investigating more pesticide modes of action. We have compared the insecticide residues achieved with low volume and standard airblast sprays and have found that the residues from low volume sprayers are lower than those from conventional airblast sprayer. We have also investigated several application parameters for optimizing low volume spraying. While certain insecticides have shown similar efficacies for spraying every row versus every other row or ground speed of 5 mph versus 5-8 mph, some chemistries have shown lower efficacies when spraying every other row or at higher ground speed. In terms of the application volume, we have not found a difference between 2 and 5 gallons per acre. However, one must stay above 2 gallons per acre to remain within the boundaries of current label guidelines. We are also currently investigating whether the rate of insecticide can be reduced with low volume spraying. Given the importance of droplet size rage in label limitations, we have further refined the technique for characterizing the droplet size range of the ULV sprays used in field application.
USDA test 1. A new block of young, HLB’free citrus (Val on Carr) was planted on May 1, 2008 in a grove with substantial levels of HLB. Three ACP control treatments (programs) are being compared in this planting: 1) a monoculture of citrus receiving monthly insecticide applications (annual chemical cost of $198/acre); 2) citrus interplanted with jasmine with a relaxed insecticide program for the citrus (annual chemical cost of $156/acre), jasmine not treated with insecticides; and 3) citrus interplanted with jasmine with a relaxed insecticide program for the citrus and regular applications of imidacloprid to jasmine (total annual chemical cost of $213/acre). For plots with jasmine, a jasmine plant was planted between each citrus tree along some rows in each plot. Program 2 is being studied because ACP may be attracted to jasmine thus reducing numbers of ACP that go to citrus, and population levels of ACP natural enemies may be enhanced by having jasmine in the vicinity of citrus. Program 3 is being studied because ACP may be attracted to jasmine and killed, reducing numbers of ACP in citrus. HLB-infected trees are removed. None of the trees tested HLB positive just before planting in May 2008. One year later, a mean of 0.9, 0.6, and 0.6% of the trees were HLB-infected under programs 1, 2 and 3, respectively. During August 2009, a mean of 9.8, 3.9, and 4.2% of the trees were infected under programs 1, 2 and 3, respectively. The rapid increase in incidence of HLB in the plots between May and August 2009 was attributed to increases in ACP infestations during June and July, particularly in a program 1 plot that was adjacent to older citrus. In February 2010, a mean of 13%, 12% and 13% of the trees were HLB-infected under programs 1, 2 and 3. USDA test 2. Three ACP control programs are being compared for preventing HLB in a block of young, HLB-free citrus (Val on Carr): 1) citrus under a relaxed insecticide program (annual chemical cost of $173/acre); 2) citrus receiving monthly insecticide applications (annual chemical cost of $198/acre); and 3) citrus treated once every three weeks with spray oil [PureSpray Foliar (470, C27) and PureSpray Green (435, C23)] from February through November (plus a December application of Danitol) (annual chemical cost of $76/acre). There are two replications of each treatment. The experiment officially started in August 2009. In January 2010, 0%, 0.3% and 0.3% of the trees tested HLB positive under programs 1, 2 and 3, respectively. These trees were removed. UF test. The ability of systemic insecticides to protect a new planting of citrus from ACP (and consequently HLB infection) is being evaluated. 112 Hamlins were planted in two rows at 15 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). On 11-Sep 2009, no significant treatment effects (Chi square = 3.61, P = 0.182) were found with respect to numbers of HLB trees. At that time, 10 of the 56 control (untreated) trees tested positive for HLB, 10 of the 28 trees treated with the granular slow release imidacloprid were positive, and 6 of the 28 trees treated with the liquid formulation of imidacloprid were positive. The same trees were resampled on 15 Jan, but all tested negative. PCR was run twice more on the DNA from both the Sep and Jan samples and the results were confirmed. Thus, the different result from Jan could only be attributed to effects of cold weather on titer and/or lack of ACP to reinoculate the trees.
1. Localization of Liberibacter asiaticus (Las) in the hemolymph and other tissues and organs of the Asian citrus psyllid (ACP). The following four methodologies have been tested and used with promising results. [A] Immunofluorescence confocal laser scanning microscopy: Two polyclonal and several monoclonal antibodies prepared against Las membrane proteins have been tested at various dilutions and incubation times. Some of the monoclonal antibodies produced positive labeling in the phloem area in sections of HLB- infected citrus leaves and in hemolymph smears of ACP collected from HLB-infected trees, but not in whole-mount ACP organs, suggesting that such potentially useful antibodies may be too slow to penetrate whole insect organs. We are working to resolve this issue with various permeabilization procedures or by immunofluorescent labeling of paraffin sections rather than whole organs. [B] Fluorescence In situ hybridization (FISH) using three oligonucleotide primers: Several FISH protocols have been tested on hemolymph smears and dissected organs of ACP and on leaf sections and extracts from HLB-infected plants. Green fluorescence, indicating Las, was detected in the hemolymph, filter chamber and midgut of field-collected ACP, but not in healthy controls. It was also detected in leaf sections from HLB-infected citrus and periwinkle plants but not in those from healthy plants. We are continuing to refine our FISH protocols to reduce the background fluorescence and to increase the permeability of insect tissues. [C] Quantitative RT-PCR of dissected insect organs: We tested two different RT-PCR procedures for detection of Las in dissected salivary glands, alimentary canals and other parts of individual ACP adults. In five successive experiments, Las was detected in 13-24% of the alimentary canals, 12-16% of the salivary glands, and in 16-25% of the rest of the body from psyllids collected from HLB-infected trees in Fort Pierce, FL. This is the first direct demonstration of Las (using PCR) in the alimentary canal and salivary glands of ACP. We are currently testing the relative concentration of Las in various ACP organs. [D] We are using 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 trees 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 immunogold-labeling TEM studies on Las pathogen-vector interactions in ACP at the cellular and tissue levels. 2. Clarification of various acquisition and transmission parameters between ACP and Las. [A] Among ACP adults field collected from HLB-infected citrus trees, averages of 37% females and 38% males have tested positive by PCR. Significantly higher percentages of field-collected adult ACP tested positive for Las during November 2009 (38.9%) than during late March 2010 (22.1%), and CT values associated with adults collected during November (mean 24.8) were significantly lower than for adults collected during late March 2010 (mean 30.5). [B] An average of 16% individual HLB-infected adult ACP (field-collected, mean CT of 26.4) transmitted HLB to young potted citrus trees. Taken together, the data in1C and 2B above support our hypothesis that transmission barriers to Las in ACP (including the alimentary canal and salivary glands) may play an important role in HLB transmission, since much higher percentages of ACP adults were PCR-positive compared to those that were actually able to transmit HLB to citrus plants. Our studies are continuing to elucidate the roles of these and other psyllid organs/ tissues in HLB transmission by ACP.
This is the final report of a three-component project on the Asian citrus psyllid. Sampling – Stem-tap sampling is preferred over sticky traps for making ACP density estimates and is considerably less expensive. Sampling to estimate ACP densities should be conducted throughout a block because there can be significant variation across a block of trees in numbers of ACP. For commercially-acceptable precision levels at means of 1 or more adult psyllids per sample, 28 stem-tap samples are required in a block of trees up to ten acres in size (grove edges are not sampled under this protocol). Larger numbers of samples were required at lower densities. Although no control threshold for ACP has been identified, this level would likely lower than than 1 ACP per sample, perhaps 0.2 to 0.3 ACP per sample – thus growers wanting to make insecticide decisions for ACP control should take larger sample sizes. Noted is that young trees do not lend themselves well to stem-tap sampling. Sticky traps are preferred for ACP detection, not density estimation. Three publications on sampling have been published (available on request) and one has been submitted. Future research efforts on ACP sampling should include a method of estimating densities for an entire block of trees including edges. A formal method is needed for sweep sampling. The relationship between mean number of ACP per tap sample and absolute ACP densities in trees needs to be clarified. Biological Control – Although this is the final report, research will continue under ARS funding. (a) Releases of three new biotypes of Tamarixia from Asia are being continued in southwest and east-central Florida. It is too early to gauge establishment. Research was initiated in east-central Florida on infestations of ACP and biological control of ACP by Tamarixia in urban plantings of jasmine. Releases of the new biotypes in these urban areas have not yet been made, but the old biotype of Tamarixia has been observed at some of these locations. Increasing biological control of ACP in urban settings could contribute to ACP area-wide management . (b) There is no evidence that releases in Florida of a new biotype of the parasitoid Diaphorencyrtus resulted in establishment. Some project funds supported a Ph.D. student, who is completing the degree and is in the process of preparing several manuscripts for publication. Seasonal HLB profile in adult ACP ‘ Although this is the final report, research will continue under ARS funding. From Feb 2008 through Mar 2010, a mean of 32% (SEM 3.1) ACP tested HLB positive. Percentages of ACP testing positive has varied widely among sample dates, with a mean minimum of 7.4% (SEM 3.7) during late Feb 2009 and a mean maximum of 70% (SEM 3.2) during late May 2009. An average CT value of 30.7 (SEM 0.2) was recorded among all infected ACP over the entire study period, or an average CT value of 26.1 (SEM 0.2) for ACP with CTs less than 32. For ACP with CT values below 32, mean CT values have ranged from 23.5 (SEM 0.6) in Aug 2009 to 29.4 (SEM 0.2) in late Feb 2009. An average of 77.1% (SEM 8.8) of the trees from which ACP are collected have tested HLB positive (mean CT 23.9, SEM 0.3). No trends in seasonality of HLB in ACP have yet been identified over the course of this two-year study. An average of 35.3% (SEM 4.1) ACP tested HLB positive and an average of 15.8% (SEM 4.2) infected ACP transmitted the disease, with means per sample date as low as 0% and as high as 38%. It is too early to determine if there are any seasonal trends in transmission rates.
This is the final report of a three-component project on the Asian citrus psyllid. Sampling – Stem-tap sampling is preferred over sticky traps for making ACP density estimates and is considerably less expensive. Sampling to estimate ACP densities should be conducted throughout a block because there can be significant variation across a block of trees in numbers of ACP. For commercially-acceptable precision levels at means of 1 or more adult psyllids per sample, 28 stem-tap samples are required in a block of trees up to ten acres in size (grove edges are not sampled under this protocol). Larger numbers of samples were required at lower densities. Although no control threshold for ACP has been identified, this level would likely lower than than 1 ACP per sample, perhaps 0.2 to 0.3 ACP per sample – thus growers wanting to make insecticide decisions for ACP control should take larger sample sizes. Noted is that young trees do not lend themselves well to stem-tap sampling. Sticky traps are preferred for ACP detection, not density estimation. Three publications on sampling have been published (available on request) and one has been submitted. Future research efforts on ACP sampling should include a method of estimating densities for an entire block of trees including edges. A formal method is needed for sweep sampling. The relationship between mean number of ACP per tap sample and absolute ACP densities in trees needs to be clarified. Biological Control – Although this is the final report, research will continue under ARS funding. (a) Releases of three new biotypes of Tamarixia from Asia are being continued in southwest and east-central Florida. It is too early to gauge establishment. Research was initiated in east-central Florida on infestations of ACP and biological control of ACP by Tamarixia in urban plantings of jasmine. Releases of the new biotypes in these urban areas have not yet been made, but the old biotype of Tamarixia has been observed at some of these locations. Increasing biological control of ACP in urban settings could contribute to ACP area-wide management . (b) There is no evidence that releases in Florida of a new biotype of the parasitoid Diaphorencyrtus resulted in establishment. Some project funds supported a Ph.D. student, who is completing the degree and is in the process of preparing several manuscripts for publication. Seasonal HLB profile in adult ACP ‘ Although this is the final report, research will continue under ARS funding. From Feb 2008 through Mar 2010, a mean of 32% (SEM 3.1) ACP tested HLB positive. Percentages of ACP testing positive has varied widely among sample dates, with a mean minimum of 7.4% (SEM 3.7) during late Feb 2009 and a mean maximum of 70% (SEM 3.2) during late May 2009. An average CT value of 30.7 (SEM 0.2) was recorded among all infected ACP over the entire study period, or an average CT value of 26.1 (SEM 0.2) for ACP with CTs less than 32. For ACP with CT values below 32, mean CT values have ranged from 23.5 (SEM 0.6) in Aug 2009 to 29.4 (SEM 0.2) in late Feb 2009. An average of 77.1% (SEM 8.8) of the trees from which ACP are collected have tested HLB positive (mean CT 23.9, SEM 0.3). No trends in seasonality of HLB in ACP have yet been identified over the course of this two-year study. An average of 35.3% (SEM 4.1) ACP tested HLB positive and an average of 15.8% (SEM 4.2) infected ACP transmitted the disease, with means per sample date as low as 0% and as high as 38%. It is too early to determine if there are any seasonal trends in transmission rates.
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