Objective 1: Assays of non-bearing trees soil drenched with the chelated copper formulations Magna-Bon (MB) and Copper Quik (CQ) confirm that copper concentration in leaves of non-sprayed field trees is well above nutritional concentrations. HLB preventative treatments of MB and CQ were applied at 3 month intervals during two growing seasons at 1,2,4,5 or 10 oz/tree in 8 oz of water as soil drenches to visually asymptomatic 1 and 2 year old Valencia trees on Volkamer lemon and Swingle citrumelo, respectively. The solid plantings, located in Sebring and Avon Park, were surrounded by adult trees with 100% incidence of HLB symptoms. After two seasons, leaf samples for determining PCR status of trees in each trial were collected in December 2013 and analysis completed in early 2014. In the 4 year old block of Valencia on Swingle, PCR positive trees across all treatments ranged from approximately 40 to 80% incidence and in the 3 year old block of Valencia on Volkamer lemon ranged from 8-15% incidence. Incidence of PCR positive trees drenched with MB or CQ drenches was not significantly different from untreated trees. Soil copper treatments will be continued in the 3 year old block of Valencia on Swingle for one more season. Second year PCR results from a trial at USDA Picos farm in Ft. Pierce treated with MB, CQ and copper or zinc phosphite drench treatments of Hamlin on Swingle trees treated since planting still has a low incidence of HLB positive trees. Drench treaments at the Picos and Sebring sites will be continued for another season.
Objective 1: Assays of non-bearing trees soil drenched with the chelated copper formulations Magna-Bon (MB) and Copper Quik (CQ) confirm that copper concentration in leaves of non-sprayed field trees is well above nutritional concentrations. HLB preventative treatments of MB and CQ were applied at 3 month intervals during two growing seasons at 1,2,4,5 or 10 oz/tree in 8 oz of water as soil drenches to visually asymptomatic 1 and 2 year old Valencia trees on Volkamer lemon and Swingle citrumelo, respectively. The solid plantings, located in Sebring and Avon Park, were surrounded by adult trees with 100% incidence of HLB symptoms. After two seasons, leaf samples for determining PCR status of trees in each trial were collected in December 2013 and analysis completed in early 2014. In the 4 year old block of Valencia on Swingle, PCR positive trees across all treatments ranged from approximately 40 to 80% incidence and in the 3 year old block of Valencia on Volkamer lemon ranged from 8-15% incidence. Incidence of PCR positive trees drenched with MB or CQ drenches was not significantly different from untreated trees. Soil copper treatments will be continued in the 3 year old block of Valencia on Swingle for one more season. Second year PCR results from a trial at USDA Picos farm in Ft. Pierce treated with MB, CQ and copper or zinc phosphite drench treatments of Hamlin on Swingle trees treated since planting still has a low incidence of HLB positive trees. Drench treaments at the Picos and Sebring sites will be continued for another season.
This research seeks to determine whether young trees infected with CLas and displaying typical HLB symptoms can be brought to maturity and produce an economically viable yield. This will be achieved by managing a 58 acre grove of 3-year-old ‘Valencia’ / Kuharske Carrizo trees using a combination of three different foliar and three different ground applied nutritional programs. Factorial AxB treatments consist of A) ground-applied: 1) Liquid/dry+Ca (BHG standard), 2) Liquid+Ca, 3) Liquid/dry-Ca B) foliar-applied: 1) BHG standard-Ca, 2) BHG standard+Ca, 3) “Prescription”(+Ca). The prescription treatment was designed to be dynamic, customized for optimization, with feedback based on frequent leaf tissue analyses, visual symptoms, and the growth of the tree canopies and yield. There are six replications of treatments, with two being pure replications. The grove still continues to look better, despite the nearly 100% HLB incidence. Foliation of canopies is dense, and leaf color going into Spring season was a healthy green. Fruit yields per plot were measured on 19 March 2014, and analyzed together with the fruit quality data determined in the packing house. Selected data is summarized below: FOLIAR FERT Fruit yield (boxes/ac) STD-Ca STD+Ca Prescrip SOIL FERT Liq/Dry 171.8* 130.2 134.9 Liq 150.6 121.3 134.5 Liq/Dry-Ca 146.4 133.9 113.0# Only the yield of the treatment marked with an * was significantly higher than the worst yield (#); all others were non-significant and can be considered the same. The best treatment yielded 1.13 boxes per tree at age 5; considerably less than healthy trees of the same age class would yield, but nevertheless showing the increasing potential of HLB-impacted young trees to recover and gradually increase their fruit yielding capacity. As previously mentioned, HLB-impacted young blocks should be planted at higher densities to offset the per tree yield losses caused by the disease. Theoretically the best treatment in this experiment could yield 410 boxes/acre at age 5 if the planting density was 363 trees/acre (8×15′) instead of the 151 trees/acre (12×24′) to which it is currently set. Statistically speaking, all the different fertilizer strategies chosen for testing in this block appear to be working equally well. However an economic analysis of the treatments will undoubtedly show that the less expensive standard fertilizer program for both soil and foliar nutrition contributes to the profitability in this grove. In fact the best treatment (*) consists of standard BHG liquid/dry (with calcium nitrate) soil fertilization and standard foliar nutrition (no Ca). Interestingly, it appears that Ca is best applied to the soil and not the foliage. The reader should understand that mitigation of HLB with plant nutrition is a strictly site-specific practice and the results obtained in this experiment are not necessarily reproducible in another grove elsewhere in Florida.
A new class of inhibitors were discovered based on discovery of enzymes present in salivary sheaths. Two of the six tested inhibitors blocked sheath formation. An additional six different proteins belonging to the same functional class of proteins were identified based on their potential to bind to salivary sheath subunits. These proteins were tested for sheath inhibition, but none caused inhibition of sheath biosynthesis or alterations in sheath appearance. The ultimate goal of this screening approach is to identify a diverse set of sheath inhibitors that can be used in combination to function as effective topically applied blockers of psyllid feeding on citrus. If feeding is inhibited at the initiation of feeding and prevents the psyllid from successfully reaching the phloem, this method of psyllid control will prevent the psyllid form acquiring or transmitting the bacterium that causes citrus greening.
As a result of our compositional structural and enzymatic analysis of psyllid sheath structure and biosynthesis, we identified a class of natural products (Generally Regarded as Safe ‘GRAS’ compounds) that could inhibit sheath biosynthesis. These compounds were selected based on the probability that their structure would be competitive with the substrates used in the sheath polymerizing process. Twelve such compounds were selected and tested for sheath inhibition. Of the twelve, the presence of 10 in the media in which these insects fed resulted in aberrant sheath formation. These are now being tested in dose studies and in combination. The doses and combinations showing the best response will be used in greenhouse application studies. The discovery of GRAS compounds that have affect sheath formation offers the possibility of a rapid commercialization process if they are shown to be effective in whole plant application trials. Some of the tested compounds are available at relatively low costs that would be compatible with commercialization if efficacy as a topically applied compound can be verified.
THis project was an extension of a previously funded CRDF grant. Funding for both overlapped during this year with just minor funds available in the base 330 project. Therefor results presented in the March 2014 report are the same for both projects and are repeated in the quarterly reports: As a result of our compositional structural and enzymatic analysis of psyllid sheath structure and biosynthesis, we identified a class of natural products (Generally Regarded as Safe ‘GRAS’ compounds) that could inhibit sheath biosynthesis. These compounds were selected based on the probability that their structure would be competitive with the substrates used in the sheath polymerizing process. Twelve such compounds were selected and tested for sheath inhibition. Of the twelve, the presence of 10 in the media in which these insects fed resulted in aberrant sheath formation. These are now being tested in dose studies and in combination. The doses and combinations showing the best response will be used in greenhouse application studies. The discovery of GRAS compounds that have affect on sheath formation offers the possibility of a rapid commercialization process if they are shown to be effective in whole plant application trials. Some of the tested compounds are available at relatively low costs that would be compatible with commercialization if efficacy as a topically applied compound can be verified.
Continued proteome analysis of isolated psyllid salivary sheaths was conducted using a more complete psyllid protein database. This analysis was performed through collaboration with Dr. Michelle Cilia, Research Molecular Biologist USDA-ARS, BioIPM Research Unit and Boyce Thompson Institute for Plant Research that has allowed the identification of a candidate enzyme that may be involved in the sheath polymerization process. The identified gene encoding the protein was then identified in the psyllid genome showing that it is indeed of psyllid origin. Further analysis of sheath formation showed that inhibitors of this enzyme blocked sheath formation. This research has allowed us to understand the nature of the sheath polymerization process and this knowledge has opened the door for natural product screening to identify natural product inhibitors to sheath formation.
In earlier phases of this study, a “Bugphone” microcontroller device that detects Asian citrus psyllid male vibrational calls and plays back female replies was tested for its ability to attract males to a sticky cone attached to a small tree. In these studies, 20-30% of tested males have responded to the Bugphone, but for various reasons, only a small percentage of the males that approached the cones were captured. In the enhancement phase of this study, considering other ways of interfering with mating that did not necessarily involve trapping, we purchased outdoor speakers that can be operated with small amplifier systems to produce signals interfering with male and female ACP duetting calls in field environments. Initial studies of the capability of the systems to disrupt mating have begun in a laboratory setting to determine the best sound pressure levels and signal timings. We have joined forces with Dr. William Eisenstadt and students at the University of Florida Department of Computer and Electrical Engineering to consider options that use low power because initial versions could operate for only 1-2 days before replacement batteries were needed.
This proposal aims to continue improvement to a novel psyllid trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing was and continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps and behaviorally active components. 570 – Grant Obj. 1: We continue to conduct field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulations of visual cues as well as trap physical components and component orientation. This within Florida research component is continuing and we now added several new locations in Puerto Rico to exploit the significantly higher ACP populations there that make the research easier. Doing this quarter we have successfully tested >20 trap prototype configurations in Puerto Rico in cooperation with Dr. David Jenkins and have now eliminated a number of configurations that are inferior as well as identified the most successful trap under field conditions. We have had no success with trap additions related to odor cues reported to be potential attractants in the literature. We are continuing this research with new ideas but now have a trap identified a “standard trap” that can be used to capture and preserve the DNA of the psyllids and their associates in situ. Obj. 2: We have initiated the areawide psyllid sampling objective to detect and develop new biological controls for use against ACP. We are continuing the sampling in the northern most citrus populations in Alachua County and around Ft. Pierce with the in Florida and Puerto Rico. The standard trap is being used for this work and performs well enough to complete this effort, i.e., where ACP occur the trap captures and preserves them in proportion to their populations. So far we have not identified any new pathogens. We have obtained cooperation with other USDA-ARS research personnel with expertise in the identification and rearing of entomopathogenic fungi and they have evaluated samples toward this end from Florida and Puerto Rico. This effort will be continued.
This proposal aims to continue improvement to a novel psyllid trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps and behaviorally active components. This enhancement grant is to further use the trap for detection and identification of biological control agents as well as to develop novel methods to manipulate psyllid behavior to improve the deployment and efficacy of biological control agents. This research was begun this quarter and is ongoing in Florida and Puerto Rico. We have have successfully solicited some producers with conventional and organic groves where we are conducting our experiments. No results to report so far but we are actively collecting field data toward the objectives that will be forthcoming in the next report.
We are evaluating and attempting to develop optimal RNA interference constructs to target Diaphorina citri, the psyllid vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening. We are using two psyllid species for our work, D. citri and Bactericera cockerelli, the latter being the potato psyllid. Using B. cockerelli offers the opportunity to use herbaceous plants and make more rapid progress that can then be applied to citrus and D. citri. So far, 7 aMIRNAs targeting the BC-ATPase gene (pAMIRA1, pAMIRA1c, pAMIRA2, pAMIRA2c, pAMIRA3, pAMIRA3c, and pAMIRA2PE) and 4 pAMIRNAs targeting the GFP gene (pAMIRG1, pAMIRG1c, pAMIRG2, and pAMIRG2c, as controls) have been generated. We cloned some of these into three expression vector systems: a TMV-based viral vector, a geminivirus-based viral vector and a binary plasmid. We are evaluating these in plants now. Both the TMV and binary plasmids appear to be functioning as expected although different amiRNA product patterns are observed after northern blot hybridization analysis. The geminivirus-based system did not work originally, we have re-generated the constructs and they are under evaluation now. We believe that the geminivirus system offers the best potential for generating high amounts of specific amiRNAs in plants. We are now preparing small RNA cDNA libraries from plants for each system and will sequence the small RNA products to assess the quantity and quality of specific miRNAs. We are also using RT-qPCR to assess target mRNA knockdown in psyllids after feeding on plants infiltrated with the above constructs. We are using psyllids per sample with replicated samples for each treatment. Simultaneously, we are testing B. cockerelli on specific transgenic plants that are engineered to generate interfering RNAs to different B. cockerelli mRNA targets. We are using a leaf disc assay and RT-qPCR to assess target mRNA knockdown, and using whole plants to assess mortality effects on adults and psyllid nymphs.
Applications of PGRs to container-grown citrus trees affected the biology and behavior of the Asian citrus psyllid. In greenhouse and growth camber experiments, prohexadione and mefluidide applications to trees reduced ACP fecundity and survivorship, whereas uniconazole affected fecundity and paclobutrazol affected survivorship. These PGRs did not affect adult ACP weight, except when paclobutrazol was applied. Dikegulac appeared to inhibit ACP egg laying, but this was likely due to phytotoxicity. Oviposition was delayed and rate was lower on PGR-treated trees, but chlormequat under greenhouse conditions had an effect. The effect of PGRs on ACP did not appear to be due to growth retardation or specific toxicity of the materials, suggesting a yet undefined effect on plant physiology that altered host quality.
The objective of this study is to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology. We have initiated this study with complementary field and laboratory experiments. We started the second year of the field experiments. We are repeating the same protocol as last year, with the difference that this year we will expand and have a second experimental field (given the additional enhancement funding that will be reported on separately). This experiment is being conducted in a solid planting of Hamlin resets that are now approximately three years old. We have already started the application of nutritional spraying, following the Keplex’ recommendations for this year. At the end of April, we will start monitoring ACP nymph and adult populations, as well as flush development. We have also planned a new screening of HLB infection status of trees with qPCR in this reset field for April to observe how infection has changed from last year, depending whether trees were supplemented with nutrients or not. We are currently performing a laboratory experiment where citrus resets are placed in a Las-infected ACP colony (55% of psyllids had been confirmed Las positive). Half of these citrus trees are nutritionally supplemented and half of them are sprayed with water (control). We are following the infection status of these trees over the course of 1 year to observe if the plants treated with nutritional supplement are more or less protected against HLB infection. Interestingly, after 4 months of exposure to infected psyllids only the controls were found to be infected by CLas, whereas all the nutritionally supplemented trees are still negative to CLas. If these results are confirmed, it may show that nutritional supplement may decrease susceptibility of resets to HLB infection. Finally, we are also conducting an experiment where we are comparing the dispersal behavior of ACP that settled on HLB-infected trees, or that settled on HLB-infected trees treated with nutrient supplements. We know that psyllids are first attracted to HLB-infected trees but subsequently disperse from these infected trees. One hypothesis for this subsequent dispersal from HLB-infected trees is that these trees are nutritionally sub-optimal to psyllids. Consequently, we hypothesize that psyllids that settled on HLB-infected trees treated with nutriment spray should disperse less than counterparts that settled on untreated HLB-infected plants. So far, our data appear to confirm this hypothesis as we have fewer psyllids dispersing from HLB-infected trees treated with nutrient supplement sprays than psyllids dispersing from untreated HLB-infected trees.
We are continuing to evaluate the new neonicotinoid, flupyradifurone (active ingredient of SivantoTM , Bayer CropSciences) against the Asian citrus psyllid in various assays in comparison to imidacloprid. We have completed a study to determine the efficacy of this insecticide to reduce feeding on plant tissue at various concentrations and found that flupyradifurone (technical grade) did not perform as well as imidacloprid. At 25 ug/ml treatment, there is no difference in efficacy between the two insecticides. However, at 2.5 ug/ml, imidacloprid continued to deter feeding at the same level as 25 ug/ml, whereas feeding behavior was no different between treatment and controls for flupyradifurone. Thus far, we have found that flupyradifurone does not perform as well as imidacloprid in topical (LD50), contact (LC50) or feeding deterrent assays. Currently, we are conducting assays to compare toxicological efficacy of flupyradifurone and imidacloprid when ingested by the insect. We are also planning experiments to investigate non-lethal effects of the insecticide including settling behavior, fecundity and longevity. We are ready to begin our annual field survey to monitor insecticide resistance in field psyllid populations and will begin those efforts in April. We have begun limited work on development of a vial assay to use in the field to test for insecticide resistance on-site and have completed the base-line susceptibility assays against the six insecticides (carbary, chlorpyrifos, fenpropathrin, flupyradifurone, imidacloprid and thiamethoxam). We hope to evaluate this tool over the course of the summer field survey. This tool would greatly improve the efficiency of insecticide resistance monitoring in the field.
The overall objective of this research is to develop a push-pull system for the Asian citrus psyllid (ACP) that can complement integrated management systems in young citrus plantings. Towards that goal, visual factors that affect psyllid takeoff into flight and landing are being examined to guide development of an optimal pull component. A vertical flight attraction assay was developed to examine response of ACP resting or in flight towards visual targets that consist of different wavelengths of filtered light. Narrowband width filters (10 nm) were used to allow clear delineation between colors tested. This assay simulates the response of ACP to visual cues that are above them such as when an adult is settled on vegetation and looks up at the sky or when having fallen to the ground looks up and sees its host plant from the ground. ACP response was lowest to red and orange in the visible spectrum. Yellow was more attractive to ACP then orange or red, but less attractive then blue, green, or lime green. Green and lime green were the most attractive to ACP in the visible spectrum. In the ultraviolet spectrum the most attractive wavelength was long UV (405nm) compared to shorter wavelengths arranged in order of decreasing attractiveness 350nm, 380nm, and 365 nm (least attractive). The vertical bioassay differed from the horizontal bioassay, in that ACP responded as strongly to UV as they did to green and lime green. This may be evidence that the ACP orient differently to vertical and horizontal cues. For example, when ACP drop to the ground following a disturbance vertical cues of UV (sky) or green and lime green (leaves) are both used to return to a place of rest (the host plant). Whereas horizontal cues are more typically used when ACP are already situated on a plant and therefore only UV (sky) cues are used to navigate towards increasingly younger and more optimal portions of the host plant (young flush), which are near the edge of the plant (near UV) versus in the center of the plant (away from UV). Ultimately, the UV cues from the horizontal axis may be causing the ACP to move to another plant in search of new host locations. Further studies are on-going with the vertical bioassay to see if there is a difference between 0-1 day old and 4-7 day old ACP in this orientation. Additionally, studies are being undertaken to see if flying ACP will be attracted to different colors then walking/flying ACP by using specially designed walking preventing chambers coated with Fluon. A horizontal walking assay was developed to stimulate walking responses of ACP on vegetation and this assay measured direction of movement as well as speed of movement. Known age studies with ACP in the horizontal bioassay revealed that 1 day old ACP were positively attracted toward green light , whereas 4-7 day old ACP remained relatively neutral to green light. Both 1 day old and 4-7 day old ACP showed more positive attraction towards long UV light. These findings indicate that newly emerging ACP may be more attracted to green, because they need to acquire nutrients from feeding on a plant. Conversely, older ACP may be more interested in movement to other plants in search of mates and oviposition sites. Further studies with the horizontal walking assay will be made between newly emerged unmated female ACP and older gravid females. Examination of the effect of different visual stimuli on flight duration of ACP as measured on flight mills is on-going. Comparisons in the duration of flight and numbers of flight bouts are being made between different colored substrates, different quality of light overhead (colored, with UV, without UV, polarized), contrast between overhead lighting and substrate coloring and presence of visual targets.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
