Entomopathogenic nematodes: Amending soils to increase biological control of insect pests We repeated an experiment at the Bartow site in which we buried caged weevil larvae in all plots and evaluated the mortality rate after 7 days in the soil. The previous trial revealed no significant effect of sulfur amendments (to reduce soil pH) on infection of buried weevils by S. diaprepesi. We obtained similar results in the second trial. S. diaprepesi infected weevils were 23% more abundant in low pH plots and H. indica were 44% more abundant in high pH plots. The trends were the same in both trials, but treatment effects were not significant in either. We assayed soils from the two treatments (sulfur amended and unamended) by adding S. diaprepesi encumbered with spores of Paenibacillus sp. After seven days spore-free nematodes were more abundant in sulfur amended, compared to non-amended soil, but the differences were not great (12% vs 5%; P=0.05) and most nematodes remained encumbered by spores. Soil pH of sulfur amended soil was 6.4 vs 7.1 in non-amended soil. The pH difference was less than observed earlier in the year and at a level approaching ineffective for removing spores. Consequently, soil in the field experiment was again treated with Tiger 90 sulfur and assays using caged weevil larvae will be reinitiated when soil pH in amended plots becomes ‘6.0. Because soil phosphorus was positively related to S. diaprepesi abundance in several field trials, we bioassayed Paenibacillus spore adherence to S. diaprepesi in solutions of 10, 50, 100 molar phosphate buffer, each adjusted to a range of pH between 5.0-8.0. As shown previously, spores detached from the nematode cuticle in inverse proportion to solution pH, but phosphate levels had no effect on spore adherence. We initiated bioassays to study effects of soil water potential on survival of the 4 most commonly encountered EPN species. Soil from the conventionally managed and advanced production system treatments (Schumann study) were oven dried and moistened to 2% (dry), 6% (
The long-term goal of this project was to develop a framework for sustainable nutrient management in South Texas Citrus orchards to ensure healthy productive trees with improved fruit quality. Specific Project Objectives for 2013 were to develop a baseline picture of native soil fertility across citrus orchards in the Rio Grande Valley, TX, and to determine which nutrients are most limiting. Procedures: Soil and leaf tissue samples were collected from thirty-six (36) orchard locations across the Valley and analyzed for mineral nutrient contents. Different soil analytical procedures were evaluated in several soil testing labs. Findings: While critical mineral nutrients such as potassium and phosphorus were found to be adequate according to the widely-used Mehlich-3 soil extraction method, they were low or marginal based on the CO2/H2O method. Although calcium, magnesium and potassium are abundant in calcareous soils in south Texas, these minerals may not be readily available for tree uptake. Potassium is readily fixed in clay minerals and rendered unavailable for uptake. Adequate calcium uptake may be limited by low transpiration since this mineral relies on a steady transpiration stream for uptake. Competitive uptake interactions among these minerals could also influence uptake. Soil phosphorus was consistently below adequacy levels, whereas boron levels seem to be excessive. It is now known that HLB and phytophtora infection severely limit root growth and uptake activity. Low soil P levels may contribute to this observation. Earlier observations from Florida indicated that Ca may play a major role in tree tolerance to HLB infection. More recent observations indicate that other nutrients, especially micro nutrients such as zinc and manganese may also be important. In the current study, zinc and manganese concentrations in grapefruit leaf tissue were consistently below sufficiency levels. There are currently no nutritional guidelines for citrus production based on local soil and weather conditions in south Texas. Fertilization guidelines adopted from other production regions are not optimized for fruit quality (solids content, color, etc.) or tolerance to pests and diseases. Nutrients are continually mined/removed from orchards each year with harvested fruits with little replacement. Higher yields are expected to remove larger quantities of nutrients, thus necessitating replacement. Future research as a continuation of this project will estimate nutrient removal amounts in order to determine which nutrients are being removed in the largest quantities and the implications for fruit quality and tolerance/resistance to biotic and abiotic stresses. Future research should also determine possible mechanisms and strategies to enhance nutrient solubilization and availability for tree uptake. In summary, while soils in south Texas seem to contain adequate levels of critical macro and micro-nutrients, most these nutrients are fixed and unavailable for tree uptake. Orchard-specific nutrient budgeting programs based on yield expectations are needed for optimal tree growth, fruit yield/quality, and resistance to biotic and abiotic stresses.
Imidacloprid (IMD) is a systemic pesticide soil-drenched to control the citrus greening primary vector Diaphorina citri Kuwayama, or Asian Citrus Psyllid (ACP). Imidacloprid fate and transport in Immokalee Fine Sand (IFS) soils was studied in SW Florida Flatwoods during citrus greening (CG) management. Soil and leaf samples have been collected in spring (dry) and summer (rainy) seasons for the past two years. Imidacloprid rates have been applied at 1X, 2X and 4X lable rates on transplants (1-2 yrs), moderate age (5-7 yrs), and mature trees (>10 yrs) each season. The 2X and 4X applications typically have proportionally greater soil concentrations in the 0-15 cm depth and decrease to less than 1 .g per mg soil. Deeper soil depths typically never increase above 0.5 .g per mg soil. These results would indicate that Imidacloprid moves with the wetting front with low soil residual concentrations. Sorption and degradation studies showed a weakly-sorbed (log Koc, 1.1-2.4) and persistent chemical (half-lives 0.9-2.3 years) with high potential for leaching. We also established field experiments in young Hamlin trees with micro-sprinkler irrigation, during 4 growing seasons between 2011 and 2013. IMD was soil-drench applied in the root zone (PA), and in a control zone not affected by roots (NPA). IMD concentrations (.g g soil-1) were higher for the NPA than PA as a function of time due to uptake by the citrus trees. IMD leached out of the root zone about 3 to 4 weeks after application in the summer, and about 6 to 8 weeks after application during spring. Nonetheless, there was effective systemic control of the CG vector Diaphorina citri Kuwayama (ACP) at about 2 weeks after application, where treated trees showed consistently lower ACP adults and immatures infestation, continuing for at least 8 weeks. A method to analyze IMD from citrus tissue (ng g-1) was developed using HPLC-MS/MS detection, and preliminary data are in close agreement with our findings in soils and insect counts on trees. Close irrigation and rainfall monitoring during IMD application is of the utmost importance to avoid leaching problems in groundwater resources of SW Florida, and to ensure systemic control of the ACP.
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. Obj. 1: We continue to conduct field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulating psyllid behavior around the trap (i.e., increase trap active distance). We have a number of positive results from our latest bioassays and continue to tweak the trap structural components to increase trap efficiency through field testing. We also have continued testing of semiochemical lures that supposedly attract psyllids provided by other scientists as well as a number of possible lures known to be behaviorally-active to other insects that we selected for testing . This work is ongoing and more experiments are being conducted but none of the lures we have tested have provided any increase in trap catch. Results from these studies and similar results from bioassays on other insects (some new research by ARS personnel notwithstanding) lead one to conclude that it is unlikely that a strong lure for ACP exists, can be made or discovered. This research is continuing and we expect to make much more rapid progress in the next quarter by exploiting the significantly higher ACP populations in Puerto Rico. Obj. 2: We have initiated the areawide psyllid sampling objective to detect and develop new biological controls for use against ACP. We have begun sampling in the northern most citrus populations in Alachua County and around Ft. Pierce with the intention of systematically sampling by working south from the northern region and outward in all direction from Ft. Pierce. The standard prototype 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 but we will continue to conduct this sampling effort Florida as well as expand it into Puerto Rico.
October 2013 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: All preliminary experiments have been completed for propiconazole, difenoconazole, and tetraconazole. Many concentrations were tested to find opitmal concentration ranges for each fungicide. We found an expected effect of light duration on the fungicide efficacy. We are exploring this further. Experiments with fenbuconazole are up-coming. Objective 2: The field trial was established in a commercial grove that was identified with ‘Valencia’ fruit with black spot. In spring 2013, the site was was scouted and rows of ‘Valencia’ were identified that had fairly even distribution of fruit with symptoms of black spot. Plots contained three trees with at least two trees serving as buffers between each plot. There were four replications per treatment arranged in a randomized complete block design. Sprays were initiated in May after fruit harvest. Applications were applied with an Air blast sprayer calibrated to deliver 126 gal per A operating at 200 psi and 3 mph with a 25 gallon mix. Applications continued until September. The grove was recently inspected for symptoms but none have developed yet. Objective 3: We are currently looking for suitable sites to conduct the spatial studies. This study will begin in earnest close to fruit maturity when symptoms become apparent. Objective 4: A contaminant was discovered in the G. citricarpa isolate used for the first set of experiments and steps taken to assure future work with clean cultures of the isolate. In addition, three additional Florida isolates were obtained for evaluation for possible use in the experiments. The government shutdown also disrupted work on the project. In an experiment repeating some of the earlier work and evaluating the effects of essential oils (Benzaldehyde, Cinnamaldehyde, Trans-cinnamaldehyde, Carvacrol, Eugenol, Citral, and Eucalypol) at two different concentrations on the organism growth in vitro, it was found that Citral (especially 100 mg/l) and Carvarol significantly inhibited mycelium growth often completely; Cinnamaldehyde, Trans-cinnamaldehyde, and Benzaldehyde inhibited mycelial growth moderately; and
Dr. Justin George assumed a postdoctoral position at USHRL in August and has begun work on this project, replacing Dr. Paul Robbins. Dr. Robbins has transferred to a position in the laboratory of Dr. Joe Patt where he will continue to work on ACP behavior and orientation to plant odors. Justin did his PhD at Pennsylvania State University in the laboratory of Tom Baker, an excellent chemical ecologist, where he worked on the behavior of mosquitoes. He brings expertise in electrophysiology and will contribute to our efforts to decipher the cues governing ACP behavior and orientation to host plants. There is no new progress to report at this time.
September 2013 The objectives of this proposal are 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3.) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. We are in the process of hiring one technician for the project. Others have been hired and will be starting shortly. Small plots were set up an Immokalee area grove with 5% urea, CaCO3, Soil set, Compost Aid, Soil Set and Compost aid, and an untreated control. Leaves were collected and treated. The samples were collected and are still being processed. The DNA sequence of the mating type locus has been established for G. citricarpa. Forty-two isolates collected from Collier and Hendry counties have been screened to identify isolates of opposite mating types. This information will be used to establish compatible pairings of isolates to investigate the environmental conditions required for pseudothecium and ascospore development. To look at the effect of bagasse, the Senior Biological Scientist and the post doctoral associate worked on assembling the necessary supplies and components to carry out the controlled experiment portion of this project. Five treatments contained leaves which had been inoculated with conidia of Guignardia citricarpa and five treatments contained un-inoculated control leaves. Treatments were as follows: 1) Bagasse; 2) Bagasse + microbial consortium + molasses; 3) Microbial consortium + molasses; 4) Urea; and 5) Untreated. In addition to the above mentioned treatments, two additional treatments were included which did not contain citrus leaves. These were: 1) Bagasse + microbial consortium; and 2) Bagasse. The microbial consortiums were Ceriporiopsis subvermispora, Cellumonas flavigena, and Azospirillum brasilense. Soil weight, leaf weight, and then the total weight of each box was recorded. Leaves were collected at time zero and at fifteen day intervals. Leaf infection by G. citricarpa was verified. At each collection time, leaves were dried and weighed. Data collection and analysis is still in process. This experiment will be repeated during the next quarter. Based on observations and preliminary results, several modifications to the experiment will be implemented. The USHRL crosses have been cut back to continue the in vitro assays and we expect to receive samples within the next month for continued experimentation. The subcontracting process with the University of Queensland is complete. In QL leaf litter samples have been collected from 4 commercial blocks for the seasonal dynamics project. Fungal fructification is being enumerated. Because the number of structures is low, monthly sampling will continue until the onset of rains. An inventory of the sour orange crosses was taken and an prototype inoculation of procedure was tried. The results won’t be known for several months.
Oct 28, 2013 The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus (Las) prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and do the flushing patterns affect the prevalence of Las in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Las on a grove scale and how does it compare the results from the citrus trees in the same grove. In 2008 and 2009 Ebert and Rogers demonstrated that the prevalence of Las in the Asian citrus psyllid (ACP) varied seasonally but the pattern between seasons was not consistent. It was suggested that perhaps the reason for the differences between the years related to the flushing patterns of citrus and the prevalence of the bacterium in the leaves where ACPs are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and ACPs. Sample collection of psyllids, phenology and plants ended in September 2013 from Conserv and Lake Wales. The grove in Lake Alfred was pushed in July therefore sampling in that location ended at that time. Plant and psyllid samples are continuing to be extracted slowly. PCR has begun to calculate the pool size for the remaining samples. This will help to clear the back log of samples and get the project back on track.
Oct 28, 2013 The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. Guignardia mangiferae sequencing is complete and the assembly is continuing. The full mating type gene has been located and the information was used to better characterize the mating gene from G. citricarpa. A manuscript is in preparation and analysis is continuing. Protocols for paraffin section of leaf tissue to look at the early infection process are being developed and finalized. Preliminary experiments to determine protocols for the field experiments are underway including maturation of pseudothecia in controlled conditions. Initial twig inoculations are underway and we are waiting for pycnidia formation on twigs. It is a 8-10 week process.
Progress on development of an acoustic trap to capture male Asian citrus psyllids (ACP) searching for females on citrus tree branches continued in the third period of 2013. An Arduino Uno microcontroller instrument, BugPhone, has been developed to detect male vibrational calls using a microphone attached to a branch of the tree. Whenever a call is detected, BugPhone immediately plays back a synthetic female reply from piezoelectric buzzer. Calling males then are attracted to a cone trap set in front of the buzzer and are captured as they try to walk through the cone towards the buzzer. During this quarter, we have focused on improving the design of the cone trap and on decreasing the power usage of the BugPhone so that the system can operate longer than four days in the field without replacing the battery. Plans are in progress to construct eight new systems of the most recent version of Bugphone for further field testing. In addition, we have published a manuscript on the design of the microcontroller system and a manuscript on streamlined procedures for rearing psyllids of known age and mating status. We have begun a manuscript that describes the operation of the BugPhone instrument.
Late summer/fall 2013: We completed 1 trial and continued monitoring multiple field trials to study citrus leafminer (CLM) attraction to pheromone blends and to test suppression of trap catch where pheromones were deployed. Trials address pheromone carrier, timing, deployment pattern, and efficacy of mating disruption and leaf damage by CLM. Trial 1: Winter and spring 2013 application of pheromone. Cooperator: Packers of Indian River. Ongoing trial to test winter and spring applications of pheromone on suppression of CLM using a factorial design with split plots in 4 replicated blocks. Rubber dispensers (DCEPT’, ISCA Technologies Inc.) loaded with triene (834 mg/ha) were deployed February 6-8 and/or April 24. Traps were placed in canopy at 1.6 m, and we also placed traps at 3.1 m to evaluate influence of height on disruption. Trap catch disruption in plots treated in winter and spring was 95% in lower traps and 75% in higher traps 19 weeks after the second application. Height of dispensers may be an important variable for optimal disruption. Flush shoots were evaluated a fourth time on 7 August. Infestation of shoots was 14% lower in treated plots. Trial 2: DCEPT dispensers (St. Lucie Co.). Cooperator: Golden River Fruit Co. We monitored a large plot trial of ~200 acres treated on 26 July with 2.5 mg triene/rubber dispenser (316 dispensers/ha; 790 mg triene/ha). Trap catch disruption was 99% after 6 weeks. Trial 3: Large plot DCEPT dispensers (St. Lucie Co.). Cooperator: Packers of Indian River. A trial established Sept 2012 compares trap catch disruption in plots (0.87 ha) treated with rubber dispensers containing ‘natural’ 3:1 triene:diene blend (330 dispensers/ha) versus untreated plots. Trap catch disruption was 92% at 49 weeks (22 August). Trial 4: Large plot DCEPT (Charlotte Co.). Cooperator: TRB Groves. Continued monitoring a grower validation trial treated 20-23 August (42 ha) with rubber dispensers loaded with pheromone (natural blend). Trap catch disruption was 95% on 17 September. Trial 5: Movement of CLM (St. Lucie Co.). Cooperator: Blue Goose. Completed trial to test how far male CLM fly to traps pheromone-baited in grassland pasture adjacent to citrus. Traps at 75, 150, 300, 600, and 1,200 meters west of a grove as a source for P. citrella captured males. Sentinel citrus plants along similar transects adjacent to the grove became infested at 1,200 m at 6 wks after placement. Trial 6: Attraction of CLM and other Phyllocnistis species to ternary and binary pheromone blends (St. Lucie Co.). We continued monitoring a trial designed to study several nontarget Phyllocnistis species that are attracted to lures containing binary and ternary pheromone blends optimized to catch P. citrella.
To further develop our research on using Wolbachia (Wol) strains to control Asian citrus psyllid (ACP), we have approached the experimental system using several approaches: 1) developing an artificial diet compatible with most life stages of the ACP, 2) isolating Wol from ACP, and 3) developing cell culture techniques to culture the ACP Wol. An artificial diet and feeding system that successfully elicits feeding between 2nd instar nymphs and adults has been developed and is being used to expose isofemale ACP lines to antibiotics for clearing them of Wol. We are currently developing a stably-infected Wol cell line that will be used for infection of ACP isofemale lines. One goal of the current project is to determine the susceptibility of native ACP populations to endemic and native Wol infections, which requires assessment of native Wol infections in ACP at population and individual levels. Using phylogenetic analysis, we have observed sequence differences in Wol isolated from native ACP populations. Ratios of these sequence variants differ by location within Florida. Currently, we are continuing to conduct multi locus sequence typing to confirm the diversity of Wol in Florida ACP. In addition, qPCR results suggest that there are gender-associated differences in Wol infection within psyllid populations. Fluorescent in-situ hybridization (FISH) and quantitative PCR are currently being conducted to determine tissue-specific infection of Wol in ACP. An additional component of this project is to determine how Las transmission is affected by environmental perturbations, such as temperature. We have completed experiments to assess how temperature may effect ACP acquisition and inoculation of Las. Psyllids pre-exposed a range of temperatures demonstrated differential Las acquisition and inoculation efficiencies. Moreover, ACPs pre-exposed to extreme temperatures exhibited significant changes in fitness and melanization. We are currently examining ACP expression of heat shock protein, defense, and melanization genes in response to temperature treatments.
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. Light produced by the sky or filtered through a leaf canopy differs in predominant colors and insect behavior may differ with exposure to the different colors. In this study, we conducted laboratory assays on comparison of light quality on directional speed of psyllids under light typical of that transmitted through leaves in a canopy (green 520 nm) or under sky light (UV/blue 405 nm). No differences in directional peed were found between males and females. Significantly more ACP moved towards UV/blue light than towards green light. Additionally, directional walking speed was greater under UV/blue light compared to green light. Preliminary analysis indicates no differences in speed or directional response between sexes or any of the color variants of males or females, however, this is continuing to be investigated. Overall, ACP under green light similar to canopy cover exhibited less directed movement and often changed direction towards or away from the light source. This movement was at lower speeds characteristic of searching behavior for feeding site location. Light that was typical of skylight (UV/blue) resulted in more rapid and directed movement towards the light source with more flights. Studies now in progress to examine how physiological stage (age, ovarian status) or rearing conditions (short vs long daylength, crowding) affect these responses. When the effect of different wavelengths of light on flight towards a target were evaluated, strong responses were obtained under UV/blue light compared to green or yellow light. Preliminary responses to UV/blue light were both more rapid and larger numbers of ACP responded. We are pursuing these studies with more wavelengths and making comparisons between different sexes, color morphs and physiological ages. Studies on flight duration under different lighting conditions are starting and flight mills, microprocessors and software have been developed to conduct these trials.
We now have five years of harvest data (2008-2013) from our replicated ‘Valencia’ trial in a 32 acre block in a commercial grove in southwest Florida evaluating the Boyd nutrient cocktail. The yield and juice data shows HLB infected trees have increased yield each year and are healthy, maintaining good growing, and maintaining production. Yield for the most productive treatments has reached 2-boxes/tree on young 7 to 8 foot tall trees. Yield has increased in all treatments each year. Preliminary economic analysis is showing the treatments with the highest fruit yield is not the most productive in dollar returns. This economic analysis takes into account the cost of the nutritional products to give the net return to the grower. The common ingredients in the most cost productive treatments during the past 5 years has been micro- and macro-nutrients, some also had phosphite (phos acid) and salicylate (salicylic acid). Juice maturity and quality factors don’t appear to be affected by HLB in trees receiving foliar nutritional therapy sprays. We are halfway through the 5th year of a replicated experiment in a 13-acre commercial block of ‘Valencia’ oranges on ‘Swingle’ to test effects two factors: (1) micro-nutrients + systemic acquired resistance inducers, and (2) Asian citrus psyllid (ACP) chemical control. Highest ACP populations were seen early summer on trees treated with nutritionals only (0.3 ‘ 0.08 ACP adults per tap) on June 26 and untreated trees (0.2 ‘ 0.05 ACP adults per tap) on July 12. ACP adult densities have not reached the established nominal threshold of 0.2 ACP adults per stem tap since April in any of the two sprayed treatments, with a maxima of 0.06 ‘ 0.04 and 0.09 ‘ 0.04, as highest densities in ‘Insecticide’ and ‘Nutritional + Insecticide’ treatments respectively on July 23. ACP densities subsequently declined throughout the summer and early fall, reaching values close to zero at the beginning of October (0.0 ‘ 0.0, 0.02 ‘ 0.01, 0.0 ‘ 0.0 and 0.01 ‘ 0.01 in ‘Insecticide’, ‘Nutritional’, ‘Nutritional + Insecticide’ and ‘Untreated’ treatments respectively). The third foliar nutritional spray application of the season is planned for the fourth week of October following the last major flushing period of the season. We have continued our leaf sampling for phloem activity in two groves in southwest Florida that have been treated with nutrient applications, foliar and under tree. As previously described some trees with specific additional applied nutrients produce new phloem and using a fluorescent dye we have tracked this phloem translocation photographically and also using a fluorescent plate reader to determine how much dye is left in the application area and how much is translocated from the area. Comparisons have been made with untreated HLB affected trees and with healthy trees. We are compiling the large amount of data and will then determine what applications have allowed the best translocation of photosynthetates.
We are developing and evaluating optimal RNA interference constructs in attempts 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 be applied to citrus and D. citri. So far, 7 pAMIRNAs 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) were constructed into the viral vector of geminivirus, and cloned into the binary vector pCB301. Our miRNA Northern blot analysis showed that in Nicotiana benthamiana, plants, the Tomato bushy stunt virus (TBSV) p19 silencing suppressor was needed in order to have high amountsof artificial miRNA (amiRNA) accumulation, while in Nicotiana tabacum, p19 triggered cell death and caused necrosis at the infiltrated area, which resulted in low amiRNA accumulation. The clones that are producing amiRNAs in N. tabacum, tested by Northern blots, are being used for psyllid feeding experiments. The target mRNA expression levels in the tested psyllids will be examined by RT-real time PCR. The amiR-TAV-pCB301 constructs will also be infiltrated into different citrus plants, and then tested by Northern blot analysis to assess for amiRNA accumulation. To mimic endogenous primary miRNAs, all the artificial primary miRNA constructs were in vitro transcribed by the mMESSAGE mMACHINE High yield capped RNA transcription kit (Ambion). The in vitro transcripts were added into a 15% sucrose artificial diet, and used for in vitro psyllid feeding tests. 11 pAMIRNAs were also cloned into the agroinfection-compatible Tobacco mosaic virus expression vector. To test if the amiRNAs can be delivered by TMV viral vector, we will do miRNA Northern blot analysis to confirm the amiRNA accumulation in plants. Using different agroinfiltration and plant viral systems to deliver amiRNAs will help us to identify the optimal versions to apply to citrus in order to target Diaphorina citri.
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