We have collected five years data in the replicated Hamlin & Valencia trials in southwest Florida evaluating the Boyd foliar cocktail. The yield and juice data shows HLB infected trees have increased yield each year and are healthy, growing well, and maintaining production. Yield for the most productive treatments was about 2-boxes/tree on 8 year-old (7 to 8 foot) trees. The common ingredient in the most productive treatments during the past 5 years has been micro- and macro-nutrients, some also had phosphite (phos acid) and/or salicylate (salicylic acid). Yield has increased in all treatments each year. Juice maturity and quality factors don’t appear to be affected by HLB in trees receiving foliar nutritional sprays. Juice quality among treatments of percent juice/fruit (52.9-58.0%), acid (0.74-0.80), Brix (10.59-11.23), ratio at harvest (13.77-14.96), and pound-solids/box (5.88-6.40) have all been in the normal and acceptable range. The unprecedented fruit drop experienced industry wide was much less. Economic analysis shows treatments with the highest fruit yield are the most expensive but yield is not significantly different from less expensive treatments that are more productive in dollar returns. Three new trials in grapefruit have been established in Indian River St. Lucie County, FL. Trial #1 is ~25 years old of ‘Flame’ on Swingle rootstock with 30 replicated experimental units, and trial #2 of ~6 year old ‘Ruby Red’ on Sour orange trees with 50 replicated experimental units are in commercial groves. Trial #3 at IRREC is to determine the effect(s) that foliar fertilizers have on young tree growth and their ability to ameliorate HLB disease symptoms in 1 year old ‘Ray Ruby’ grapefruit on Kuharske rootstock. Foliar fertilizer applications have been started since February 2014. Tree growth data, leaf nutrition data, and CLas titer measurements have been made. Fruit drop monitoring will begin in September 2014 in trials #1 and #2. In prosecution of these efforts, 2-part-time OPS employees have been hired to do our foliar sprays and collect data observations. We completed the 6th harvest in a commercial block of ‘Valencia’/Swingle testing effects of: (1) foliar nutrition and (2) chemical control on ACP populations, HLB titer, and yield. Results from the first 4 years yrs have been published. The ACP threshold to trigger insecticide application was lowered in 2014 from 0.2 to 0.1 due to diminished ACP populations in the area. Since the last report, ACP exceeded threshold seven times in nutrition treatments and twice in insecticide treatments according to biweekly monitoring. We applied Mustang @4.3oz/ac on 23 Apr and Exirel @20oz/ac 7 Jul. Insecticide treatments significantly (P < 0.05) reduced ACP adult counts 14 Apr to 26 Jun. HLB samples were taken Nov 2013; no treatment effects were detected for reset trees. Lower (P<0.05) Ct values were detected in insecticide treatments than without insecticide. Treatments with insecticide had higher (P < 0.05) yields. Fruit from nutrition-alone treatments had higher Brix values than untreated controls. No other measures of fruit quality differed among treatments. Fluorescent leaf assay to determine if phloem translocation was enhanced by the use of extra nutritional supplements continued. As reported previously dye translocation from samples at SWREC and the Orange Hammock grove in Felda were similar to healthy samples from our greenhouse. In an organic grove operation where good fruit size was produced on HLB qPCR positive trees, some of the trees had good translocation while others did not. Visual assays showed that the leaves from the HLB affected trees had uptake similar to healthy leaves. In all cases visual assays were easier to see differences than readings the fluorescent plate reader, due to leaf extracts having other fluorescent compounds released.
For this enhancement study, we conducted experiments to assess ACP gene expression in response to simulate cool temperature challenges to determine the impact of cool temperatures, and expression of melanization genes, on ACP vector competence. We have recently completed inoculation and transmission assays with ACP pre-exposed to a range of temperatures typically experienced by this pest under field conditions. Following transmission assays, qRT-PCR was used to assess changes in the expression of heat shock and melanization protein genes associated with immune defense and thermal protection. Preliminary data analysis suggested that pre-exposure to cool temperatures resulted in up-regulation of several target genes and reduced the vector capacity of ACP. Both acquisition and inoculation of Las by ACP were reduced following exposure to cool temperatures. These data support our hypothesis that the prevailing climatic conditions during Florida winters may have a reductive effect on Las transmission. Cool temperatures, ranging from 15-22’C, resulted in the production of dark-colored ACP adults. Altered coloration was correlated with up-regulation of melanization genes. Insect development slowed following exposure to cooler temperatures, as expected; however, there were no discernible impacts on insect longevity or reproductive output. Due to the short-term nature of this project, it was not possible to complete assays to assess the susceptibility of ACP to challenge with entomopathogenic fungi following exposure to cool temperatures, although we expect that susceptibility to fungi will be altered as a result of the changes in the expression of melanization genes. Similar studies of insect immunity indicate that individuals with a strong melanization responses are more likely to clear microbial infections. These findings suggest potential novel gene targets for disrupting ACP vector competence that could be exploited as part of an integrated HLB management program. Furthermore, these targets may be harnessed to improve the efficacy of entomopathogenic fungi as biological control agents for ACP.
The goal of this project was to enhance our understanding of resistance mechanisms for Asian citrus psyllid (ACP). The entire voltage-gated sodium channel of ACP was cloned and mapped back to the genomic sequence from the genome database. What was discovered is that ACP does not have predisposition to develop the typical L1014F mutation associated with pyrethroid resistance that is observed in many other insect species, at least without multiple changes to the nucleotide sequence. However, it does have the propensity for other changes that are associated with as high, and in some cases, higher, pyrethroid resistance (M918T and L925I). These two changes have been observed in other hemipteran species. Taken together, these results suggests that with sufficient pyrethroid pressure, target-site changes are likely to be observed. In addition, we found significant potential for alternative splicing, another mechanism that has been associated with pyrethroid resistance in field populations of insects. In our studies, we identified two isoforms in nymphs, demonstrating that alternative splicing occurs in this insect within this channel, again underscoring the importance for judicious use of pyrethroids. These results indicate that target site resistance for pyrethroids is a distict possibility and judicious rotation is very important to keep these tools useful.
Citrus greening disease, also called Hu’ngl’ngb’ng (HLB) is a severe threat to the growth and sustainability of the FL citrus industry. HLB infection is caused by C. liberibacter spp transported by the Asian Citrus Psyllid (ACP) during feeding between infected and non-infected trees. The objective of this research is to develop and optimize a promising film-forming ACP repellent material for preventing HLB infection. The film-forming material has been named hereafter organo-silica based composite film (OSCF). A series of OSCF materials has been synthesized using a variety of polymers, silanes and crosslinkers, as proposed. Preliminary phytotoxicity (plant tissue damage) studies has been conducted using Vinca sp. and calamondin lime (purchased from the local Home Depot). Phytotoxicity studies indicated that the first series of OSCF materials are safe to plants. In our next report, we will include OSCF material characterization data and results obtained from rainfastness studies.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuabe for monitoring CLM but need further evaluation to optimize use for management. The objectives of the proposed project include: (1) assess species composition of trap catches, (2) optimize spatial and temporal distribution of pheromone traps. Trapping of wild populations of Phyllocnistis citrella, congeners and other similar moths continued. Species composition in trap captures also continued to be evaluated by analysis for Cytochrome Oxidase 1 (CO1) sequence generation incorporating an anchored hybrid enrichment probe set from Illumina HiSeq data with the Lemmon Lab at FSU. Each sample is associated with locality, collection date, type of storage (dry or ethanol), and trap number. Samples collected in 2013-2014 and received in March 2014 are being processed. Photographs of the specimens will help elucidate morphological characters in order to provide more accurate identifications. David Plotkin’s work on photographing moths through a microscope and generating COI sequence data in the Kawahara Lab as a part-time assistant continued. Almost two years of CLM monitoring for a study to evaluate trap densities and lure efficacy decay under field conditions demonstrated that densities of 1 trap per 2 ha (5 acres) were sufficient to obtain reliable moth counts. Lure efficacy of the two pheromone brands tested declined by 25% after 3-5 weeks and 50% by 6-10 weeks during the spring through fall growing season in Florida. Therefore, large correction factors need to be applied to trap catch readings if traps are replaced at 8-13 weeks. Results obtained will help growers to optimize CLM monitoring programs. A manuscript submitted to the Journal of Economic Entomology retunred with useful comments from reviewers and editor that will allow us to make improvement to the article for publication and dissemination of these results. Mark-recapture work continued. A release of 1500 CLM adults in a naturalized and therefore low-citrus region of Southwest Florida (Picayune Strand) was conducted using the fluorescent powder marking technique. No released moths were recovered in the three week duration of this trial. Pyridine marking has also been attempted without captures of released moths. Efforts to improve the marking technique and study design continue.
Citrus greening is a devastating disease of citrus that has cost the Florida citrus industry over $3.64 billion. ‘Push-pull’ strategies because of their multi-component approach are often synergistic and have been successful in controlling insect vectors. The overall long-term objective of this research was to develop a push-pull system for the Asian citrus psyllid (ACP) that can complement integrated management systems in young citrus plantings. To achieve this goal, studies were conducted to optimize the attraction of psyllids to visual targets utilizing previously discovered wavelengths of light that were most attractive to ACP. ACP showed strong preferences to two choice visual targets. ACP were most attracted to unfiltered light that possessed UV. Polarized white light was found to be slightly more attractive then unpolarized white light. ACP were as attracted to yellow targets compared to green targets as to white light with UV. However, excluding UV from yellow targets caused a marked decline in ACP attraction to yellow compared to green. Contrast patterns using a black mark increased attraction of both yellow and green hues compared to the same hue that was unmarked. The results of the vertical assays provide a better understanding as to the various levels of attraction of ACP to different visual targets. An unexpected finding was that ACP were more responsive to light following a 12 hour decline in pressure. Elements of these findings were incorporated into cage assays containing two types of sticky traps. Standard yellow and green sticky traps were more attractive then translucent traps of the same wavelength. Black stripes on the green sticky traps did not enhance attractiveness towards ACP, while black stripes and black borders on yellow sticky traps did increase the attractiveness towards ACP by 10%. Polarization did not affect trap catch on clear or colored traps significantly on sticky traps, except for yellow translucent traps, which showed improve catches with polarizing filters present. Lastly, both green and yellow sticky traps were more attractive in the vertical rather than the horizontal plane. Effects of adding UV reflectance to yellow and green sticky traps with and without contrast patterns are in development to be tested in cage assays. Results from these studies represent crucial steps in the development and optimization of traps that can be incorporated into a push-pull management system. Furthermore, these results improve efficiency of passive traps to better serve stakeholders by providing a more accurate count of ACP in the field for management purposes.
Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops (such as Bergera koenigii) to attract and kill. Objective 1. Bioasssays using a wind tunnel to determine ACP response to different stimuli have shown thus far a plateau effect in light intensity and air temperature. Movement increases incrementally with these two parameters to a point and then levels off. Also, ACP tend to move up wind as wind speed increases within the range of the fan. Objective 2. Dr. Stelinski’s lab ahs continued to investigate the movement capability of ACP using laboratory flight mills as a tool to quantify duration and distance of movement using a technique that was recently published as part of this investigation (Martini, X., A. Hoyte, and L.L. Stelinski. 2014. Abdominal color of the Asian citrus psyllid (Hemiptera: Liviidae) is associated with flight capabilities. Annals of the Entomological Society of America. 107: 842-847.). they are trying to understand the factors that may influence flight capability/capacity/or motivation in the Asian citrus psyllid (ACP). Currently, they are testing the hypothesis that application of a juvenile hormone analogue, fenoxycarb, may increase flight activity of ACP. One to four day old ACP were chilled for 3 min at -20 C’ and then 0.2 .L of a solution of fenoxycarb at 2.5 .g/.L in acetone was applied onto the abdomens of treated psyllids with a micro-injector. 0.2 .L of acetone only was applied onto psyllids used as negative controls. The psyllids were then maintained in Petri dishes with citrus leaf discs for 3 days. After 3 days, psyllids were tested on the flight mill to determine the effect of treatment on flight capability. The results collected to date indicate an increase of ACP flight activity following treatment with fenoxycarb. If the results are confirmed in subsequent replicates, we will perform biochemical bioassays to measure the titer of juvenile hormone in the green-blue morphotype of ACP (this morphotype is capable of long distance flights as documented in the above mentioned publication) versus in the gray-brown morphotype of ACP (this morphotype performs short flights). Our goal is to understand the underlying physiological mechanisms that allow a certain proportion of ACP to move long distances, while other proportions of the population do not appear to disperse. Objective 3. Mark release trials have been put on hiatus due to extremely low recapture rates. Objective 4. Yellow sticky traps set at three canopy heights continued with mixed results after 1784 captures. More ACP captures in the upper canopy in the older Charleston grove and a more even distribution at the younger and more uniform Bob Paul grove that still slightly favor the higher sticky card locations. However, the east side of the trees captures more than the west side at both locations. At both locations and also the English grove, ACP captures are greatest adjacent to a hammock or wind break. At Bob Paul, traps set up to assess ACP movement between insecticide sprayed and unsprayed blocks with low and high populations of ACP respectively indicate no significant differences with 153 captures to date. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting has been published in the refereed journal Pest Management Science. Subsequent results indicate that the 5 ft strip of metalized mulch is not effective on trees more than 5 ft tall. Trials to determine the impact of reflective mulch on the height of ACP flight were resumed with warmer spring weather during which ACP movement increases and final results are pending.
The objectives of this project are: 1) Improve effectiveness of CLM management strategies with the assessment of the use of pheromone traps for monitoring CLM populations. 2. Monitor susceptibility of CLM field populations exposed to intensive versus modest insecticide use; 3) Relate CLM and canker incidence and severity and 4) Provide recommendations for management of CLM and citrus canker disease. Work on CLM LD50s and LD80s for some commonly used insecticides for management of citrus pests in Florida continued to be calculated for CLM from a susceptible colony. These values are being used to evaluate possible resistance to the tested insecticides in CLM field populations. Since the last report, larvae from the susceptible colony were exposed for 48 h to different doses (0; 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100, 300, 600 and 1000 ppm) of the following insecticides: Agri-Mek 0.15EC and Delegate WG. These data contributed to the results associated with the following active ingredients (CLM larvae): Actara (thiamethoxam) LD50=2.813 (CL95: 1.07-6.46) ppm, LD80=619.176 (CL95 199.10-3100.06) (n = 681; .2 = 3.81; d.f = 7; Heterogeneity = 0.76); Agri-Mek (abamectin): LD50= 0.259 ppm (CL95: 0.104-0.527), LD80=18.348 ppm (CL95: 7.083-85.113) (n = 1182; .2 = 9.681; d.f = 6; Heterogeneity = 1.613); Cyazypyr (cyantraniliprole): LD50=33.289 ppm (CL95: 15.2-69.1), LD80=8882.7 (CL95: 2905.3-44518) ppm, (n = 954; .2 = 1.749; d.f = 5; Heterogeneity = 0.35); Delegate (spinetoram): LD50=2.589 (CL95: 0.729-6.352), LD80= 95.606 (CL95: 33.676-553.88) ppm (n = 859; .2 = 10.501 d.f = 5; Heterogeneity = 2.100); for Dimethoate (dimethoate) LD50=1.56 ppm (CL95: 0.15-56.31) and LD80=497.45 ppm (CL95: 22.27-0.37E+09) (n = 546; .2 = 13.07; d.f = 6; Heterogeneity = 2.18); and Micromite (diflubenzuron): LD50=88.32 (CL95: 20.69-246.69) LD80=1800.5 ppm (CL95: 522.84-75810.00), (n = 714; .2 = 10.31; d.f = 5; Heterogeneity = 2.06). A novel bioassay technique is currently being developed for Intrepid and will be evaluated in the coming weeks. LD50 and LD80 evaluations for CLM adults continue. Further collections from field populations for laboratory evaluations of insecticides are planned for this summer. A manuscript “Placement Density and Longevity of Pheromone Traps for Monitoring of Citrus Leafminer, Phyllocnistis citrella (Lepidoptera: Gracillariidae)” is being prepared for submission to Economic Entomology.
Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops (such as Bergera koenigii) to attract and kill. Objective 1. Bioasssays using a wind tunnel to determine ACP response to different stimuli have shown that ACP movement stops when temperature dips below 67 F, and when there is no light. Wind speeds above 4.25 mph reduce movement greatly, as does humidity over 70%. ACP tend to move with the wind when lighting is even and increased light intensity does not increase ACP movement. Objective 2. Experiments were conducted with a laboratory flight mill in the Stelinski laboratory to determine the effect of applying Fenoxycarb, a juvenile hormone (JH) analogue, on flight behavior of ACP. No significant differences were observed between the controls and the psyllids treated with Fenoxycarb, suggesting that JH may not impact flight capability. Following their recent finding that Clas infection increased flight propensity of ACP, they are now testing the hypothesis that Clas infection may impact other movement patterns, such as male attraction toward female odor(s). In a laboratory olfactometer assay, non-infected ACP males were attracted by non-infected ACP female odors. However, if nymphal development was completed on HLB-infected plants, only infected ACP females were attractive to males. These data will be used to develop a predictive model for spread of the CLas pathogen causing HLB. Objective 3. Mark release trials were not successful and discontinued, but environmental effects on ACP movement was studied by hourly monitoring from sunrise to sunset of sticky cards placed between 3 year old orange trees with a final observation next day at sunrise. Captures increased in the morning with two peaks seen at mid-day followed by a gradual increase until dusk. Thus far we have not captured any ACP during the overnight period which was expected due to the results of the no light variable in the wind tunnel. This study is a good companion to Objective 1 allowing us to confirm the wind tunnel results in the the field. Objective 4. More ACP were captured on sticky cards in the upper canopy compared to the lower canopy of mature trees in a 2 year study at two locations, Bob Paul (‘Pineapple’ orange) and Tanner Road (‘Valencia’ orange). Sticky cards on the east side of the trees captures more ACP than the west side at both locations. At both locations and also the English grove (‘Valencia’), ACP captures are greatest adjacent to a hammock or wind break on the east and west sides. No differences were seen in trap captures facing insecticide sprayed or unsprayed blocks at Bob Paul. Two-sided clear and yellow sticky cards have been placed in several groves to compare directional responses in an attempt to determine whether ACP adults flying in one direction may turn back and be captured on the opposite side of the yellow card. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting has been published in the refereed journal Pest Management Science. Subsequent results indicated diminished repellency from the 5 ft strip of metalized mulch after trees exceeded 6 ft in height. In another trial, repellency was demonstrated by the result that fewer ACP were captured on sticky traps set at 1 m in reflective mulch compared to 2 m, where as the opposite was true over white mulch or no mulch (grass) where more ACP were captured more at 1m than 2m.
This is a three-year project. The goal is to determine the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can registered for use in field citrus and control HLB. The project started in April 1, 2014. The first field citrus grove at Pico farm of USHRL-USDA-ARS was evaluated for ACP and HLB based on past records of ACP and HLB incidence, visual observation and qPCR quantification. A randomized block complete design was carried out to combine nutritional and chemical treatments. The nutritional treatment was applied to three rows (#39, 41 and 43) and the other three rows (#40, 42 and 44) with normal nutrition. The chemical treatment was two antimicrobial compounds, which were loaded into the nano-emulsion formulations of W/O (Water in Oil) for foliar spray or O/W (Oil in Water) for bark application. Blank nano-emulsions without antimicrobial compounds were used for controls. The chemical treatments were applied separately or in combination with the nutrient treatments. All trees involved in this experiment have been confirmed to be Las-positive via a real-time quantitative polymerase chain reaction (qPCR) assay in this quarter. They were also pruned and fertilized. Chemical treatment applications will be made every month when flush is present starting with the spring flush in 2014. The nutrition will be applied as the protocol of Quantum Growth by Ecological Lab. INC. ACP populations will be recorded by stem-tap sampling. Citrus effects of the combined treatments will be investigated, including: a) reduction in fruit drop; b) increased canopy density; c) reduced HLB symptoms and Las titers; d) increased root uptake; e) residues and phytotoxicity. Additional groves, chemical treatments, nutreint treatments, and heat treatments will be added during the next quarter.
In order to speed up their application and registration for use by the citrus industry, this one-year enhancement project immediately tested antimicrobial compounds in the field. The research focused on evaluating five antimicrobial compounds, that were effective against Las in our citrus graft assay test, against HLB in field citrus. The antimicrobials were SDX (a sufonamide), ZS and VA (Chinese antibiotics) and CAR and PCY (natural oils). Positive control was ampicillin and negative control was water. Application methods were foliar-spray, basal bark application and gravity bag infusion. Trees will be evaluated at 2 month intervals. The four month assay, which is still preliminary, showed that the SDX, ZS and VA treatments were more effective in reducing Las titers than water, but not as good as the positive control (AMP). All treatments, except the water control, decreased the Las titer in the new leaves at 4 months after initial treatment. Temporal phytotoxicity was observed in some gravity bag infusion treatments, but all treated trees recovered after four months. After 4 months, trees treated with SDX, ZX and VA still had high titers of HLB bacteria in the older leaves, but less titer in the young leaves. CAR and PCY treatments have not yet been analyzed for Las.
This is the second year of a 3-year project. The overall goal is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. This quarter’s (From April to July 2014) research continued to evaluate the effect of novel nano-emulsions against HLB bacteria, and optimize nano-emulsions by particle size analysis and evaluation of thermal stability. HLB-affected potted citrus trees treated with W/O and O/W nano-emulsion containing several antimicrobials by foliar spray and basal bark application respectively, were free of HLB bacteria by PCR and recovered from any disease symptoms, after 8 months and 4 months from initial treatment in 2013. Based on pesdo-ternary diagram phase in our pervious study, several O/W and W/O nano-emulsion were selected for optimizing the formulation by analyzing the particle size and evaluating the thermal stability. The particle size of five O/W nano-emulsion formulations ranged from 3.81 nm to 30.30 nm and the formulations were stable at temperatures from -20 ‘C to 45 ‘C. And the particle sizes of 16 W/O nano-emulsions formulations ranged from 2.95 nm to 35.3 nm and they were also stable at temperatures from -20 ‘C to 45 ‘C. Thus, we have developed stable formulations that will effectively deliver antimicrobials to citrus and control HLB under greenhouse conditions. The future work will be focus on: 1) Evaluation of compound loading using the optimized nano-emulsion formulations; 2) Evaluation of effect of the combination of optimized nano-emulsion and ideal penetrants on controlling HLB disease in the field by foliar spray and basal bark application.
The goal of this enhanced project of CRDF#584 is to evaluate effective compounds which were coupled with nano-emulsions and penetrants, against HLB bacteria in the field. It is one year project started on June 30, 2013. HLB-affected trees were pruned and fertilized before treatment. Two experiments were carried out in the field. Experiment I: HLB-affected citrus was treated with two effective compounds which were coupled with nano-emulsion and ideal penetrant, respectively,by foliar spray and bark painting. Experiment II: Four formulations of two effective compounds were applied on HLB-affected trees by gravity bag infusion. In experiment I, the tested compounds were delivered into citrus phloem through leaves by foliar spray and bark by bark painting. The HLB bacteria titer was significantly reduced 2 months after initial treatment. Furthermore, HLB symptoms also were reduced, and the number and height of summer flush was significantly increased under the nano-emulsion treatment when compared to the water control. No phytotoxicity appeared in the treated citrus trees. However, Las bacterial titer increased a little in the treated citrus trees four months after initial treatment. We will continue to test for one year. In experiment II, HLB bacteria titer was significantly reduced by gravity bag infusion with a penicillin solution and summer flush number was much greater, compared with other 3 formulations and water control 4 months after initial treatment. No phytotoxicity was observed in these four formulation treatments. We will continue to test for one year. In summary, effective compounds against HLB bacteria can be delivered into citrus phloem by foliar spray and bark painting or gravity bag infusion. Therefore, combining one or more of these delivery systems with an effective antimicrobial compound and controlling psyllids may be effective strategy to combat the HLB disease in the field. We will continue the field trial for more than one year. ACP populations will be recorded by stem-tap sampling. Citrus effects of the combined treatments will be investigated, including: a) reduction in fruit drop; b) increased canopy density; c) reduced HLB symptoms and Las titer; d) increased root uptake; e) residues and phytotoxicity.
This is the second year of a 3-year project. The overall goal is to evaluate the efficacy of 11 compounds individually and in combination for control of the HLB disease based on reducing titers of HLB bacteria in HLB-affected citrus. Based on our optimized delivery system, during this quarter (April to July, 2014), all 72 treatments of 11 compounds and their combinations were applied by bark-application to greenhouse citrus using nano formulations of the antimicrobial agents. Several compound formulations, such as Amp and ACT+VA, significantly reduced HLB bacteria titer 2 months after the initial treatment. The treated, greenhouse plants recovered normal growth. No phytotoxicity was observed in the treated citrus, except the treatment of STZ and VA. The treatments will be applied each month, and samples will be continue to be analyzed for Las every two months. The effective treatments will be tested in the field.
We have completed development of tools for in planta transient (and in the long term, transgenic) expression of dsRNAs and specific artificial micro RNAs (amiRNAs), and expression of siRNAs via using plant viruses. Our intent is to compare specific and non-specific RNAi effects by expressing these various forms of interfering RNAs in plants as such information will be critical to effectively design and even implement RNAi approaches targeting D. citri. So far these experiments have worked very well for N. benthamiana and tobacco plants which can be used to test against B. cockerelli, but we have not have good success in transiently expressing RNAs directly in citrus by using Agrobacterium tumefaciens (but see below for objective 2). However, we are further experimenting with this strategy as it offers great opportunities for rapid and efficient screening of interfering RNAs against D. citri, directly in citrus. We have compared the qualitative and quantitative expression of amiRNAs by directly expressing them from A. tumefaciens, by using Tobacco mosaic virus (TMV) and by using a modified begomovirus expression system (TAV). So far we have generated seven different interfering RNA sequences targeting the B. cockerelli ATPase mRNA. We assessed interfering RNA expression by using RNA hybridization and by using Next generation sequencing of small RNAs. Our data show that by using the TAV system, we can specifically express very high numbers of the exact 22 nucleotide artificial microRNA that we have engineered to target psyllids. Objective 2 is to evaluate and optimize in planta expression of anti-psyllid interfering RNAs. We are on track here with experiments towards B. cockerelli. We have generated transgenic plants using two different promoters (the 35S promoter for general tissue expression and the AtSuc2 promoter for phloem-specific expression). These plants will be characterized this year for expression of interfering RNAs and for efficacy towards B. cockerelli. The results from these experiments will provide critical information for use in objective 3. As noted above, we have not had good success yet with Agrobacterium tumefaciens-directed transient expression of specific interfering RNAs in citrus. However, with our collaborator, Dr. William Dawson of the Univ. of Florida, CREC, we have citrus plants that are infected with different recombinant Citrus tristeza virus (CTV) constructs engineered to express specific D. citri RNAs in citrus plants. The recombinant CTVs are engineered to express RNAs targeting D. citri mRNAs for HSP70, CLIC and/or V-ATPase, alone or in combination. We obtained budwood (under APHIS PPQ and Biotechnology permits) and grafted buds to C. macrophylla plants in the UC Davis CRF. These plants are only one month post grafting, we are pushing buds now and will use these for direct RNAi studies towards the Asian citrus psyllid in the upcoming year. This general approach offers the most rapid way to deploy RNAi strategies for D. citri, and could even be applicable to existing, non-transgenic trees. The CTV being used by us is the same as is being used in Florida for field tests and anti-psyllid peptide approaches. We anticipate that we will use the Florida CTV experimentally (it likely could be used in the field in Florida), and in the long term we will collaborate with Dr. James Ng of UC Riverside to use a mild California CTV for RNAi studies in California.
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