Our research on use of double stranded RNA (dsRNA) targeting specific psyllid genes has previously shown that, when dsRNA targeting either a psyllid cathepsin or a psyllid vacuolar ATPase gene are fed in artificial diets to the Asian citrus psyllid, an increase in psyllid mortality is realized. Associated with the elevated mortality, we observed that mRNA transcripts corresponding to the dsRNA being fed were reduced, strongly supporting an RNAi-induced mortality. This mortality was observed within a range of fed dsRNA concentrations between 3 ng/uL and 45 ng/uL present in the diet. Based on known accumulation levels of plant mRNAs in their cells, these concentrations are presumed to be physiologically relevant to what could be produced in transgenic citrus phloem. Therefore, we worked in association with the lab of Dr. Bill Dawson at the University of Florida (CREC) to construct plants infected with a recombinant CTV vector containing these psyllid sequences that produce dsRNA within citrus phloem. We have analyzed these plants and shown that dsRNAs of each of these sequences are present. These plants have been propagated and are being used for psyllid feeding studies. In leaf feeding assays, where psyllids are allowed to feed on excised leaves, leaves from plants containing the CTV-cathepsin construct showed greater mortality to adult psyllids than leaves from control plants. These experiments are currently being replicated to determine statistical significance. We are also pursuing research on what appears to be general toxicity of higher levels of dsRNA to psyllids when it is fed in artificial diets. It is hypothesized that the toxicity is due either to cryptic homology of the control dsRNAs to critical psyllid genes, or the result of a general yet uncharacterized dsRNA-induced toxicity This phenomenon has not been identified in insects and may be an important phonemenon that could be utilized in developing optimal psyllid control strategies based on dsRNA-mediated psyllid toxicity.
Progress on a new special regulatory trap for capturing and preserving citrus psyllids in situ has been advanced enough to develop some prototypes for further field testing and refinement. Laboratory bioassays that examined psyllid behavior in response to physical details on the surface of the traps is ongoing. Our premise is that eventually some types of chemical attractants will be identified and deployed with trap visual cues optimized in yellow to enhance the numbers responding to the vicinity of traps. We have to maximize trap efficiency by exploiting psyllid behavior. We are testing various trap physical modifications to optimize trap capture rates using lab and field experiments. We are also evaluating various structural designs of the trap to simplify mass production. So far we have developed a working prototype that is ready to use with chemical lures and other psyllid attractants as they come available.
Progress on a new special trap for capturing and preserving citrus psyllids, their associates and DNA in situ is now advanced enough to compare to conventional yellow sticky traps and combine with other behavioral tools such as chemical attractants. Using trial and error methods as well as behavioral observations under laboratory and field conditions, we have investigated Asian citrus psyllid behavior in response to trap physical parameters. Our premise is that eventually some effective chemical attractants will be identified and deployed with trap visual cues optimized in yellow to enhance the numbers responding to the vicinity of traps. However, the trap capture efficiency will depend on psyllid behavior on the trap relative to their willingness to orient, land and enter the trap. We have been concentrating on testing and improving trap components to optimize these behaviors to improve trap capture efficiency. We have also submitted the required paperwork concerning the technology to the University of Florida Technology and Licensing Office. We have located a commercial company that will manufacture the new traps for further research.
One of the objetives of this project was to determine how long Diaphorina citri should feed on healthy citrus plants in order to inoculate Ca. Liberibacter asiaticus (Las). Here we describe transmission trials in which we varied the duration of the inoculation access period (IAP) of infective psyllids on healthy citrus seedlings and measured inoculation efficiency. Early transmission experiments with variable IAP. In the beginning of this project we established seven transmission trials of Las by D. citri in which the following IAP durations were tested: 0.5, 1.5, 6, 12, 24 and 96 h. Before the IAP treatments, healthy 3rd-4th instar nymphs were submitted to an AAP of 14 days on Las-infected plants, followed by a 14-day period on healthy citrus seedlings. In each trial, 7-10 test plants (sweet orange seedlings) were inoculated per IAP treatment, using five psyllids per test plant, except in trial I, in which only 1 insect was used per test plant. Inoculated test plants were assayed for Las infection by qPCR 12 months later. Despite the large number of test plants inoculated in all trials (57-59 plants per treatment), only four of them turned out positive for Las, for IAPs of 6 h (1 positive plant), 24 h (2 plants) and 96 h (1 plant). Therefore, transmission of Las by D. citri was very inefficient in this experiment. A possible reason for the very low transmission rates was the experimental design, with a rather long AAP (14 days) on Las-infected plants, followed by another long period on healthy intermediate plants (14 days).Thus, the insects were submitted to the IAP treatments only at 28 days after the beginning of the AAP, when they were probably too old. We used a long AAP and latency period in this early transmission experiment because at the time we did not have the information that shorter AAPs (48-96 h) could result in high acquisition efficiency (Study 1); in addition, there was no clear information in the literature on the duration of the latent period of the pathogen in the vector. Recent transmission experiments with variable PAI. Based on recent information on the minimum latent period of Las in the psyllid vector (around 10 days) (see a previous report), two new transmission trials with variable PAI were carried out with some modifications in the experimental design. Nymphs were submitted to an AAP of 7 days on Las-infected plants (source plants), followed by 7 days on healthy citrus seedlings (intermediate plants); the psyllids were then transferred to test plants for the following IAPs: 0.75, 1.5, 6, 12, 24, 48 and 96 h. Overall, the transmission efficiency was still low (4.7-11.7%) in the modified experimental design, but higher proportions of infected plants were observed, especially for trial VIII, whose PCR evaluations have already been concluded (12 months post inoculation). For trial IX, we have only one PCR evaluation and need to reevaluate the plants with 12 months after inoculation. Interestingly, the psyllids transmitted Las during the shortest IAP tested (45 min) and the estimated transmission probability did not increase significantly with longer IAPs. These results indicate that infective D. citri adults do not require long periods of exposure to healthy citrus to successfully transmit the pathogen, and the minimum IAP is shorter than 45 min.
Objective 1: To evaluate topical applications to the trunk of bactericides and Pentra-bark, a penetrant proven effective for trunk application of systemic insecticides. In the greenhouse, 0.5-1.0 cm dia trunks of Hamlin orange trees (1 yr old) were painted with a Magna-Bon (Copper sulfate pentahydrate), Cop-R-Quik (copper nitrate), a copper phosphite (CP), an experimental copper (EXP) or oxytetracycline (OTC) applied with 0.1% Pentra-Bark or left non-treated. After for 2-3 weeks, the leaves were observed for phytotoxicity. No phytotoxicity was observed, potential bactericidal activity in the plants was assayed with detached immature leaves inoculated with Xanthomonas citri subsp. citri (Xcc) as a Gram negative bacterial surrogate for non-culturable Canidatus Liberibacter asiaticus (Las). Canker disease control effect was be measured as the number and size of the canker lesions with an in vitro assay. Canker lesion no. per leaf was reduced for trees treated EXP, and Cop-R-Quik but not, OTC, CP or MB. In July, the bactericide treatments were scaled-up in a field trial by painting 1-2 yr-old nursery trees exposed to highly infected psyllid populations at USDA-ARS Picos farm in Ft. Pierce. The trees will be assayed for PCR status in Jan 2012.
The objective is to evaluate soil-applied neo-nicotinoids and other SAR inducers on HLB disease progress in newly planted citrus trees subjected to psyllid-mediated infection or graft-inoculation. One yr-old Hamlin trees were planted in May 2009 and treated as follows: 1) untreated check (UTC), 2) foliar insecticide to control psyllids, 3) soil-applied imidacloprid/thiamethoxam (IMID/THIA) to induce SAR, 4) soil-applied IMID/THIA plus foliar insecticides, 5) graft-inoculated UTC, 6) graft-inoculated with IMID/THIA. There were 50 trees per treatment (5 blocks of 10 trees). In 2009, the effect of SAR inducers on HLB infection progress was inconclusive perhaps attributable to the interaction of IMID/THIA with psyllid control which may have an uncontrolled effect on psyllid transmission. In 2010, the SAR inducer acibenzolar-S-methyl (ASM, Actigard 50WP) which does not control psyllids was substituted in treatments 3, 4 and 6. At 24 months after treatments began, 105 trees were PCR+ (35%) in the trial. Higher number of PCR+ trees occurred in the UTC (20), the UTC with graft inoculation (22), and the IMID/THIA/ASM with graft-inoculation (28). A lower number of PCR+ trees occurred in the treatments with SAR inducers (11), foliar insecticides (12), and foliar insecticide plus SAR inducers (12). Two years after treatments were initiated, the effect of SAR on HLB disease progress has been minimal, which indicates a lack of promise for SAR inducers in HLB management. These findings were communicated to citrus growers in a Citrus Industry magazine article entitled “The ABCs of SAR” (May issue).
Under Objective 1 and 2 the following was submitted for presentation at a canker workshop in Ribeirao Preto, SP, Brazil: Soil applications of SAR inducers at various rates and application frequencies were evaluated for control of canker in a field trial of 3- and 4-yr old ‘Ray Ruby’ grapefruit trees in southeastern Florida. Reduction of foliar incidence of canker produced by one, two or four soil applications of the neonicotinoids, IMID and thiamethoxam (THIA), or ASM was compared with 11 foliar sprays of copper hydroxide and streptomycin applied at 21-day intervals. In 2008 and 2009 crop seasons, canker incidence on each set of vegetative flushes was assessed as the percentage of the total leaves with lesions. By the end of the 2008 season, despite above average rainfall and a tropical storm event, all treatments significantly reduced foliar incidence of canker on the combined Spring-Summer-Fall flushes. Sprays of copper hydroxide and streptomycin were effective for reducing canker incidence on shoot flushes produced throughout the season compared to the untreated control, whereas soil applied SAR inducers reduced foliar disease depending on rate, frequency and timing of application. Except for the treatment of four applications of ASM at 0.2 g a.i. per tree or two applications of imidacloprid, SAR inducers were ineffective for reducing foliar disease on the flushes that were present during the tropical storm. In 2009, all treatments significantly reduced the incidence of foliar canker on the combined Spring-Summer-Fall flushes but not all treatments of Spring-Summer flushes with SAR inducers were effective compared to the untreated control. Hence, depending on rate, frequency and timing of application, soil-applied SAR inducers reduced incidence of canker on foliar flushes of young grapefruit trees under epidemic conditions. While soil application of systemic neo-nicotinoid insecticides has been demonstrated in our field trials to induce SAR and provide long lasting canker control for non-bearing trees, use of these systemic insecticides at higher rates for young fruiting trees is restricted due to perceived risks for soil leaching and insecticide residues in flowers. An objective of current field research is to develop more effective suppression of Xcc and fruit infection using trunk applications of neonicotinoids as well non-insecticidal SAR inducers. A trial with 5 yr-old fruiting grapefruit trees trees showed the efficacy of trunk application as an alternative to soil application for IMID, THIA and ASM at 3.5X the label rate per season to compensate for the larger tree volume produced canker control on foliage that matched that of 11 sprays of copper. Trunk application was as effective for canker control as soil application. Under Objective 3, The integrated use of ASM, THIA and IMID soil applications was evaluated to increase and/or extend canker control in 3-yr-old grapefruit and 2-yr-old Vernia orange trees. The highest incidence of disease trees and/or leaves was in the untreated check in each trial compared with a very low incidence of canker in the integrated SAR treatments. A field trial with soil applied neonicotinoids in Parana, Brazil was evaluated. IMID (Confidor) as a soil drench and IMID (Winner) applied to trunk gave comparable in disease control activity on 2-yr old Valencia orange trees, as well as, the other neonicotinoids tested, THIA and Clothianidin. Clothianidin (Belay) is now registered for use on non-bearing citrus in Florida, hence all of neonicotinoids registered for non-bearing citrus in Florida have been shown to have SAR-inducing activity against canker as well.
Under Objective 1 (define rates and formulations of copper sprays for more effective control) and Objective 2 (establish the period of fruit susceptibility, residual activity and phytotoxicity of copper) The following has been accepted for publication in the proceedings of the Florida State Horticultural Society: Protection of ‘Hamlin’ orange fruit from infection by Xanthomonas citri subsp. citri, the cause of citrus canker, is necessary to reduce premature fruit drop. The objective was to evaluate copper formulations for control of fruit infection and drop in 6- to 8 year-old ‘Hamlin’ trees. Copper sprays were applied at 21-day intervals after fruit reached 0.5 to 1.0 cm (0.25 to 0.50 inches) diameter. The period of susceptibility of fruit to canker infection and fruit drop was established by increasing the number of applications through the fruit growth period. Separate treatments ended at each 21-day interval so that there were four to seven applications per season. In 2008, early season infection occurred during rains before copper sprays commenced in late April. Subsequently, five sprays of copper formulations at rates exceeding 0.5 kg /ha (1.1 lb/acre) metallic copper significantly reduced incidence of lesions on fruit. Early season fruit disease and cumulative fruit drop were highly correlated among copper treatments (r = 0.95). Although a tropical storm in early August promoted disease on fruit late in the season, late season fruit disease and fruit drop were less well correlated (r = 0.78). In 2008 and 2009, additional sprays after the period of early fruit susceptibility did not further reduce canker incidence or fruit drop. In 2009, copper sprays were initiated before significant spring rainfall and the incidence of fruit disease and fruit drop were lower and the correlation of early season fruit disease was less positively correlated with fruit drop (r = 0.52) compared to 2008. In 2010, disease on fruit and premature drop were not significantly different from the untreated checks although fruit disease and early season infection were still significantly correlated (r = 0.70). Overall, there was little difference in efficacy among copper formulations, although control was reduced for treatments with copper sulfate pentahydrate at lower rates of metallic copper. In each season, copper treatments controlled fruit drop by ~50% compared to the untreated check, however as ‘Hamlin’ trees grew from 6 to 8 years of age, canker incidence dropped due to the development of hedgerows, which reduced windblown rain penetration into the grove. Hence, fewer copper sprays will be necessary after canopy closure promotes an internal windbreak effect. Under Objective 4 (To define risk for development of bacterial resistance to copper and streptomycin in FL citrus groves) the results were submitted and accepted for publication in European Journal of Plant Pathology. Briefly summarized: Copper resistance determinants from a citrus epiphytic strain of Stenotrophomonas maltophilia (Stm) were cloned and expressed in Xcc and other Xanthomonas strains. Copper resistance genes in Xcc were determined to be present on a large (~300 kb) conjugative plasmid. Cu resistance was transferred via conjugation from two copper resistant citrus strains, Xcc and X. alfalfae subsp. citrumelonis (Xac), and two tomato pathogens, X. euvesicatoria (Xe) and X. perforans (Xp), to Xcc. PCR analysis revealed that two CuR strains from citrus, an epiphytic Xanthomonas ssp. and a strain of Stm, harbored homologs of the copper resistance genes found in CuR Xcc. The introduction of copLAB gene cluster from Stm into different xanthomonads conferred copper resistance to sensitive strains of Xcc, Xac, Xe and Xp. Under Objective 5 (rapid transfer improved canker management technology to the Florida citrus industry), the results from Objective 1 were presented at the annual meeting of the of the Florida State Horticultural Society in St. Petersburg.
Objective 1 is to conduct a field evaluation of nutritional sprays for control of HLB and HLB symptom expression and yield. The field study was set up May 2010 in Southern Grove, Hendry Co., FL. Six treatments were located in 4 plots of 150 trees per treatment (interior 10 trees in each block were identified for PCR, leaf nutrition sampling, tree health and yield evaluation). Treatments were 1) non-treated check; 2) Nutri-Phite sprayed 4 times bimonthly; 3) N-Sure sprayed bimonthly; 4) Agra Sol Mn/Zn/Fe plus Nutri-Phite plus triazone urea sprayed bimonthly; 5) Keyplex 1400 DP plus Nutriphite plus triazone urea sprayed bimonthly; 6) Wettable powder nutrients (Diamond R #2) plus Nutri-Phite P+K sprayed bimonthly. The materials were applied to both sides of the tree in 125 gallons per acre with an airblast sprayer driven at 2 mph to obtain thorough coverage. Four disease ratings have been taken so far and a slight decline in tree health has been observed, but no significant treatment effects have been observed. There were no significant treatment differences in yield at the first harvest, after the initiation of treatments the previous April. The second harvest was just completed and analyses are currently underway. Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application, Rep 1 using Hamlin sweet orange trees inoculated with HLB and treated bimonthly with the nutritional sprays treatments 1, 2, 3, and 5 from objective 1 has finished. Monthly monitoring of Infection rate and disease development did not show obvious treatment differences except a possible increased rate of decline in treatment 2 compared to all other treatments. Trees that were only PCR+ in root tissue showed an unexpectedly fast decline across treatments. After pruning trees at 6 MPI for canopy management, sampling at 7 MPI showed a slight reduction in titer in the new flush of all treatments except treatment 1, where no Las was detected until 8 MPI. This suggests that treatments 2,3, and 5 may potentiate movement of Las to new flush where psyllids are most likely to feed and acquire Las. Sectioned midrib samples were observed by light microscopy at 6 and 8 MPI and 9 MPI. At 6 MPI reduced phloem plugging and necrosis was observed in treatments 3 and 5, however these treatments had some symptomatic leaves without detectable Las. These leaves had abnormal starch buildup preferentially in phloem tissue instead of mesophyll cells. At 9 MPI there was significant variation in plugging between midribs within a treatment even with highly similar symptoms and Las titer. All treatments had a full range of phloem damage observable in midribs from similarly symptomatic leaves ranging from severe plugging and collapse to apparently healthy phloem. Rep 2 has been inoculated and monthly samplings of leaf and root tissue are underway. Root samples are split for qPCR Las quantification and starch analysis for a quantitive measure of phloem function throughout the plant. Microscopy will be continued, however the high variability of phloem plugging and collapse even within the same midrib from a symptomatic leaf makes interpretation of results difficult. We will pursue methods to quantify phloem plugging in microscopic images to improve interpretation that does not involve selecting representative images, which can be prone to bias. We are also pursuing other methods to quantify phloem function throughout the tree, since leaf to leaf variability in phloem plugging is high.
The host specificity testing for Tamarixia radiata, a parasitoid of the Asian citrus psyllid sourced from the Punjab of Pakistan has been completed. A 60 page Environment Assessment Report was prepared for USDA-APHIS and submitted for evaluation in November 2011. Safety tests conducted by Dr. Raju Pandey in Quarantine at UC Riverside clearly demonstrated that this parasitoid posed no undue risk to California’s environment, other species of insects, or humans. A 60 page Environment Assessment Report on Tamarixia that summarized the results of these studies was prepared by Mark Hoddle and Raju Pandey for review by USDA-APHIS. On 7 December 2012, APHIS issued a permit (P526P-11-04159) authorizing the release of Tamarixia from Quarantine for establishment in California for the biological control of ACP.On 20 December 2011 at 11:00am, 12 glass vials containing 186 female Tamarixia and 95 male Tamarixia (total 281 parasitoids) were opened to release the parasitoids in the Biocontrol Grove at UC Riverside. The eight colonies in Quarantine from which these parasitoids were sourced for release were tested using DNA analyses to ensure that they were free of the bacterium that causes HLB. All tests were negative for HLB indicating that the parasitoids were free of this bacterium. The UCR Biocontrol Grove is a repository for natural enemies that have been imported for the biological control of citrus pests (e.g., scales, mealybugs, whiteflies, etc) in California over the last 50+ years. With the releases of Tamarixia in the Biocontrol Grove, one more natural enemy is being established here to combat an invasive pest that threatens California’s agricultural prosperity. Since this initial release ~ 2,000 Tamarixia have been released in urban areas in LA where ACP infestations are highest. Two recoveries of ACP nymphs with Tamarixia emergence holes have been made in Bell Gardens, LA County. This is a very promising result given the relatively low numbers of parasitoids that have been released at ~15 sites during the winter in southern California. More details on the Tamarixia program at UCR can be found at these websites: http://cisr.ucr.edu/blog/news/first-release-of-tamarixia-radiata-in-california-for-the-biological-control-of-asian-citrus-psyllid/ http://cisr.ucr.edu/blog/invasive-species/tamarixia-radiata-release-video/
This is a one-year project. The overall objectives are to rapidly screen and evaluate chemical compounds for the control of citrus HLB using a graft-based screening method. We have made significant progress in the project in 2011 as summarized below: 1. Screening criteria: A total of seventy-four files were received in May 10 and Sept. 15, 2011. Based on the rank of an expert panel and the mode of action of the compound, 60 compounds from 41 of these files (ranked higher than 0.33) were purchased and tested. Compounds from 33 files were not tested when their ranks were lower than 0.17. Fifteen additional compounds were suggested to be tested by our team. The treatment concentrations of each compound were decided based on previous use of the compound. If the phytotoxicity of a compound was observed, the compound will be retested at a lower concentration. 2. Screening chemotherapy compounds against Las bacterium: A graft-based screening method was used based on our previous studies. The primary results showed that more than 60% of the scions survived and grew when treated with all forty compounds in the first round, except actidione (cycloheximide) at 50 mg/L (0%) and benzyl isothiocyanate at 50 mg/L (54.2%), which were retested at the lower concentrations of 25 mg/L. From Sept. 2011, the leaf samples from the seventeen treated compounds were taken for determining the Las bacterial titers by qPCR. The primary results showed that Ampicilin (Amp) and Rifampicin (Rim) were effective in reducing the Las bacteria to undetectable levels in HLB-affected citrus; Rifamycin (Rif) and Carbenicillin (Carb) were highly effective with lower bacterial titers and transmission rates; The other 13 tested compounds were not very effective in eliminating Las bacterium, but partly suppressed the Las bacterium, including Gentamycin (Gent), Kasugamycin (Kasu), Kanamycin (Kana), Colistin (Col), polymixin B (PMB), Hygromycin (Hyg), Isonicotinic acid hydrazide (INH), Rifaximin (Rix), Cycloserine (Cys), Amikacin (AMK), Quinoline (QUI), Chloramphenicol (Chlo) and Vanomycin (Van). The other grafted material will be tested soon. The twenty compounds in the second round have been grafted and will being tested later. 3.Problems and suggestions: As the contested files were available in September and all providers didn’t submit the contested compounds, some compounds such as peptides in Folder 9932744_044 and lead compounds in Folder 9932744_066 and 9932744_070 could not be purchased and tested. Scions treated with all other compounds have been grafted in December 6, 2011, so we hope to finish this project by June 6, 2012.
This project consists of 5 studies about factors that influence acquisition and inoculation of Candidatus Liberibacter asiaticus (LAS) by th Asian citrus psyllid (ACP), Diaphorina citri. This knowledge is basic to understand the pathogen transmission process and determine more efficient management strategies to manage Huanglongbing (HLB). The results of the first 3 studies on effects of vector development stadia and duration of acquisition access period (AAP) (study 1), leaf age (study 2), pathogen titer and symptom expression in infected citrus (study 3), were shown in the earlier reports of this project. The project is progressing as planned. Studies 1-3 are concluded and most experiments related to studies 4 and 5 were already set up. This report describes partial results of study 4, in which we are investigating the relationship between duration of the inoculation access period (IAP) and transmission efficiency of CLAS by ACP. First we set up an experiment to determine when ACP starts inoculating the pathogen after acquiring it from source plants (latent period), which is important to study the effects of insecticides on transmission. Groups of 3rd-instar nymphs and 1-week old adults obtained from our healthy laboratory colony were confined on LAS-infected sweet orange plants for a 48-h acquisition access period at 25oC. Next, 25 insects of each age group were individually and serially transferred to healthy sweet orange seedlings (test plants) for successive inoculations access periods (IAP) of 48 h at 25oC, until 37 days after the beginning of the acquisition. To assess successful inoculations, TaqMan’ Real-time PCR detection assays with LAS-specific primers were performed on DNA extracts from test plants 10 months later. First transmission events were observed after 11 and 13 days from beginning of the AAP by 1st instar nymphs and adults, respectively. This experiment was repeated and the first transmission event was observed after 9 days from the beginning of acquisition by nymphs, whereas no transmission was detected at anytime following acquisition by ACP adults; first symptoms in inoculated test plants were observed at 5 months after inoculation. In a second latency experiment, we used a longer AAP (96 h) of 3rd-instar nymphs and 1-week old adults on LAS-infected plants and subsequently transferred larger numbers of insects to test plants, in order to increase transmission rates and obtain a better estimate of the minimum latent period. Ten insects of each age group were serially transferred to healthy sweet orange seedlings for successive 48-h IAPs at 25oC, until 33 days after beginning of the AAP. The first transmission event occurred at 11 days after the onset of acquisition by nymphs for most groups tested (14 out of 15 inoculated plants were RT-PCR positive and symptomatic). For ACP groups that acquired LAS as adults, first transmission was detected at13 days from beginning of acquisition, but for only 1 out of 15 groups tested. After the first transmission event, transmission by each group of insets was intermittent, following a random or aggregated pattern. Overall, the results indicate that the minimum latent period of LAS in ACP is approximately 10 and 13 days at 25’C, after acquisition by 3rd-instar nymphs and adults, respectively. It also confirms that the pathogen is transmitted more efficiently by nymphs, as shown in study 1.
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. To date, we have cloned sequences at least 400 bp in length from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. In a side project, we have cloned and characterized two novel IP-free SUS1 promoter alleles (CsSUS1-1 and 2) from Citrus sinensis cv. valencia and have found them to drive phloem-specific expression in both Arabidopsis and tobacco when fused to a reporter gene. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, we evaluated the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). In this objective, Gateway-based vectors were used to express the selected insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We successfully generated Gateway vectors that will result in constitutive (35S promoter) or phloem-specific (CsSUS1 promoter) expression, respectively, of M. persicae-specific Coo2, GSTS1 and S4e dsRNA, as well as a control derived from GFP. Our results suggest that the M. persicae-specific dsRNA expressed in planta has a negative effect on both the lifespan of the insects and the number of offspring generated. In the fall of 2010, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. To date, we are in the process of transforming and regenerating citrus with the D. citri-specific gateway vectors for evaluation and use by the Florida citrus industry. Initial attempts to transform and regenerate Citrus sinensis ‘Valencia’ and ‘Hamlin’ containing reporter gene constructs were successful. Currently we have completed transformation of citrus callous tissue using gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. We are in the process of regenerating transformed lines, and are preparing to generate additional lines with the other transcript/promoter combinations. In summary, we have cloned a number of transcripts from both D. citri and our model organism, M. persicae, and have analyzed a subset of derived dsRNAs to test their effect on M. persicae using in vitro assays (objective 1). We have also cloned and characterized several novel phloem-specific promoters from C. sinensis, and have evaluated their expression patterns. We have also created new Gateway-derived vectors bearing a native citrus phloem-specific promoter, for use in RNAi in our M. persicae model and evaluated them in planta (objective 2). Finally, we’ve now generated similar vectors specifically designed against D. citri (objective 3), and are now generating transgenic lines expressing D. citri dsRNA for evaluation.
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. To date, we have cloned sequences from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, we evaluated the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). In this objective, Gateway-based vectors were used to express the selected insect dsRNA (Coo2, GSTS1 and S4e) either constitutively (35S promoter) or in a phloem-specific manner. Our results suggest that the M. persicae-specific dsRNA expressed in planta has a negative effect on both the lifespan of the insects and the number of offspring generated. In the fall of 2010, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. To date, we are in the process of transforming and regenerating citrus with the D. citri-specific gateway vectors for evaluation and use by the Florida citrus industry. Initial attempts to transform and regenerate Citrus sinensis ‘Valencia’ and ‘Hamlin’ containing reporter gene constructs were successful. Currently we have completed transformation of citrus callous tissue using gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. We are in the process of regenerating transformed lines, and are preparing to generate additional lines with the other transcript/promoter combinations.
1) Evaluation of screens impregnated with insecticide barriers. As explained in previous reports, the experiment is being conducted in two farms located in the Sao Paulo State. On the farm located in Sao Manuel 21 evaluations were performed. In both areas (with and without screen impregnated with insecticide barriers)13 insects were collected. In Descalvado 17 evaluations were performed, in a total of 90 and 94 psyllids collected in areas with and without barrier, respectively. On both farms, about 80% of the catch occurred from August to October, this probably happened due to favorable climate conditions to D. citri, observed in this period. Due to the no difference in the psyllids capture in areas with and without barrier, sample screens from both farms were taken to determine the insecticide amount present. Initially the screen showed 4 g of deltamethrin / kg of the screen. After 8 (Sao Manuel) and 5 (Descalvado) months in the field, the insecticide amount was detected: 0.28 and 0.53 g deltamethrin / kg of the screen, respectively. In this same period, samples were also taken from the screen to perform a bioassay in the laboratory to determine whether the screens were still effective against D. citri. After a 24-hour contact with the screens, it was observed an efficiency (Abbott, 1925) of 63 and 80% to the screens from San Manuel and Descalvado, respectively. The D. citri capture assessment using yellow sticky traps are being performed and they will be maintained until the experiment completes one year. New assessments for inseticide quantification (deltamentrina) and effectiveness of the screens are planned. 2) Evaluate the treatment impact of plants with systemic insecticides on the Ca. L. asiaticus trnsmission by starved psyllids. The application of insecticides has been performed and the transmission trials are underway.
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