New outbreaks of invasive fruit flies (Diptera: Tephritidae) continue to threaten agriculture world-wide. Establishment of these pests often results in serious economic and environmental consequences associated with quarantine, control, and eradication programs. Early fruit fly detection and eradication in the United States requires deployment of large numbers of traps baited with the highly attractive male specific parapheromone lures trimedlure (TML), cue-lure (C-L), and methyl eugenol (ME) to detect such pests as Mediterranean fruit fly, Ceratitis capitata (Wiedemann), melon fly, Bactrocera cucurbitae (Coquillett), and oriental fruit fly, B. dorsalis (Hendel), respectively. The current study compared the performance of solid single lure cones and plugs in conjunction with DDVP insecticidal strips; liquid lure with naled formulations; and single, double, and triple solid lure wafers impregnated with insecticide. Treatments were placed in AWPM and Jackson traps under Hawaiian climatic conditions in habitats where B. dorsalis, C. capitata, and B. cucurbitae occur together. The overall goal of this study was to develop a more convenient, effective, and safer means to use male lures and insecticides for improved detection and male annihilation of invasive fruit flies. In survey trials near Kona, HI captures of C. capitata, B. cucurbitae, and B. dorsalis with Mallet TMR wafers were equal to those for the standard TML, ME, and C-L traps used in Florida and California. A solid Mallet TMR wafer is more convenient to handle, safer, and may be used in place of several individual lure and trap systems, potentially reducing costs of large survey and detection programs in Florida and California, and male annihilation programs in Hawaii. Trials of the new TMR wafers have now moved to evaluations in typical citrus growing areas of California. Mallet TMR dispensers are being weathered inside Jackson traps in citrus trees in Tulare, Ventura, Riverside, Kern, and Orange Counties of California. Two series of trials are being conducted in conditions representative of summer (August-September) and winter (January-March) conditions in California. Each week weathered wafers are sent to Kauai Island, HI or Hawaii Island, HI (USDA-ARS-PBARC) for fruit fly bioassays and to Farma Tech International, North Bend, Washington for chemical analysis using conventional gas liquid chromatography methods. Currently, approximately 30,000 sets of TML, ME, and C-L traps are maintained throughout the state. From a worker safety, convenience, and economic standpoint, Farma Tech TMR Mallet solid wafers with DDVP may be more cost effective, convenient, and safer to handle than current liquid lure and insecticide formulations (e.g. naled) used for detection programs for TML, ME and C-L responding flies in California. Cost/benefit analyses of Mallet TMR vs. standard trapping systems will be done.
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. Screening of psyllid performance on plants infected with a recombinant CTV vector containing psyllid DNA sequences from these genes that produce dsRNA within citrus phloem was continued. 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. This was confirmed in triplicate studies and the effect of this feeding on psyllid gene regulation is now being characterized. Enough plants have also now been propagated to initiate whole plant challenge studies with psyllids to look at effect on psyllid nymph survival.
The strategy was to identify Asian citrus psyllid genes as targets for dsRNA-mediated RNA inhibition induced by feeding the insects dsRNAs that match coding regions of the selected genes. There was a bias placed on the selection of genes that would be necessary in the digestive tract epithelium lining since dsRNA molecules introduced by feeding would be presumed to be present at the highest concentrations in these cells. These cells are considered to be highly metabolically active in support of the production of oxidative enzymes used in digestion and the transport of nutrients, ions and numerous organic molecules from the digestive tract lumen into the endothelium cells, and from there, into the hemolymph (the insect circulatory system). Figure 1 shows a graph depicting the effect of feeding single gene sequence specific double stranded RNAs (dsRNAs) to the psyllids through the use of an artificial diet. Double-stranded RNA molecules matching eight gene targets were shown to induce significant mortality in adult Asian citrus psyllids when fed to the insects in artificial diets. Mortality (indicated as frequency: percent mortality divided by 100) is presented by normalizing to mortality seen in diets without any dsRNA. We define significant mortality as greater than 30% mortality ( a frequency of 0.3 on the x-axis). This is mortality above what is seen in diet without dsRNA after the insects have fed on the artificial diet for 6 days. A control dsRNA molecule used in this study targeted the sequence of a gene for a protein within another insect (the Diaprepes root weevil- DRW) and is listed in the graph as ‘DRW average’. We consistently saw mortality with the high concentrations of DRW control dsRNA (48 and 24 ng/uL) and this was also observed with dsRNA’s matching other presumed negative controls (i.e. dsRNAs cognate to the green fluorescent protein, GFP). The nature of this non-specific mortality is not yet understood but has been observed by other researchers working with the potato psyllid and the whitefly (Bemisia tabaci). Clearly, not all dsRNAs induced statistically significant mortality when added to diets, so at this time, the nonspecific mortality is not believed to be a general dsRNA toxicity. However, continued experimentation is needed to determine the nature of this toxicity. Interestingly, three of the eight mortality inducing dsRNAs target genes potentially involved in the electron transport pathway (ATP production from oxidative metabolism) and a forth, Ferritin, is involved in regulating iron availability which is a key cofactor in many electron transport pathway protein complexes. Therefore, of the dsRNA molecules that induced significant mortality, 50% could be influencing electron transport functions (ATP production). Of the other four, one is a membrane transporter of unknown substrate specificity, one is a regulatory of Ca2+ ion availability, one is involved in phenylalaline metabolism and the final one is a member of a unique family of Osiris proteins that are dose sensitive lethal and thought to play a role in development.
Two 3-year field experiments are being conducted in two commercial orange blocks in Hendry County (southwest Florida). One of the groves is planted with ‘Earlygold’ oranges and the other with ‘Valencia’ oranges. Average HLB incidence estimated in both groves at the beginning of the experiment based on PCR analysis of a random sample of 160 trees was 98% in ‘Earlygold’ and 42% in ‘Valencia’. Experimental design is randomized complete block with 4 replicates and 4 treatments: (1) No insecticide, (2) Calendar applications, in order to drive vector populations close to 0, (3) nominal threshold of 0.2 psyllids per tap, and (4) nominal threshold of 0.7 psyllids per tap. Calendar applications are being applied every month. In January, treatments (2) and (3) recieved a second dormant spary with zeta-cypermethrin (Mustang) at 4.3 fl oz/ac. In February, treatment (2) was sprayed with spirotetramat (Movento MPC) at 16 fl oz/ac and horticultural mineral oil 2%. All treatments received a foliar nutritional spray according to established protocols. Juice quality analyses from the “Earlygold” block fruit included estimates of Brix/acid ratio and the Lbs solids per box from samples take in each plot. In these analyses it was found significant differences for the Brix/acid ratio between treatments but not for the Lbs solids per box (F = 3.86; df = 3, 15; P = 0.0382 and F = 1.06; df = 3, 15; P = 0.4486 respectively). The highest Brix/acid ratio was obtained for treatment (2) (33.26 ‘ 0.25) and the lowest for treatments (1) and (3) (30.22 ‘ 0.23 and 30.25 ‘ 0.54 respectively). For all treatments, the Brix/acid ratio was higher during 2010 harvest (31.55 ‘ 0.49) than during 2011 harvest (26.05 ‘ 0.55). No differences were found between years for the Lbs solids per box. In the last week of November, the “Valencia” block was harvested and yields, yield increments with respect to the 2010 harvest as well as several fruit quality parameters were measured for each treatment. Yield values were correlated to the cumulative number of ACP adults obtained by stem tap sampling during the last season. A trend toward higher yields in the Calendar treatment (271.88 ‘ 57.75 boxes/acre) and lower yields in the treatment that did not received any sprays (232.03 ‘ 7.85 boxes per acre) was found but differences were not significant (F = 0.32; df = 3, 15; P =0.8086). Similarly, yields did not vary significantly with treatment compared to the previous harvest (F = 1.48; df = 3, 15; P = 0.285) although a trend for decreasing yields was found in the treatments where higher ACP populations was observed (-59.22 ‘ 39.78 and -20.92 ‘ 22.61 boxes/acre for treatments (1) and (4) respectively). On the other hand, a significant treatment effect on fruit diameter (F = 7.37; df = 3, 639; P < 0.0001), weight (F = 7.92; df = 3, 639; P = < 0.0001) and the fruit weight/volume ratio (F = 4.8 df = 3, 639; P < 0.0001) was seen, with higher values for the first two parameters in treatment (2), and for the last parameter in treatment (1). ACP cumulative numbers during 2011 were lower in treatment (2) than in treatment (1), but differences were only marginal (P = 0.063). In addition, no significant correlation was found between cumulative ACP numbers and yield (P = 0.1942). To evaluate the incidence that calendar sprays have on natural enemies an exclusion experiment on ACP infested flushes was conducted during the first flushing period of the year. Mortality in ACP cohorts due to predation was higher in treatment (1) than in (2) (38.29 ' 10.76% and 12.71 ' 10.11% respectively) (F = 3.94; df = 1, 21; P =0.0637). These results show that ACP natural enemies have been negatively affected by a year of calendar sprays.
At the beginning of this project growers were aggressively scouting for and removing HLB infected citrus trees with heavy duty front end loaders. This project had two objectives, the first was to evaluate chemicals that would kill individual citrus trees affected with HLB so they did not have to be removed and the second was to evaluate killing general areas of abandoned citrus trees that are present in Florida and present a large reservoir of the HLB bacteria for vector transmission to healthy commercial trees. For the first objective, canopy spray methods and trunk applications were used to test several chemicals. None of the usual citrus herbicides were effective by any application method, but the herbicide imazapyr effectively killed citrus trees by all application methods. It was consistent in effect at a reasonably low application rate (Albrigo et al, 200?). An alternative method of using the soil fumigant Midas (Iodomethane) was also tested and worked well on smaller trees, 8 or less inch diameter, but was very inconsistent on larger trees (Albrigo et al, 200?). The company manufacturing the fumigant choose not to continue evaluation. As part of this first objective, two machines were designed to apply the imazapyr to the tree trunk in machine cuts followed by the machine spraying the chemical. One machine was a hydraulically operated three saw blade system that moved around the trunk to cut and spray the cuts and was mounted on a small tractor. The second machine was a modified weed cutter with a slide guide to hold the cutter at a uniform distance from the ground and a backpack sprayer with nozzle mounted to the end of the cutter tube. A paper is in preparation describing the design and efficacy of these application machines. Because growers have mostly shifted to nutritional-SAR maintenance of HOLB infected trees no commercial development of the machines or clearance of the herbicide imazapyr for this use. Cooperating with the South Florida Water Management District we monitored aerial application of imazapyr to 560 acres of citrus in an area intended for water retention. The application was very successful in killing the citrus trees in most blocks and appeared as a good way to kill these abandoned stands. Material costs were about $50 per acre and would appear to be cost prohibitive.
In 2011, we started a new project on mandarins, which we conducted at a commercial grove in Bakersfield. The project is of interest for two main reasons. First, mandarins are often planted at a higher density than other varieties; and second, we were able to compare the uptake of imidacloprid into trees that were grown under different irrigation systems ‘ drip (average of 3 drippers per tree) versus sprinkler (placed centrally between trees) whereas other conditions (tree age and size, soil type, volume of water per tree) were similar in adjacent blocks with the two types of irrigation. The significance of tree density is important when the imidacloprid application rate is taken into account. The ‘per acre’ amount of insecticide allowed does not change when the tree density is increased. In short, changing the tree density will alter the amount of imidacloprid delivered to each tree. Our study on mandarins permitted us to determine how effective field rates of imidacloprid were on a high-density citrus planting. The impact of different irrigation systems on the efficacy of imidacloprid uptake is unclear. The manner in which trees are irrigated will likely affect the root pattern. With a drip system, the roots will be trained to a very precise area around each dripper, whereas with a sprinkler system the roots may be more dispersed. In our study, we did not evaluate the rooting patterns for the different systems, but followed the progress of imidacloprid uptake once the treatments had been applied. We also compared 3 application timings, beginning with the first application on June 9 soon after the honey bee netting had been removed from the trees. Additional treatments were applied on July 13 and Aug. 24. The trees used in our study were W. Murcott on ‘Carrizo’ rootstock, and were planted in blocks that were either drip-irrigated (11 years old) or sprinkler-irrigated (9 years old). As with all our trials, we strive to collect the youngest tissue available on the trees because this will be the target for the ACP adults to feed and oviposit on. Excellent uptake of imidacloprid occurred under both irrigation systems. In all but one of our 6 treatments, the 200 ppb threshold was achieved within 1 week of the application. The exception was the June treatment timing to the sprinkler-irrigated block, which took 2 weeks to reach the threshold. Overall, uptake was more rapid with the drip system although there was little to separate the two systems with the mid-summer timing. Peak imidacloprid levels were very high for all treatments, exceeding 1,800 ppb for the three drip applications, and at least 1,200 ppb for the sprinkler applications. In terms of persistence, the efficacy of the June application was 12 weeks; we are still processing data for timing 2 and timing 3 applications, but so far we can see that the persistence of the July applications (timing 2) has extended to 8 weeks, and the August applications (timing 3) to 6 weeks.
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. Unfortunately, the the boron and zinc nutrient deficiencies started to revert in the last quarter of 2011. We continue to truck distilled water to the greenhouse to water the trees. Since we have begun the distilled water treatment, the trees have slowly begun to revert to a nutrient deficient state. The results of the leaf analysis in February showed that the zinc levels have again become deficient but the boron levels had not dropped sufficiently. The rest of the project is waiting on nutrient symptom development. HLB symptom development continues in our plants with the different HLB strains. As soon as strong deficiency symptoms develop, then psyllid testing for objective 1 and 2 will commence and is expected to move rapidly. Tree maintenance has been continued but funds have not been received so all other activities have been curtailed.
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. Data collection for Las prevalence in psyllids and branches continues in three sites (Lake Alfred, Conserv II, and Lake Wales) for the second year. Phenology is also being collected for all three sites. Processing the backlog of samples for PCR starting with DNA extraction continues but at a slow pace. We are continuing with the reduced total number of psyllids per sampling date because we determined that the smaller samples were sufficient for a robust statistical test. We missed some psyllid sampling dates due to a miscommunication with collaborators but pysllid collection has resumed.
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. Unfortunately, the the boron and zinc nutrient deficiencies started to revert in the last quarter of 2011. We continue to truck distilled water to the greenhouse to water the trees. Since we have begun the distilled water treatment, the trees have slowly begun to revert to a nutrient deficient state. The results of the leaf analysis in February showed that the zinc levels have again become deficient but the boron levels had not dropped sufficiently. The rest of the project is waiting on nutrient symptom development. HLB symptom development continues in our plants with the different HLB strains. As soon as strong deficiency symptoms develop, then psyllid testing for objective 1 and 2 will commence and is expected to move rapidly. We are also interested to determine if strains of Las will have any effect on the attractiveness of trees to D. citri. It has been reported that Las strains have varying levels of virulence and symptomatology (Tsai et al. 2008). We have analyzed DNA samples from HLB positive trees from Polk and Highlands counties as well as the ‘Smoak Grove’ CREC greenhouse strain by PCR and sequencing. Three putative strains of Ca. Liberibacter asiaticus (Las) were found with 5 (CREC greenhouse isolate), 13, and 15 tandem repeats of DNA in the LAPGP locus described by Chen et al. 2010 and have identified sources of budwood. Cloning and sequencing of loci including the b-operon, OMP (outer membrane protein) gene and phage DNA polymerase to support the differentiation of the three strains is complete (Bastianel et al. 2005; Lin et al. 2008; Okuda et al. 2005; Tomimura et al. 2009). Results from sequence analysis clearly defines two strains based on conserved mutations in the b-operon sequence, matching strains from Japan and Vietnam and a strain from Vietnam for the northern and southern Florida strains, respectively.
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. Data collection for Las prevalence in psyllids and branches continues in three sites (Lake Alfred, Conserv II, and Lake Wales) for the second year. Phenology is also being collected for all three sites. Despite the departure of the post-doc responsible for the project. We have begun to process our backlog of samples for PCR starting with DNA extraction but hope to resume the qPCR soon. We are continuing with the reduced total number of psyllids per sampling date because we determined that the smaller samples were sufficient for a robust statistical test.
During winter/spring 2012, we established 2 field experiments in St. Lucie county and participated in 4 validation trials in Charlotte, DeSoto, Okeechobee and St. Lucie counties in collaboration with ISCA Technol. (manufacturer of SPLAT CLM), Fly Masters International (custom applicator), grove managers, owners and crop consultants. These trials address various issues related to timing, coverage, formulation, longevity and efficacy of mating disruption (MD) under various crop phenologies. 1. Effect of winter applications on CLM suppression. Cooperator: The Packers of Indian River, St. Lucie county. SPLAT CLM provided by ISCA. A split-plot design was established to test the value of SPLAT CLM application when CLM populations are low during winter months. First application was made on Feb. 6, followed by a second application in mid-April. Trap catch disruption, CLM mining activity and citrus canker incidence will be documented. 2. Effect of formulation on longevity and efficacy across a treatment gap. Cooperator: The Packers of Indian River. SPLAT CLM provided by ISCA. This trial, established April 2012, will compare the current SPLAT CLM pheromone formulation (an off-ratio blend consisting of the major triene pheromone component) with a formulation that incorporates the ‘natural’ 3:1 triene:diene blend of two pheromone components. This trial will focus on evaluation of longevity of MD and the ability of the formulations to affect MD across treatment gaps (skipped rows) of varying width. 3. Validation trial, 200 g/acre nominal rate with treatment gaps (effective rate 140 g/acre). Cooperator: The Packers of Indian River. SPLAT CLM purchased by grower. 40 acres of grapefruit were treated in mid-April (1300′ rows, 10T 4S 8T 6S 8T 4S 11T where T=treated row, S=skipped row). Longevity of MD, mining activity and canker incidence will be documented. 4. Validation trial, 200 g/acre, no treatment gaps. Cooperator: W.M. Marsh, DeSoto county. SPLAT CLM purchased by grower. Approximately 100 acres of pomelos treated, late February. Longevity of MD, mining activity and canker incidence will be documented. 5. Validation trial, 200 g/acre, no gaps. Cooperator: Travis Murphy, Okeechobee county. Initial SPLAT CLM supplied by ISCA. SPLAT applied by hand to ~12 acres of an irregularly-shaped grove of 3-yr-old grapefruit trees with a corresponding control in early March, 2012. Trap catch disruption to date suggests MD can be established under these conditions. Longevity will be documented. 6. Validation trial in 100 acres of grapefruit, 200 g/acre nominal rate with treatment gaps (effective rate 140 g/acre). Cooperator: TRB Grove, Charlotte county. SPLAT CLM purchased by grower. First application 22 March 2012. Longevity, mining and canker incidence and severity will be documented.
One of the goals of this project is to determine how long the Asian citrus psyllid (ACP) should feed on citrus plants in order to be able to acquire or inoculate Candidatus Liberibacter asiaticus (CLas). We previously showed that acquisition efficiency was related to the duration of acquisition access periods (AAP), increasing with time available for feeding on infected plants (study 1). We also observed that almost 30% of the nymphs and adults were PCR positive for CLas after a short AAP (1.5 h), suggesting that the minimum AAP required for acquisition is <1.5 h. In this report we describe further transmission experiments coupled with Electrical penetration graph (EPG) analyses, in which we explore in greater detail the feeding site and time required for acquisition and inoculation. We used the EPG technique to determine stylet penetration phases and probing periods required for pathogen acquisition and inoculation in citrus. In a first experiment designed to evaluate acquisition, healthy ACP adults were connected to the EPG system and placed on CLas-infected plants. Probes were artificially terminated by pulling the insects out of the infected plant after the following stylet penetration periods (treatments; n ' 50 insects per treatment): I) 20 min in waveform C (pathway phase through epidermis and parenchyma); II) waveform C + 30 s in waveform D (first contact with phloem tissue); III) waveforms C+D + 90 s in waveform E1 (penetration and possibly salivation/ingestion(?) in the phloem sieve elements); and IV) waveforms C+D+E1+E2 (phloem sap ingestion). After each treatment, the insects were kept on healthy sweet orange seedlings (test plants) for an inoculation access period (IAP) of 3 wks and then tested by Real time (RT)-PCR for CLas infectivity. As a negative control, 25 insects from the same rearing batch were transferred directly to the IAP on test plants. A second EPG experiment was carried out to evaluate inoculation, in which 3rd-instar ACP nymphs were first submitted to an AAP of 2 weeks on CLas-infected plants and then connected to the EPG system on healthy test plants for the same stylet penetration periods evaluated in the first experiment (treatments I, II, III and IV; n ' 50 insects per treatment). As a negative control, 25 insects from the same batch were transferred individually to test plants, without a previous AAP on infected plants. All inoculated plants were assayed for CLas infection by RT-PCR 8-12 months later. The results of the first EPG experiment showed that ACP acquires the pathogen (CLas) only in the phloem phase of the stylet penetration, mostly during waveform E2 (27 insects positive by RT-PCR out of 54 tested), when sustained phloem sap ingestion takes place (treatment IV). A significant proportion (49%) of these insects were able to subsequently transmit CLas to test plants, confirming their infectivity. Two of 52 insects (3.9%) exposed to infected plants until waveform E1 (treatment III) were positive, but none of them transmitted the bacterium to test plants. In the second EPG experiment, transmission of CLas (by 9 out of 50 insects tested) was observed only when the insects were allowed to perform waveform E2 (treatment IV) on the test plants, showing that inoculation takes place only during the phloem phase. Previous EPG observations have shown that nymphs and adults take an average of 63 min (range of 16 - 241) and 75 min (range of 16 - 241), respectively, to start phloem sap ingestion (waveform E2). Altogether, these EPG data indicate that at least 15 min of exposure to citrus plants is required for psyllids to reach citrus sieve elements, where it is able to acquire or inoculate the pathogen.
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (March 2013 Update) This quarter, method validation continued for selected pesticides of interest. The LOD and LOQ of selected pesticides was examined for the pesticide extraction method used for citrus leaf tissues. Focus then turned to analysis for potential residues in citrus nectar. Due to the different substrate being evaluated, new methods were obtained from literature and collaborating registrants and work began comparing different methods of analysis for residues in citrus nectar.
This is a one-year project. The overall objective is to rapidly screen and evaluate chemical compounds for the control of citrus HLB using a graft-based screening method. In this quarter from Jan. to April 2012, the research was focused on the continual evaluation of the compounds by qPCR. The leaf samples of scions and inoculated plants treated with 56 compounds were taken for determining the Las bacterial titers by qPCR. The scion survival rate (%), the pathogen transmission efficiency (%) and the scion infected percentage (%) were also recorded. Based on the scion infected percentage, Las transmission efficiency (rootstock infected percentage) and the Las bacterial titers in the inoculated plants (including rootstock, scion1 and scion2), the tested compounds wiere divided into four groups: 1) Highly effective (O): The compounds eliminated the Las bacterium with low scion infection and transmission and the titers in the inoculated plants were less than 15,000 cells per gram plant tissue (CT>32.0). They were Ampicilin and Penicillin; 2) Effective (I): The compounds eliminated most of the Las bacteria with low scion infection and transmission, and the Las bacterial titers in the inoculated plants were less than 200,000 cells per gram plant tissue (CT>28.0). They are Carbenicillin, Validoxylamine A, Oxytetracycline and Rifampicin; 3) Partly effective (II): The compounds suppressed the Las bacteria with medium scion infection and transmission, and the Las bacterial titers in the inoculated plants were more than 200,000 cells, but less than 3,000,000 cells per gram plant tissue (24.0
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 have completed one round of transformation and regeneration of citrus lines with the D. citri-specific gateway vectors for greenhouse evaluation. This first round generated several lines containing gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. We had regenerated at least one line for each of the constructs of interest, but realized that the transformation process produces few plants for evaluation. In response, we attempted to develop an alternative transformation/regeneration process for citrus that would generate numerous shoots (10-20) per transformant. This would serve as material for in vitro micro propagation to produce many copies of each line for evaulation. Thus far this process appears to be working with Citrus on the second round of transformants/regenerants. We are also establishing an evaluation pipeline with collaborators in Florida, and are beginning a third round of transformations for additional lines with other transcript/promoter combinations such as a B tubulin subunit from D. citri.
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