This proposal aims to continue improvement to a novel psyllid the trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing was and continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps. Research by others to discover and identify semiochemicals that actively attract or repel ACP is ongoing. While a number of plant volatiles and ACP-produced compounds have shown their presence or activity in laboratory bioassays, only low level capture increases (less than or equal to a 25% increase in trap captures over unbaited traps) and inconsistent results have been manifested in field bioassays. To date psyllid visual is the only behavioral response reliable enough to attract psyllids. Therefore, we have conducted a large number of field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulating psyllid behavior around the trap or farther away so that they are moved close enough to the trap to perceive it (i.e., increase trap active distance). This work is continuing. We have also initiated conversations with several private companies to get a trap prototype mass produced.
This proposal aims to continue improvement to a novel psyllid the trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing was and continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps. Research by others to discover and identify semiochemicals that actively attract or repel ACP is ongoing. While a number of plant volatiles and ACP-produced compounds have shown their presence or activity in laboratory bioassays, only low level capture increases (less than or equal to a 25% increase in trap captures over unbaited traps) and inconsistent results have been manifested in field bioassays. To date psyllid visual is the only behavioral response reliable enough to attract psyllids. Therefore, we have conducted a large number of field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulating psyllid behavior around the trap or farther away so that they are moved close enough to the trap to perceive it (i.e., increase trap active distance). We have a number of positive results from our bioassays with which to attempt to increase trap efficiency but have yet to reach a level of trap capture rate which is a satisfactory representation of ACP populations in the vicinity of the trap. This work is continuing and we are conducting a series of experiments to further exploit the success from the addition of a LED light source to the trap. These include tests of a number of available narrowband bulbs on psyllid attraction. We have also in this quarter begun developing the procedures and components of the final prototype trap with a third party distributor to enable mass production of the traps in preparation for the field sampling objectives. This areawide intensive sampling will begin in late first or early second quarter of 2013 and should provide a unique data set from which to detect and determine the natural infection rate of adult vectors by known and novel entomopathogens. The genome sampled will provide a benchmark for future research as appropriate and remain available for continued use in posterity.
This proposal aims to continue improvement to a novel psyllid trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing was and continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps and behaviorally active components. Research by others to discover and identify semiochemicals that actively attract or repel ACP is ongoing. While a number of plant volatiles and ACP-produced compounds have shown their presence or activity in laboratory bioassays, only low level capture increases (less than or equal to a 25% increase in trap captures over unbaited traps) and inconsistent results have been manifested in field bioassays to date. To date psyllid visual is the only behavioral response reliable enough to attract psyllids. Therefore, we continue to conduct field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulating psyllid behavior around the trap or farther away so that they are moved close enough to the trap to perceive it (i.e., increase trap active distance). We have a number of positive results from our bioassays with which to attempt to increase trap efficiency but have yet to reach a level of trap capture rate which is a satisfactory representation of ACP populations in the vicinity of the trap. This work is continuing and we are conducting a series of experiments to further exploit these ideas. During this quarter we have cooperated with several USDA-ARS personnel to develop, test and incorporate other trap features that affect psyllid visual response but also involve other sensory modalities including sound productions during psyllid courtship. This work is ongoing and more experiments will be conducted in the next quarter. This quarter we continued developing the procedures to manufacture components of the final prototype trap with a third party distributor to enable mass production of the traps in preparation for the field sampling objectives. We have developed and tested a number of different prototype trap components including ones made with 3-D printing to reduce the cost of the enterprise. In preparation for the areawide psyllid sampling objective, preliminary field tests were conducted with the trap prototype under South Florida conditions to determine how traps endure more intense field conditions of heat and moisture. This areawide intensive sampling will begin in soon.
June 30, 2013 Update: Activities of the CHMA program assistant (Brandon Page) March 31 – June 30, 2013: During this reporting period, the CHMA assistant provided support to the CHMA effort by attending various local CHMA meetings as well as statewide meetings. Specifically, Mr. Page attended 9 meetings where he either gave an oral presentation, setup CHMA displays such as posters, or participated in group discussions. Venues included Hardee Co CHMA meeting (2x), Florida CItrus Growers Institute, Gulf CHMA meeting, All Florida Ag Show, Volusia CHMA meeting, Immokalee growers meeting, and the Florida State Horticultural Society meeting. Time was also spent out of the office giving a presentation on the CHMA program to the Extension Dean of IFAS. In addition to attending meetings, daily updates were made to the CHMA website based on communication with CHMA grower leaders. Time was also spent signing growers up for the mapping program that went live online at the end of December 2012. Grove visits were also made to followup on scouting reports for some CHMAs that appeared to not follow the trends observed by growers in the field.
June 2013 update: As part of this grant, we are investigating the use of kaolin clay as a supplemental tool to protect both young and mature trees from becoming colonized by Asian citrus psyllid. The fact that kaolin can deter insects from colonizing plants has been previously demonstrated by other researchers which have shown reduction (not elimination) of certain pests, including psyllids, on kaolin treated plants. Our goal of the ongoing investigations described below is to determine the feasiblity of kaolin to deter psyllids from colonizing citrus trees, thus serving as a repellent that could be used in conjunction with pesticide sprays which we have previously reported as providing very short residual activity. In laboratory trials, choice tests were conducted by releasing psyllids into arenas with plants treated with different rates of kaolin applied. Three of the four rates evaluated demonstrated a repellent effect with psyllid choosing the untreated over kaolin treated plants. The lowest rate evaluated did not deter psyllids from landing and feeding on treated plants. In young tree field trials we previously reported on, we have shown reductions in psyllid populations on young trees treated with kaolin as well as a subsequent decrease in HLB infection rate that appeared to not increase until the rainy season when it is more difficult to keep leaves coated with the kaolin residues. In order to determine how rapdily rain can degrade the repellent effects of kaolin, laboratory trials were conducted to quantify the dislodgeable kaolin residues based on different rates of material applied. Through this study, we were able to predict dislodgeable residue levels that should deter psyllid feeding across a range of concentrations tested. Based on the results obtained from our laboratory studies, we intiated three field trials this year (two of which are still ongoing) to examine the efficacy of kaolin applied to mature citrus in order to first determine the efficacy provided by different rates of product applied and secondly determine how long such residues will last when exposed to rainfall. THe results from this work will be presented in future reports.
As a more sustainable and environmentally friendly method of psyllid control, we evaluated a RNA interference (RNAi) approach based on psyllid oral uptake of dsRNA molecules that target specific psyllid genes. This approach is based on the finding that cellular uptake of dsRNAs, that match the sequence of essential genes, results in down regulation of those genes and can lead to cell/organism death. These dsRNA molecules were introduced into the psyllids through feeding on citrus engineered to express the dsRNA using a Citrus tristeza virus as a paratransgenesis vector. Increased toxicity was observed when adult psyllids were fed on citrus producing dsRNA targeting two gut protease genes. No increased psyllid toxicity was observed in psyllids fed on citrus producing green fluorescent protein (GFP) dsRNA. These results suggest that RNAi-based control may be a viable alternative to current pesticide use for control of psyllids and all phloem feeding pests.
Xambr’ Paran’ Brazil Plots: To quantify the effect of windbreaks, copper sprays, and insecticide sprays individual and combined effects on citrus canker management. In 2010 replicated field plots were established. I was soon evident that the screens used as windbreaks were not strong enough to provide the required effect. Thus in addition to the screens, we established natural windbreaks using Casuarina. Casuarina trees that are currently ~6 m high. There are a few canker-affected plants in the experimental area that will be pruned every 3 weeks. We forecast that in September/October 2013, trees will have developed enough canopy to allow inoculation and to start the experiment. During May, 2013 inspection, 25 plants showed HLB like symptoms. St Lucie Co. windbreak studies: Data from the windbreak system at the first location (5-y old Ray Ruby grapefruit, Estes Orchard, 11 acres, surrounded by 25-ft Corymbia torelliana) demonstrated a windbreak effect on canker incidence on fruit in August 2012. The weather stations (deployed in east-west and north-south directions) measured the effect of proximity to the windbreak on wind speed expressed as the number of wind gusts ’11 mph. The relationship with incidence of fruit canker lesions was explored. In the period May -August 2012, the greater frequency of wind gusts ‘ 11 mph, the higher the canker fruit incidence. The number of wind gusts ‘ 11 mph increased with distance from the windbreak whether measured within the north to south rows or across the rows from east to west, resulting in a relationship between wind and disease incidence on fruit (R2=0.70). In August, the highest incidence of fruit disease (3.5%) was in the center of the 11 acre bock and the lowest incidence (0.4%) was on the end of the eastern most row nearest to the windbreak. Programmable leaf wetness controller (PLWC): During the previous quarter we developed a complex sensor that closest emulates a leaf. This pin sensor, is a flat surface, made from wax. Contact wires are then connected together in a pattern, so that a drop of water will bridge a ‘+’ to a ‘-‘, creating a connection. The more drops of water, the more connections; the more connections, the more current flow. Results from tests of the new leaf wetness ‘pin sensor’ show that this arrangement is way more sensitive to water amount than any previously tested sensor. After some tweaking of the gain circuit on the DC-AC-DC board, we were able to show that with each successive drop of water, the voltage increased until there was a saturation of water. This is what we have been looking for as this will produce a scalar approximation of leaf wetness as opposed to the current Boolean systems. The drying time is a bit faster than a citrus leaf. To correct this we will add layer of silk to the top of the pin sensor. Previous work with the silks has shown that it dries in a similar amount of time to a leaf. In theory, this thin layer of silk will hold water to the sensor for a more realistic time. Project publications: (In addition to the 7 previously reported publications relative to this project, the following was recently submitted as well) Bock, C. H., Gottwald, T. R. and Graham, J. H. 2013. A comparison of the bioassay test and culture to detect Xanthomonas citri subsp. citri . Plant Pathology 62: xx-xx.
During this quarter, particular attention was given to the three primary objectives of the project: 1) Develop a generic epidemiological model that can be used to compare control scenarios and to optimize the probability of controlling and managing high-risk pathogens of agricultural significance. 2) Development and testing of efficient methods of statistical inference to estimate epidemiological parameters from maps of emerging epidemics. 3) Develop user friendly model ‘front ends’ that can be used by researchers and regulatory agencies. Following an extensive period of testing methods to parameterize stochastic models for HLB and citrus canker, and the development of flexible models for spread of epidemics through heterogeneous landscapes, we have focused on (i) the practical use of the models to compare control scenarios at the landscape scale; (ii) to allow for uncertainties in the location of hosts at different scales in the landscape and in a policy makers knowledge of parameter estimates (iii) to incorporate meteorological dynamics in the models, particularly to allow for real weather in dispersal events. One of the principal challenges ‘ also addressed in the development of user-friendly models – is to be able to run the models sufficiently fast to enable the user to compare a range of ‘what-if’ control scenarios with different levels of uncertainty, allowing rapid presentation of results within real time. Substantial progress has been made through the development and testing of efficient computational methods (to allow finely-resolved computation of multi-county and state-wide spread of disease/ Two approaches have been used, the familiar individual-tree–based models and meta-population models. We have also analyzed the effectiveness of greatly increasing the speed of execution of stochastic, spatially-extended model using dedicated multi-core computers available to the Epidemiology and Modeling Group. The additional computing power has also allowed the incorporation of meteorological dispersal models into the epidemiological toolbox in objective (1) above. These models can be used to track intermediate and long-distance movement of vectors: providing measures of primary introduction of inoculum. Turning to detailed models within plantations, analysis of the effects of host planting age on the transmission of HLB has been completed. We have also completed the analysis of how to allow for removal or treatment of infected trees in a region in which the dispersal and transmission are being estimated for an emerging epidemic. We are continuing to test the risk-based method to show where disease is most likely to spread in order to optimize control that involves removal of more susceptible hosts around key infected sites. Recent work on citrus canker has examined weather-driven variability in the transmission rate of infection by extracting signals from Markov chain Monte Carlo estimates of parameters averaged over different time-scales of one to several months and correlating these with weather variables. The method suggests that it will be possible to use Florida data to estimate the spread of disease under different environmental conditions, for example those typical of Texas or California and to compare the risk of spread under different runs of months/years of favorable or unfavorable weather conditions. Building on a model for HLB within individual plants, we have compared the relative importance of pathogen transmission within the vascular system of the tree and transmission between leaves via psyllid vectors. The model is being used to compare the efficacy of roguing, application of insecticide and the use of nutritional products and thermotherapy for disease control and mitigation.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests We sampled the Bartow experiment in which soil pH was reduced with elemental sulfur. Soil pH was significantly reduced in December 2012, but the treatment had no effect on population density of Paenibacillus sp. or Steinernema diaprepesi (December report). By February-March the bacterial populations in control plots (high pH) were nearly 18-fold greater (P=0.02) than in sulfur treated plots. The S. diaprepesi populations were more than twice as large in sulfur treated plots as in untreated ones (P=0.04). Therefore, we have increased an indigenous entomopathogenic nematode population by modifying the soil chemistry, and a corresponding reduction in levels of a natural enemy of the nematode suggests a mechanism for the response we observed. We observed an additional potential benefit of reducing soil pH at this site, because citrus nematode numbers from December through March were 4-fold greater (P=0.02) in control plots than in plots treated with sulfur. However, there was a slight non-significant trend toward fewer fibrous roots on sulfur treated trees that requires further study. We will continue to evaluate the effect of this treatment quarterly throughout 2013. We are also evaluating the effects of soils from the two treatments at Bartow on the EPNs S. diaprepesi and H. indica independently of the bacterium in controlled studies in the laboratory. The manuscript ‘New citriculture system suppresses native and augmented entomopathogenic nematodes’ Campos-Herrera, R., Pathak, E., El-Borai, F., Schumann, A., Abd-Elgawad, M.M., Duncan, L.W. was accepted for publication in Biological Control. Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. We Initiated a nematicide trial to evaluate the tree response to dagger nematode control in an 8 acre block of 3-year-old Flame grapefruit on Cleopatra mandarin rootstock. Valves were installed variously at north and south ends of 8, double-row beds in order to created 12 untreated plots of 8 trees each, paired with 12 Vydate-treated plots of 8 trees each. Valves were also installed on additional beds to prevent Vydate treatment of trees. Some of these beds were plumbed to receive a dose response trial of an experimental DuPont compound that has shown excellent efficacy against sting nematode, Belonolaimus longicaudatus. The DuPont compound has an added benefit of a much lower mammalian toxicity than does Vydate. Other beds were plumbed to receive treatments to evaluate 2 experimental nematicides being developed by Bayer. The site had very regular and abundant levels of dagger nematodes across all beds and HLB is evident throughout the block. The 3 trials initiated here should provide important information about the effects of these nematodes on HLB affected trees and also generate data concerning the efficacy of urgently needed additional nematicides for use in Florida.
This research seeks to determine whether young trees infected with CLas and displaying typical HLB symptoms can be brought to maturity and produce an economically viable yield. This will be achieved by managing a 60 acre grove of 3-year-old ‘Valencia’ / Kuharske Carrizo trees using a combination of three different foliar and three different ground applied nutritional programs. The trial block is located in Ben Hill Griffin (BHG) commercial groves near Venus, Florida and was planted in December 2008 at a density of 151 trees/acre with tree rows oriented north-south. The grove has been scouted for HLB four times in November 2009, November 2010, April 2011 and August 2011, and 8, 11, 3 and 1258 infected trees were found in each scouting cycle, respectively. These finds represent infection rates of 0.09%, 0.12%, 0.03% and 13.89%, for a cumulative infection level of 14.13%. Psyllid control has been provided with soil drench applications of the systemic neonicotinoid insecticide imidacloprid up to the maximum per acre annual limit. Supplemental foliar applications of insecticides have been made using pyrethoid and organophosphate pesticides. The treatments were initiated in March 2012, before the grant funding cycle began, in order to capture the full growing season. By July 2012 when the project was officially started, the HLB incidence in the block was so widespread that it was impossible to separately identify and sample uninfected trees, so that the separate measurement of “HLB” versus “healthy” for comparison was dropped. Factorial AxB treatments consist of A) ground-applied: 1) Liquid/dry+Ca (BHG standard), 2) Liquid+Ca, 3) Liquid/dry-Ca B) foliar-applied: 1) BHG standard-Ca, 2) BHG standard+Ca, 3) “Prescription”(+Ca). The prescription treatment was designed to be dynamic, customized for optimization, with feedback based on frequent leaf tissue analyses, visual symptoms, and the growth of the tree canopies and yield. There are six replications of treatments, with two being pure replications. Leaf tissue analysis conducted in June 2012 indicated that N was high in all treatments, P and K were optimum, Mg was marginally low, Ca was low in all treatments, and S, Zn, Mn, Fe were all optimum. B was marginally low in all treatments, and Cu was optimum to marginally high in all treatments. We will monitor the tree growth in each plot to compare canopy growth rates, fruit yield and quality. These data will be correlated with leaf and soil analyses in an effort to determine the factors contributing to tree growth and yield in the presence of HLB.
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 three rounds 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 have now regenerated at 2-3 lines for each of the constructs of interest. In order to boost the number of regenerant shoots for micropropagation we developed an alternative transformation/regeneration process for citrus that would generate numerous shoots (10-20) per transformant. The process appears to be working with Citrus plants containing most of the constructs developed to date. We have formalized an evaluation pipeline with collaborators in Florida for regenerant evaluation and attempting to develop larger numbers of lines.
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 work toward the transformation of citrus (objective 3) 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 three rounds of transformation and regeneration of citrus lines with the D. citri-specific gateway vectors using all of the aforementioned constructs in combination with various promoters. We generated several lines containing gateway vectors with the vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter and under constitutive control. Overall, we regenerated at 2-3 lines for each of the gene and promoter combination of interest. Given that the traditional transformation/regeneration process produced few plants for evaluation, we attempted to implement 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 evaluation. This process appears to be working with transgenic Citrus plants from the second and third rounds of transformants/regenerants. We are in process of reaching out to an evaluation pipeline with collaborators in Florida. In summary, we have identified RNAi candidates that appear to negatively impact the lifespan and reproduction of phloem-feeding insects. We have cloned analogues of the candidates from D. citri and have we completed three rounds of transformation and regeneration of citrus lines with the D. citri-specific gateway vectors using several promoters including citrus CsSUS1p. In the absence of an evaluation pipeline for D. citri in NY, we used developed lines to optimize an alternative transformation/regeneration process for citrus that would generate numerous shoots (10-20) per transformant. We are in discussion with collaborators in Florida in regards to participating in an evaluation pipeline.
Soil and leaf samples have been collected in spring (dry) and summer (rainy) seasons for the past two years. Imidacloprid rates have been applied at 1X, 2X and 4X lable rates on transplants (1-2 yrs), moderate age (5-7 yrs), and mature trees (>10 yrs) each season. Bromide has been applied with each application to serve as a tracer for the wetting front. Psylid populations were surveyed weekly following applications. soil and leaf extracts have been analyized using IMD. Water movement has been monitored using soil capacitance sensors with all measurements ranging between field capacity and 40 percent depletion for all irrigation rates. Bromide concentrations fell to zero at all applications by 8 days after application Soil Imidicloprid concentrations for the 1X rate start at less than 2.5 .g per mg soil in the upper 15 cm of soil. Within five days after application, imidaploprid concentrations in the 0-15 cm depth are reduced to less than 0.5 .g per mg soil by tree uptake or movement to deeper depths. Soil Imidacloprid concentrations at 15- 30 an 30-45 cm remain below 0.5 .g per mg soil for the duration of the study. The 2X and 4X applications typically have proportionally greater soil concentrations in the 0-15 cm depth and decrease to less than 1 .g per mg soil. Deeper soil depths typically never increase above 0.5 .g per mg soil. These results would indicate that Imidacloprid moves with the wetting front with low soil residual concentrations. Pysillid populations fell to near zero for approximately 15 days after application for all application rates for all tree ages. The Pysillid populations of the 1X application rate increased to pre-treatment counts within 21 days of application. Pysllid populations in the 2X and 4X applicaitons remained below 0.5 Pysillids per tree to 70 days after application for the transplanted trees, and increased to more than 1 pysllid per tree by 35 days after application for the moderate age trees and 57 days for the mature trees. These data would indicate that higher application rates will be required for trees more than 5 yrs of age. .
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 two rounds 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 have now regenerated at 2-3 lines for each of the constructs of interest, but have to terms with the fact 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 evaluation. Thus far this process appears to be working with transgenic Citrus plants from the second round of transformants/regenerants. We are formalizing an evaluation pipeline with collaborators in Florida, and have completed a third round of transformations and are regenerating additional lines with other transcript/promoter combinations such as a B tubulin subunit from D. citri.
The Unforbidden Fruits project goal is to undercut the demand for smuggled budwood and product by making available disease-tested propagating material for citrus and citrus relatives in demand by ethnic and religious communities in California. Smuggled citrus and citrus relatives present the danger of introducing pests and diseases of quarantine significance. Two major focuses of the project are curry leaf (Bergera koenigii) and bael (Aegle marmelos). We have distributed several hundred seeds of curry leaf and bael to four interested citrus nurseries. Growers are concerned about planting curry leaf or bael next to commercial citrus because it is a prime host of the Asian citrus psyllid (ACP), the vector of Huanglongbing (HLB) disease (Candidatus Liberibacter spp.). Recent ACP discoveries in the San Joaquin Valley have made growers reluctant to plant these citrus relatives. The quickest and most efficient way to propagate curry leaf and bael would be by tissue culture. One nursery has expressed interest in this, but has not yet started the process. The ideal situation for growing curry leaf and bael in California would an insect-protected greenhouse in a non-citrus growing area. Curry and bael trees would grow faster in a climate-controlled environment and with the proper regulatory updates could be shippable, like certified nursery stock, even from an ACP or HLB quarantine zone. The major obstacle is cost. We continue to guide aspiring growers of citron (Citrus medica) ‘ both Orthodox Jews on the East Coast and hands-on farmers in California ‘ to fulfill the applicable phytosanitary and religious laws in starting up citron plantings. In the past three years at least four new plantings have gone into the ground or been planned in California, in Coachella, Valley Center, Orange Cove, and Fresno; there is also one in Yuma, Arizona. All these plantings so far have been established by seed propagation. Trees of ‘Assads’ citron (a never-grafted line brought from Morocco) in the UCR Citrus Variety Collection will fulfill the three-year Orlah requirement on July 1, 2013, at which time trees planted from its seeds will be fully kosher for Jewish ritual use. ‘Morning Song Temoni’, another never-grafted line, will fulfill Orlah in 2015. Seeds from these trees may not meet the standards of the most exigent buyers and commercial growers, but will provide a supply for moderately religious Jews who want ungrafted trees. Woodapple (Limonia acidissima) is a citrus relative whose fruits are much sought and sometimes smuggled into California. It rarely fruits in Riverside, but a tree at the UCR Citrus Variety Collection is bearing fruits this year that will provide seeds for further propagation. Pummelo (Citrus maxima) is perhaps the most important of Citrus species from the epidemiological standpoint. We have requested exotic pummelo germplasm from local sources on multiple occasions, so far without success; we will continue to pursue this objective.
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