Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. These include Polyamines such as putrecine, spermine and spermidine; and Antioxidants such as lipoic acid, glycine betaine and glutathion. Lipoic acid continues to yield the best results. A carrot suspension culture overlay procedure is also being evaluated. Experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency are also underway, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publication from work on alternative transformation systems: Dutt, M. and J.W. Grosser. 2010. An embryogenic suspension cell culture system for Agrobacterium mediated transformation of citrus. Plant Cell Reports. 29(11): 1251-1260. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ A field trial was established in collaboration with Mr. Orie Lee to evaluate sweet orange seedlings from six selected somaclones of precocious ‘Vernia’ sweet orange under commercial conditions. Juvenile Vernia trees are less thorny than other commercial sweet oranges, and our plan is to girdle the trees to induce early flowering and fruiting once the trees reach adequate size. The goal is to quickly establish a producing grove from juvenile budwood – as necessary to have a system for comparable transgenics. Significant progress was also made to identify rootstocks to enhance early production from juvenile scions, including subsequent transgenics. The 2.5 year old field trial using a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks is showing significant rootstock affects on precocious bearing – the best selections from this trial will be tested with juvenile transgenics, based on yield and fruit quality data to be taken in February. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious ‘Vernia’ sweet orange (including somaclones) were regenerated and successfully micrografted for further study of early flowering and transgene expression. 31 transgenic ‘Vernia’ trees were produced containing four different gene constructs. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
After stabilization in the hydroponics system, greenhouse plants have been subjected to the three levels of boron fertilization (high, normal, and deficiency). As the nutrient levels stabilize to the new fertilization treatments we will inoculate the seedlings with HLB and monitor for Las titer and symptom development. Determining the mechanism of this possible HLB symptom escape will provide important information about how Las causes disease and provide a possible strategy for HLB management by citrus growers.
In cloning the three SA genes, EDS1, SID2, and WIN3, we currently confirmed the cloning of the full-length ctEDS1 and are in the process of moving the sequence to the binary vector for plant transformation. We showed in the last progress report that we obtained 3′ end RACE sequence for ctWIN3 and 5′ end RACE sequence for ctSID2. In order to amplify the other ends of the two genes, we tried to design different primers for RT-PCR. We also performed TAIL PCR, in which we used citrus genomic DNA as a template in a series of PCR in order to obtain the missing regions of the two genes. However, these attempts were unsuccessful. With Carrizo sequence database (http://citrus.pw.usda.gov/) recently available, we have been doing bioinformatics analysis and have identified additional SA genes that have citrus homologs with available sequence. We are currently design primers to further amplify these additional SA genes. For ctEDS5/pBINplusARS transformation, we obtained 5 Col-0 and 5 eds5-1 carrying the transgene. We are in the process of screening T0 seeds for additional independent transformants. In the meantime, we planted these 10 transgenic plants for disease resistance assay with Pseudomonas infection. We continue to characterize the transgenic plants overexpressing ctNDR1/pBINplusARS, ctNPR1/pBINplusARS, or ctPAD4/pBINplusARS. We obtained 4 homozygous ndr1 + ctNDR1/pBINplusARS and performed disease resistance assay. The recent data confirmed our earlier report that ctNDR1 complemented Arabidopsis ndr1 mutant. Additional analysis will be conducted to verify this result and to further characterize the defense phenotypes of the transgenic plants. For plants overexpressing ctNPR1/pBINplusARS or ctPAD4/pBINplusARS, we did not observe complementation of npr1 or pad4 mutant with transgenic plants currently obtained. We reason that overexpression of these two genes may be toxic or citrus cDNA clones may not be well expressed in Arabidopsis. We are currently trying to clone the genomic fragments of these two genes. We will repeat npr1 or pad4 complementation once we obtain the genomic clones.
The main objective of the project is to identify all the microorganisms in the phloem cells of HLB affected citrus trees. To this end, we carried out extensive microscopy imaging of vascular tissues from Candidatus Liberibacter asiaticus (Las) infected citrus and periwinkle plants. The following are conclusions from our investigation. 1) Las is the sole type of bacterium morphologically identified in the phloem of Las infected (Las+) citrus plants. Neither mollicutes (bacteria without cell wall) nor flagellated bacterial cells were detected. Occasionally, Citrus tristeza virus was seen in companion cell nuclei in the Las+ phloem of citrus plants. 2) Phage particles were observed in Las+ periwinkle phloem cells. The phage particles were detected only in Las+ periwinkle plants and they appear to lyse Las cells. Based on their structural features, the phage particles seem to be a member of Podoviridae. To the contrary, no phage particles were identified in Las+ citrus phloem cells. 3) In the phloem cells of Las+ infected leaves, excessive amounts of callose accumulate around the plasmodesmata (PD) and sieve pores. 4) The leaf chlorosis symptom of HLB is due to starch over-accumulation in the leaf chloroplasts and callose deposition in the phloem takes place prior to leaf chlorosis. 5) Rate of photoassimilate export from leaves is reduced in Las+ plants. These results provide evidence that Las is responsible for HLB symptoms and that callose formation around PD and sieve pores of Las+ plants could disrupt phloem transport. The low titers of Las and inactivation of symplastic transport in HLB infected trees suggest that the HLB symptoms are self-inflicted by the plant’s response to Las infection rather than caused by direct injury to the phloem by Las cells or by physical occlusion of the phloem by bacterial proliferation. We have submitted a manuscript to Phytopathology reporting conclusions 1, 3, 4, and 5. Conclusion 2 will be published in a manuscript from Dr. Dean Gabriel’s lab.
During the 2nd quarter of funding, the Core Citrus Transformation Facility (CREC) continued it’s mission of producing transgenic Citrus plants according to the orders from multiple clients. The demand for genetically transformed citrus plants remains high. Most recently, CCTF received three new orders to produce transgenic grapefruit carrying genes harbored in following vectors: p19-5; p20-7; and p21-1. The bulk of the work presently revolves around orders placed in the previous quarter but work also goes on to complete older orders. Out of presently serviced orders, all except two are concerned with resistance of different citrus cultivars to diseases, primarily HLB and canker. The following transgenic citrus plants were delivered to various researchers: Resistance to bacterial diseases-canker and HLB: 1) N1* gene: two Duncan plants; 2) pCIT108P3 vector: two Flame plants; 3) NPR1: three Flame plants and superNPR1-four Hamlin plants; 4) AS7 gene: eight Duncan plants and A13* gene: four Duncan plants; 5) pMOG800 vector: two Duncan plants. Resistance to CTV: 1) Gene in p33 vector: 18 Mexican limes, 16 C. macrophylla, and seven Hamlin plants. Orders not associated with citrus disease resistance: 1) CL1 gene: one Duncan. 2) pHK vector: 12 Mexican limes. During this quarter, more than forty recovered new transgenic plants were soil-adapted, and are ready for PCR testing to confirm the presence of the trasngene of interest. Please be informed that the person directly managing the CCTF (and co-PI) is Dr. Vladimir Orbovic.
The objectives of the first year’s research project are focusing on: 1. Conduct genome-wide sequence analysis to identify Simple Sequence Repeat (SSR) loci from genomic sequences of ‘Candidatus Liberibacter asiaticus’ (Las). Design and develop PCR-based multi-locus SSR molecular diagnosis assay. 2. Analyzing Las population structures, assessing the genetic diversity of Las in Florida populations. We assessed Florida Las population and compared it with a global genetic diversity of Las populations using a multi-locus SSR marker system. A total 293 HLB samples used for population analysis were composed of isolates obtained from the US (177 isolates), China (42 isolates), India (35 isolates), Brazil (22 isolates), Cambodia (11 isolates), Vietnam (3 isolates), Taiwan (1 isolate), Thailand (1 isolate), and Japan (1 isolate). This sample population had an abundance of sequence types, consistent with the sampling diversity and the type of marker (SSR) used for this study; a total of 147 sequence types (STs) were identified from the 293-member sample pool (Supplemental Table 1). All of the STs identified within the sample population were restricted to the boundaries of their country of origin; in other words, we found no individual ST in more than one country. Profile frequencies and allele frequencies were calculated for this sample population. Based on allele frequencies of SSR loci among these populations, a genetic similarity matrix that consisted of 71 alleles and 293 samples was generated. The genetic distance analyses were performed using Neil’s coefficient with 1,000 bootstrap and 95% confidential intervals. A pair wise population genetic analysis indicated that among four Las populations, the genetic distance between Florida vs China Las populations is 0.61, while the genetic distance between Florida vs India is 1.35, Florida vs Brazil is 0.96, China vs Brazil is 0.84, India vs Brazil is 0.95 and China vs India is 1.72, respectively. The results indicated that the genetic distance between Florida and China have the closest distance (0.61) as compared with other populations. Analysis of genotypic profiles present amongst an HLB sample population allowed us to make inferences about the relationships between L. asiaticus isolates associated with HLB. From this work, we conclude that at least two major clonal complexes of L. asiaticus are associated with HLB worldwide and that the recent introduction of HLB into the southeastern US probably occurred as a result of at least two separate events. The panel of markers we introduce here will be useful for future population studies of HLB and may also aid in the identification of segments of the L. asiaticus chromosome associated with bacterial virulence. High percentage of commonly-shared allele types detected in both populations suggests that Las populations in Florida and China are more related. This leads to a hypothesis that Las populations in Florida could be possibly derived from China. It is not clear if the introduction was from single or multiple times and/or from multiple sources as well. Our results demonstrate the useful of multi-locus SSR marker system for genetic analysis of Las. Bacterium has very compact genome. Sequencing variations of SSR loci located within or near the coding and/or gene regulatory regions could have profound effect on gene expression and functions. We have mapped SSR loci genome containing genes of interest. Our next step is to examine the functionality of the candidate genes using a standard in vitro heterologous expression system. This type of study will facilitate the link of DNA-based genotyping to phenotyping of Las. We have accomplished objective 1 and 2 during the first year of the project. Research had been summarized and reported in 2009 and 2010 American Phytopathological Society meetings.
We want to obtain a pure culture of Ca. Liberibacter asiaticus (LAS) by first co-culturing the bacteria with insect cells. The strategy consists of primocultures of the bacteria in insect cell cultures used as feeder cells. For this annual report our project objectives are described and discussed below. Objective 1: LAS inoculum. LAS source materials are from infected symptomatic citrus trees from Vietnam. Transmissions from citrus to citrus or from citrus to periwinkles are performed via the insect psyllid Diaphorina citri and subsequent grafting. A maceration method was found to be the most appropriate way to release the bacteria in the insect cell cultures. Antibiotics are used to select for Gram negative bacteria. Objective 2: Primo-cultures. Various insect cell cultures in various culture media were tested. -We didn’t detect any Las in Mamestra (hemocyte, ovarian cells) or Spodoptera (hemocyte cells) cell lines after inoculation. -We detected LAS in two lines of drosophila cell cultures by direct PCR after inoculation. One line lost the detection after 6 subcultures. For the second line, positive PCR signal was obtained up to the 20th subculture, then LAS detection troubles occured due to high insect cell density and we detected the presence of another bacteria (Bradyrhizobium). We started new LAS primo-cultures with much less drosophila cells and got LAS positive cultures for some combinations of cell line/culture medium, still positive after 5 weeks and successive dilutions (confirmed by sequencing). -First attempt to co-culture LAS with an Aedes albopictus insect cell line failed. However, adding an antioxidant and osmoprotectant at the time of inoculation allowed us to get LAS positive Aedes cultures still positive after 9 weeks and successive dilutions (confirmed by sequencing). We are currently adapting conditions of described LAS qPCR detection to quantify LAS in insect cell cultures. We can detect LAS bacteria by qPCR but more efforts are needed to quantify bacteria:insect cell ratio with a multiplexed qPCR approach. Our objectives 3 and 4 are aiming to improve culture conditions to get a higher bacterial titer and to free the co-cultures of insect cells (axenization). Objective 3: Axenization. LAS/Aedes primo-cultures were obtained at a low insect cell concentration. We are progressively diluting the concentration of insect cells through each new passage . Objective 4: Medium optimization. To maintain the bacteria for a longer period of time and to reach higher bacterial concentration, we started complementing the primo-cultures with various sugars, vitamins described in citrus/periwinkle phloem. In parallel we analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to define limiting factors and/or growth inhibitors. Of the complements tested we selected sodium pyruvate, proline and fructose for their positive effect on the bacteria detection and they now are systematically assayed on our new inoculations. We are analyzing sugar, amino-acids and minerals and trace elements variations in insect cell culture media over culture time to identify potential LAS growth limiting factors. We are reaching our milestones for the first year of this project with significant progress on objectives 1, and 2 (inoculums, primo-cultures). For objective 2, we will test new insect cell lines. As at least two laboratories involved in the FCPRAC program established Diaphorina citri cell lines, we will request for these lines to trial our inoculation protocol and try to obtain LAS/ D. citri co-cultures. We are now fully involved in objectives 3 and 4 (axenization/medium improvement). If they succeed in time, we will attempt to inoculate cultivated bacteria through Diaphorina citri to Citrus trees (objective 5).
Physiological changes in HLB fruit: Global gene expression in HLB-infected fruit tissues was evaluated using an Affymetrix array containing 30,279 Citrus spp. ESTs. Flavedo (FF), vascular tissue (VT), and juice vesicle (JV) tissue of symptomatic (SY), asymptomatic (AS) and healthy (H) fruit harvested from sweet orange ‘Hamlin’ and ‘Valencia’ cultivars was investigated. Transcriptional profiles indicated the number of genes impacted by HLB was highest in FF and VT and least in JV. In ‘Hamlin’, over 860 genes were changed in SYVT and SYFF compared with H tissues. In ‘Valencia’, fewer genes were changed in SYVT and SYFF, but approximately 50% additional genes (397 genes) were changed in SYJV compared with ‘Hamlin’. As in SY tissues, ASVT and ASFF were strongly impacted and had similar gene changes in ‘Valencia’. However, only 28 genes were altered in ASJV compared with HJV. The top five gene groups with high numbers of changes included transporters, carbohydrate metabolism, genetic information processes, phytohormone metabolism and defense responses. Highest titer of the HLB bacterium was found specifically located in SYVT. Gene changes in SYVT were associated with host-pathogen interaction and seed abortion, and those in SYJV with fruit size, juice sac maturity and morphology, and juice quality. Similar to SY fruit, girdled fruit were small and could abscise prematurely. Comparison of gene expression between HLB-infected and girdled treatments is ongoing to differentiate HLB-mediated gene changes from those of girdling-related carbohydrate loss. We studied carbohydrate and phytohormone changes in HLB affected fruit to explain symptom development since 1) carbohydrate shortage has been linked to fruit growth arrest and eventually abscission and 2) hormonal signals regulate, at least partially, fruit set and development. SY, AS and H fruit were harvested from ‘Valencia’ trees (Citrus sinensis L. Osbeck) infected (SY and AS) or not (H) with the HLB pathogen, as verified by PCR. Mature SY fruit weighed less, had lower ‘Brix, were smaller, had more aborted seeds, and were greener than AS or H fruit. Starch and sucrose contents were lower in SY mature fruit flavedo compared with H and AS. SY and AS fruit harvested 7 and 12 months after full bloom produced significantly less ethylene than H fruit. Indole-3-acetic acid (IAA) and abscisic acid (ABA) contents in flavedo removed from the stylar-end, middle section, or stem-end of fruit, generally were higher in SY fruit than in AS and H. Flavedo excised from the misshapen area of SY fruit had significantly higher IAA content when compared with the normal-sized area of the same fruit on the opposite side. This increase corresponded to an increase in hypodermal cell area in S fruit flavedo. Overall, these data reveal an imbalance of carbohydrate and hormonal status in fruit from HLB-infected trees and suggest a role of such changes in fruit symptom development. Extension and education: The effects on Huanglongbing on Florida Orange Juice brochure by Danyluk, Spann, Rouseff, Goodrich Schneider, and Sims has been submitted to EDIS for publication and was distributed to all attendees at the 2010 International Citrus and Beverage Conference (ICBC) in Clearwater Beach, Florida. An educational display was also created and displayed at the 2010 ICBC meeting. ICBC is an annual meeting with an average attendance of 350 individuals from the citrus industry.
n FL nurseries, rootstock seed trees are located outdoors and only protected from psyllid transmission of Candidatus Liberibacter asiaticus (Las) by insecticide applications. In 2008, a survey detected two Carrizo citrange trees as HLB+. Given the potential risk for seed transmission and introduction of Las into nurseries by seed from source trees, assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008 seed were collected from mature Pineapple sweet orange trees in Collier Co. and in 2009 from Murcott tangor trees in Hendry Co., FL. For Pineapple orange, 415, 723 and 439 seedlings and for Murcott, 332 seedlings were tested at least twice by qPCR using 16S primers. In 2007, a single Pineapple seedling was suspect HLB+ but upon repeated testing was negative. From nurseries in 2008, 290 seedlings were recovered from fruit located on symptomatic branches of 2 Carrizo trees, and in 2009, 125 seedlings were recovered from 2 trees of Swingle citrumelo, 649 from 4 trees of ‘Kuharske’ Carrizo, 100 from 1 tree of Cleopatra mandarin and 100 from 1 tree of Sekwasha mandarin. In 2008, one suspect HLB+ Carrizo seedling was detected but HLB+ status was not confirmed after repeated testing. In 2009, a single questionable PCR detection for Cleopatra mandarin was obtained. Subsequent detection occurred in only 75 and 33% of repeated 16S runs from two DNA extractions and the assay was negative using beta-operon primers. Despite the occasional HLB+ test results, no plants have ever developed HLB symptoms and repeated testing has never confirmed anything other than the transient presence of Las in seedlings grown from seed obtained from Las-infected trees.
Objective 1 (confirm biofilm formation by X. citri subsp. citri in comparison with other bacteria that are well known to form biofilms). Using stable and labile Gfp expressing strains, aggregates (biofilms) were confirmed to be formed by viable cells. Xcc-A is the wide host range strain attacking most citrus cultivars, whereas Xcc-Aw is restricted to Mexican lime and Xac to Swingle citrumelo. The three strains aggregated on the leaf surface of Swingle citrumelo and Mexican lime. However, higher aggregation on the leaf surface was observed for Xcc-A than for Aw or Xac leaf. In addition, higher aggregation by Xcc-A developed on Mexican lime than on Swingle citrumelo. Xcc-A formed large complex aggregates on these hosts whereas Aw or Xac formed filaments. A similar relationship among strains and hosts was observed for biofilm formation on fruit. Xcc-A strain was able to aggregate and form biofilm on the surface of both Mexican lime and grapefruit, but Aw produced biofilm only on Mexican lime and none on grapefruit. Xac did not form biofilm on either grapefruit or Mexican lime. Based on differential aggregation among these strains on compatible and incompatible hosts, biofilm formation on the plant surface may play a role in the disease causation process. What role biofilm formation has in the infection process is still unknown. However, we hypothesize that Xcc-A strains may be more virulent in part because they are a better colonizer of compatible hosts than less virulent Aw and Xac. gumD is involved in biofilm formation. A gumD transcript of 445 bp is only amplified from active cells, therefore its presence may be used as a viability marker. gumD transcript in Xcc-A was higher in cells 72 h after biofilm formation (aggregated state) than in cells in suspension (planktonic state) signifying higher cell activity. rpf gene is expressed during quorum sensing in the biofilm formation process. rpf is involved in synthesis of a DSF (diffusible signal factor) related to quorum sensing and is expressed at a higher level in Xcc-A during biofilm formation. Other studies are currently in progress using genes expressed during chemotaxis (cheZ, motA, fleN), and cellular division (Rsp, SulA) to evaluate their role in the Xcc-A colonization process. Objective 2. A wide host strain of Xcc-A with stable and labile Gfp was grown in the presence of different concentrations of the bactericidal compounds, CuSO4, NaCl and SOPP. The treated bacteria were then sprayed on Swingle citrumelo leaves and biofilm formation on the surface observed at different days after inoculation (dpi). At 1 dpi, aggregates were detected in all the treatments but a larger area of aggregation occurred after Xcc-A was exposed to NaCl. After SOPP exposure, aggregates were restricted to certain locations on the leaf surface proximal to stomata and in depressions. At 3 dpi, NaCl treatment showed extensive and thick biofilm formation while, in the case of SOPP, living bacteria were mainly found inside in the sub-stomatal chamber and in the apoplast. At 9 dpi SOPP biofilm on the leaf surface was almost non-existent although a substantial bacterial population was present in the sub-stomatal chamber and apoplast. In summary all bactericides do not reduce the bacteria’s ability to form biofilms i.e. neither NaCl nor SOPP prevented bacterial infection.
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. Objective 1 of our proposal intended to evaluate candidate genes for dsRNA-induced lethality of Diaphorina citri and our model organism, M yzus persicae, using artificial 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. While our results suggest that the M. persicae-specific dsRNA has a negative effect on both the lifespan of the insects and the number of offspring generated, additional independent assays are needed to enhance treatment replication to better resolve statistically significant differences among dsRNA treatments. Since the annual report, we have nearly completed our evaluation of the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, this approach may prove to be critical to the success of this study. This research requires the use of Gateway-based vectors that express the selected insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We have cloned and characterized two 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. Deletion analyses have shown that two separate phloem-specific enhancer regions (nucleotides -1153 to -462, and -410 to -268) exist in CsSUS1-1. Conversely, only the latter enhancer region promotes phloem-specific expression in CsSUS1-2, while the former region drives constitutive expression. We have also successfully generated Gateway vectors that will result in the 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. We have also commenced objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Initially, 3′ rapid amplification of cDNA ends was utilized to decipher additional nucleotide sequence from vacuolar ATP synthase subunit G, S4e and .-tubulin transcripts from D. citri. We are currently in the process of inserting sequences of the aforementioned transcripts into Gateway vectors downstream of both the constitutive 35S and phloem-specific CsSUS1 promoters. The resulting D. citri-specific gateway vectors will subsequently be transformed into citrus for subsequent feeding assays with D. citri. Initial attempts suggest that transformed Citrus sinensis cvs. Valencia and Hamlin can be regenerated. 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 developed new Gateway-derived vectors bearing a native citrus phloem-specific promoter, for use in RNAi and are evaluating them in planta (objective 2). Finally, we are in the process of generating similar vectors specifically designed against D. citri (objective 3), and preparing use them with our developing citrus transformation infrastructure.
The objectives of this proposal are to address some strict regulatory needs concerned with reducing the spread of citrus psyllid and greening. We propose to develop a citrus psyllid trap that will capture adult psyllids at very low population levels in an urban retail environment (and in other venues as applicable) and preserve them such that the citrus greening bacteria can be extracted from the psyllids to determine the psyllid infection rate at a later date; and to do so in a secure manner. This type of trap has never been developed for any insect to our knowledge and requires an indepth understanding of the behavior of the target insect. As a result we have executed a number of laboratory and field experiments to determine the specific behaviors exhibited by psyllids to a broad range of visual and odor cues. These have been broken down into fine grain and very specific behavioral responses and investigated in simple choice comparisons. the second year of the proposal we will complete the development of the trap though continued refinement of its parameters, complete and test several prototypes, and modify a final prototype that will be tested for its efficiency under the regulatory conditions for which the trap is to be used. This will include evaluation of the usefulness of the trap and comparison of various preservatives to enable determination at various post capture intervals of vector infection rates. We have made excellent progress toward our stated objectives through a combination of laboratory and field experiments looking at the behavior of Asian citrus psyllids. We have determined the following: 1) The 2-sided flat yellow trap used as the standard psyllid monitoring and detection tool is extremely inefficient and will not serve in any manner as the basis for meeting our objective, therefore, it was necessary to start from scratch to develop a trap. 2) In laboratory bioassays, we have determined that psyllids are highly attracted to yellow and orange objects and yellow and green LED-produced light; psyllids will orient visually to and follow dark lines, raised ridges and shadowed areas (apparently similar to their response to leaf edges). This aspect of their behavior can be exploited to direct their movement on a surface. Psyllids will respond readily to sunlight in an effort to escape confinement. Psyllids cannot walk without falling from a surface covered with fluon (liquid teflon used to confine flightless insects inside open containers). 3) In field bioassays we have determined which configurations (trap sizes and shapes) of various parameters have promise to serve as the most efficient and attractive visual cues. We also have tested the trap attraction angle or degree of slant and horizontal and vertical orientation of traps to optimize trap efficiency once Asian citrus psyllids arrive on the trap’s attracting surface. These remain to be optimized under target conditions along with development of the security portion of the trap to prevent tampering by the public. Testing of semiochemicals reported to be attractive to psyllids by other researchers were tested at various concentrations in the lab alone or in combination with lights and traps. The results were inconclusive but promising and maybe an important incremental component of the final trap since it will not rely on stickem to capture the psyllids. Various procedures for psyllid collection, shipping and processing were evaluated during the year. In preliminary experiments, over 1000 psyllids from a model system (Bactericera cockerelli from tomato carrying Candidatus Liberibacter psyllaurous) were used. Over 2000 single psyllid extractions of Asian citrus psyllids were conducted using 25 different extraction methods for finding out the best method for detection of Liberibacter in Asian citrus psyllids. An efficient high throughput method of extraction has been developed. This method will be used for comparing different collection reagents in the second year.
1) Barriers with screens insecticides: Areas already defined (there are three areas in Descalvado, Bebedouro, and Botucatu). The support structure is already ready (40 rods of iron). The implementation and early evaluation is planned for week of 25/10/10 days. 2) Evaluation of the impact of treatment of plants with systemic insecticides on the transmission of Ca. L. asiaticus by starved psyllids, will be started in November, 2010. 3) translocation insecticides: only starts when we get the amount to purchase stylectomy equipment.
This report covers the period July 1, 2010 through September 30, 2010. This project was funded July 1, 2009. This program now operates on a no-cost extension. Two general coordinating meetings were held during this period. One presentation to the Indian River Citrus League was made during this period. The number of sites being monitored was: Indian River 265, St. Lucie 315, and Martin 150. This is the same as the last reporting period. 1. There were 3,445 trees surveyed in Indian River County, 4,095 in St.Lucie County, and 1,950 in Martin County. 2. There were 1,873 psyllids coaught in Indian River County, 542 in St. Lucie County and 400 in Martin County for a total of 2,815 psyllids caught. Intensive sampling for the aerial spray program began on Sept. 2, 2010. This pre application sampling ended on Sept 10. An application of 1 quart of malathion per acre occurred from Sept. 11 through Sept. 18. The post application sampling began on Sept. 23. The data from these activities will be detailed in the next report. Participants in this program intensively reviewed the data collected to date. Based on that review and after consultation with key members of the Indian River community, an areawide application is being coordinated for the last two weeks in January 2011. This application and associated monitoring will be the last field activity of this project.
Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica were subjected to psyllid inoculation using the psyllid, Diaphorina citri. As reported all of these hosts were new ones for the psyllid and in transmission experiments the psyllid did survive and in some instances layed eggs on these hosts. To date only Choisya ternata again was found positive for Liberibacter asiaticus. Similar transmission tests in quarantine using dodder to infect Choisya ternata and Choisya arizonica with L. africanus but no positive results have been found. Graft inoculation tests with these hosts also showed that they were graft incompatible with citrus. As previously reported Severinia buxifolia has been determined to be a host for the L. asiaticus and transmission to citrus was obtained. Some difficulty in getting repeated positive results have recently occurred. This is under study. It may be similar to results found with Murraya paniculata. Primers to a different region have been produced and will be used to verify results. A new primer set and probe to detect all Liberibacter species including the potato bacterium was developed and successfully tested. Further tests are underway by various labs to confirm. Work with IAPAR73 is ongoing using psyllid transmission tests rather than grafting to determine if this cultivar is tolerant or resistant to L. asiaticus. Numerous propagations of this cultivar have been made and are ready for testing against L. americanus and L. africanus.
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