Recently we identified several sulfur chemicals from guava that repel Asian citrus psyllid (ACP) in the laboratory, but are difficult to formulate into controlled release devices for field use because of their high volatility. As we continue to work on formulating these sulfur compounds into devices that will have practical application, we have also investigated several potential “of-the-shelf” essential oils for their repellency against ACP. These were chosen based on their known repellency to many insects and based on their perceived similarity to guava in chemistry. Also, we have found that volatiles from essential oils of coriander, lavender, rose, thyme, tea tree oil and 2-undecanone, a major constituent of rue oil repelled ACP adults compared with clean air. Also, coriander, lavender, rose and thyme oil inhibited the response of ACP when co-presented with citrus leaves. Volatiles from eugenol, eucalyptol, carvacrol, .-caryophyllene, .-pinene, .-gurjunene and linalool did not repel ACP adults compared with clean air. Chemical analysis of the headspace components of coriander and lavender oil by gas chromatography-mass spectrometry revealed that .-pinene and linalool were the primary volatiles present in coriander oil while linalool and linalyl acetate were the primary volatiles present in lavender oil. Coriander, lavender and garlic chive oils were also highly toxic to ACP when evaluated as contact action insecticides using a topical application technique. The LC50 values for these 3 oils ranged between 0.16 to 0.25 ‘g/ACP adult while LC50 values for rose and thyme oil ranged between 2.45 to 17.26 ‘g/insect. Our current efforts are focusing on quantifying the airborne concentrations of these essential oils found to have behavioral activity against ACP that are required to induce the effect. Our current results suggest that garlic chive, lavender, and coriander essential oils should be further investigated as possible repellents or insecticides against ACP. Also, these repellents may be useful in organic citrus production, which currently has few available tools for management of ACP. We have also developed a method with which to sample and quantify the airborne concentrations of sulfur violates directly in the field. This has allowed us to precisely measure the concentrations of repellent chemicals needed in the field to affect psyllid behavior which is helping guide development of practical release devices. Our field results with DMDS released from SPLAT in 2010 were mixed. While some trials with the initially developed formulation appeared to show reductions of ACP populations, others did not. However, we have now completed analysis and processing of data from an investigation of four new (advanced) SPLAT formulations of DMDS that were designed for longer field longevity. Two of these four formulations produced very good results, suppressing psyllid populations better than the previous formulations and longer than was previously achieved. These two formulations lasted longer than four weeks, but their full potential could not be investigated because the trial was unfortunately interrupted by a pesticide application. We plan to further investigate these two formulations in 2011 and hope to replicate these results.
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. ‘ A protocol for the accelerated production of transgenic plants has been published:Dutt M., Vasconcellos M., Grosser J.W. (2011) Effects of antioxidants on Agrobacterium-mediated transformation and accelerated production of transgenic plants of Mexican lime (Citrus aurantifolia Swingle). Plant Cell, Tissue and Organ Culture 107:79-89. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Seeds of precocious rootstocks (based on data from the St. Helena project) were harvested and planted for subsequent budding with transgenic precocious sweet oranges (Vernia and OLL series). Plans are underway to build a PVC-pipe scaffolding structure/rapid evaluation system (RES) in our transgenic greenhouse, similar to our successful RES in the field. This will allow horticultural manipulation of the precocious transgenic germplasm to demonstrate the reduced juvenility. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL and Vernia sweet oranges were successfully micrografted to Carrizo citrange or experimental Tetrazyg rootstocks and are growing well in the greenhouse. Clonal propagation of these transgenic oranges onto the available liners of the precocious rootstocks mentioned above is underway. Transformation with early-flowering genes: ‘Duncan grapefruit plants transgenic for the poplar ft1 gene have been produced and are being tested for precocious and/or induced flowering using 2 different promoters. More experiments with the citrus ft constructs are also underway. Progeny plants of transgenic tobacco are being assayed for phenotype and transgene segregation.’ 122 transgenic Carrizo trees were generated following a co-transformation experiment using two vectors. The first containing 35S-cft1 and the second containing AtSUC2′ gus. The objective is to rapidly evaluate transgene expression in the fruit. PCR analysis revealed that 16 lines contained both cassettes. Plants have not flowered 12 months after transformation. Plants are currently being evaluated in an unheated greenhouse for cold stress in order to hopefully initiate early flowering in spring 2012.
Over the past quarter, we have made progress in the following areas: 1. We have made progress with our Nicotiana benthamiana – GUS test system to examine effector specificity for induction. We have developed improved vectors and used these to demonstrate specific promoter activation by three distinct TAL effectors thus far. We have also tested a RACE method to map which UPT boxes in our test promoters are used. This method is still in development. 2. Novel TAL effectors from additional citrus canker strains have been isolated, and sequence analysis has been initiated. 3. Transformation of Duncan grapefruit has continued. At present, we have over 330 new candidate stable transgenic lines in soil, with six different promoter-gene constructs. We are systematically characterizing these on a molecular basis to confirm presence of the specific genetic elements transformed. Pathogen testing will begin shortly, and we will seek to identify the best performing transgenic lines. New Fall seeds will be available soon for additional transformations. 4. Transformed lines of sweet orange and Ruby Red grapefruit are at the rooting stage.
Over the past quarter, we have made progress in the following areas: 1. We have analyzed the contributions of individual PthA proteins by knocking out specific pthA genes from X. citri strain 306 from Brazil and testing them with GUS reporter gene fused to our super promoter Bs3 construct containing binding sites for 17 X. citri TAL effectors. Strains in which the genes pthA 1 and 2 were disrupted activated the reporter gene at levels nearly comparable to the wild-type strain. These results suggest that there is little significant contribution of these effectors to gene regulation. Strains disrupted for (i) pthA 2 and 3 showed a 25% reduction in GUS activity. (ii) pthA 1 and 3 showed a 50% reduction, pthA 1,2 and 3 showed a 60% reduction, and deletion of all four pthA genes showed a 98% reduction in GUS activity, similar to a type three secretion deficient strain, 306.hrpG. This results suggest that pthA4 is the principle effector in the activation of gene expression, with additional smaller contributions from the other three TALEs. These results confirm that combinations of UPT boxes allows triggering of engineered resistance promoters by more than one TALE which should reduce pressure on individual TALEs to evolve to evade detection. 2. A robust transformation system using either epicotyls or cotyledons has produced a large pipeline of transformed plant material. More than 800 Duncan grapefruit lines have been transformed, made shoots, and roots, and been transferred to soil, with six different promoter-gene constructs. 153 Ruby Red grapefruit transformants and 63 pineapple sweet orange transformants are now in soil. It is slow work, and further attrition occurs following molecular characterization to identify the desired lines. 3. Once plants are transferred to 4 inch pots and reach adequate size, the final line selection process can take place by pathogen testing. We have now begun analyzing new stably transformed Duncan plants containing the super promoter:resistance construct using the avrGf1 gene. Two candidate lines showed reduced pustule formation by pin prick inoculation, relative to a non-transformed control line, and a hypersensitive reaction to infiltration inoculation, compared to a water-soaked lesion in the susceptible control. These reactions suggest that the test construct is successfully conferring canker resistance in these stable lines. We are continuing to study these plant lines and additional stable lines that are reaching adequate size for pathogen testing. Lines identified from this analysis will be candidates for grafting and filed testing. These results support our hypothesis that a resistance construct based on a promoter containing multiple citrus TALE binding sites can confer transcriptional activation and disease resistance to canker strains.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, we have generated 20 transgenic lines of Duncan grapefruit, and the transgenic plants are currently under canker resistance test. After canker resistance test, we will identify transgenic lines overexpressing AtMKK7, then chose 4 to 6 lines that highly express the transgene AtMKK7 for propagation. Six plants from each line will be used for greening resistance test. For objective 2, we are repeating the screen with gamma ray-irradiated Ray Ruby grapefruit seeds. Another two quarts of seeds have been treated with gamma-ray irradiation. All seeds were irradiated at 50 Gy, as we previously found that this dose will not significantly decrease the germination rate of the seeds. Both untreated and irradiated seeds were plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots formed on the seeds previously plated were transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Shoots formed on these gamma irradiated seeds will be screened again on the selective medium. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test.
The objectives of this project include: (1) Characterization of the transgenic citrus plants for resistance to canker and greening; (2) Examination of changes in host gene expression in the NPR1 overexpression lines in response to canker or greening inoculations; (3) Examination of changes of hormones in the NPR1 overexpression lines in response to canker or greening inoculations; (4) Overexpression of AtNPR1 and CtNPR1 in citrus by using a phloem-specific promoter. We have transformed the cloned CtNPR1 (also named CtNH1) into the susceptible citrus cultivar ‘Duncan’ grapefruit. After survey on transgene expression, we now focus on the three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts. The three lines were inoculated with Xac306. They all developed significantly less severe canker symptoms as compared with the ‘Duncan’ grapefruit plants. To confirm resistance, we carried out growth curve analysis. Consistent with the lesion development data, as early as 7 days after inoculation (DAI), there is a differential Xac population in the infiltrated leaves between CtNH1-1 and ‘Duncan’ grapefruit. At 19 DAI, the level of Xac in CtNH1-1 plants is 104 fold lower than that in ‘Duncan’ grapefruit. These results indicate that overexpression of CtNH1 results in a high level of resistance to citrus canker. We have propagated the CtNH1 line by grafting. We are in the process of inoculating the CtNH1 lines with Candidatus Liberibacter asiaticus (Las). We have completed the SUC2::CtNH1 construct, in which CtNH1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. The construct were transformed into ‘Duncan’ grapefruit. Five transgenic lines have been obtained.
Funds for this project have not yet been received by Dr. McNellis. Penn State has assigned a fund number, but the Office of Sponsored Programs has not yet finalized a budget for the funds. Once funds are received, the development of the NodT antibody will be initiated immediately.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Discussions have been ongoing with Eliezer Louzada of Texas A&M to plant his transgenics wihc have altered Ca metabolism to target canker, HLB and other diseases. Jude Grosser will be planting ~250 additional trees on the test site next week. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants will be monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding.
One of the most recent challenges with deploying DMDS in the ISCA release device called SPLAT (specialized pheromone lure application technology) has been the phytotoxicity of the treatment to leaves and branches. A substantial amount of the DMDS active ingredient is needed in order to affect psyllid population densities. Unfortunately, we have found the DMDS active ingredient burns tree foliage and can even kill entire small tree branches, if applied directly to the wood surface. Therefore, we have been developing alternative release devices that would allow deploying the DMDS active ingredient within trees without touching tree surfaces. Our most recent prototype is a sachet that is hung in trees with a wire hanger. The sachet contains the SPLAT-DMDS, which is allowed to evaporate through a porous membrane. Therefore, the DMDS dispenser is deployed in trees; however, the active ingredient does not come in direct contact with tree branches or leaves. We are currently investigating whether these devices will be effective. The experiment was initiated in September and we are still analyzing data. The results of an earlier experiment conducted in August with a newer formulation of SPLAT-DMDS have been analyzed. The results of this test did not replicate the success we observed with the same formulation last fall. Psyllid populations were not significantly reduced by deployment of this formulation in this mid-summer test (as compared with control plots)as was observed last fall. We are unsure why we were unable to replicate the earlier success. We are trying to determine if the inconsistent results are because of differences in psyllid population densities between these tests or differences in environmental conditions when the different tests were conducted. It is possible that the formulation is not holding up to the intense temperatures and rainfall experienced in the summer as compared with cooler and dryer conditions in the fall. Finally, we have initiated testing of three new formulations of SPLAT that contain repellents other than DMDS that have proven effective against psyllids in laboratory tests. These experiments have only recently been initiated and we should have initial results in 3-4 months.
We have been investigating the mechanisms underlying why Asian citrus psyllid (ACP). We have found that ACP are attracted to common volatiles released by citrus such as .-ocimene and D-limonene, implicating these as general host selection cues. Both .-ocimene and D-limonene are predominant citrus volatiles released by citrus flush, foliage and fruits. However, the release of methyl salicylate (MeSA) was increased (presumably induced) in Las-infected plants as compared with uninfected plants. Correspondingly, this induced compound attracted ACP, suggesting that it may be a chemical cue that facilitates spread of pathogen. Insect herbivory can change the volatile profile of host plants. Insects that use a piercing/sucking mode of feeding have long-lasting interactions with plants cells and/or phloem. It may not be surprising that plant responses to phloem-feeding are distinct from that of chewing insects and that phloem-feeders often induce salicylate-dependent defense pathways commonly activated by bacterial, fungal, and viral pathogens. Such salicylate-dependent associations are implicated in the current investigation given detection of MeSA release from plants infected with the HLB causal pathogen. We hypothesized that psyllids (phloem-feeding insects) could also induce a plant response similar to that caused by the pathogen. Therefore, we investigated whether release of MeSA may be induced by ACP feeding and also used by psyllids as a cue for locating established conspecifics. Volatile collections from psyllid-infested plants detected induced release of MeSA, suggesting a coincidental convergence on a single cue that may simultaneously benefit the pathogen by deceptively attracting its vector, which also uses this cue to locate conspecifics. Movement of psyllids from infected to uninfected plants after initial selection of infected plants suggests that their initial response to olfactory cues may not be directly linked to the most beneficial host plant. Relatively more ACP responded to Las-infected plants under light than dark conditions. This suggests that visual cues may have also played role in attracting ACP to diseased plants. Yellowing of leaves is a typical initial symptom of Las infection on citrus plants. Furthermore, ACP adults are strongly attracted to yellow color. Las-infected plants were initially more attractive to ACP adults than uninfected plants; however, psyllids dispersed subsequently to uninfected plants to make them their final location of settling rather than diseased plants. The mechanisms underlying ACP host acceptance are currently under investigation. Specifically, we are trying to understand why leave infected plants after they initial prefer infected plants over uninfected plants. The duration of initial setting on infected plants was sufficient for ACP to acquire the Las pathogen. Thus, the pathogen is modifying the behavior of the vector by inducing changes in the attractiveness of the host plant through both olfactory and visual cues. This scenario suggests a mechanism for spread of the pathogen in the field because initial attraction and feeding of ACP on diseased host plants should facilitate acquisition of the pathogen while subsequent movement to uninfected plants should facilitate inoculation of uninfected plants. Overall, this behavioral manipulation of the vector by the action of the pathogen on the plant appears to favor spread of pathogen-induced disease. Our results indicate that MeSA may be the specific chemical cue mediating initial psyllid attraction to Las-infected plants. At low dosages, synthetic MeSA is an ACP attractant and at high dosages it is a repellent. We are investigating whether this can be used for possible management applications for ACP. To determine why ACP leave infected plants after after acquiring the HLB bacterium, we are more analyzing psyllid feeding preference between infected and uninfected plants as it relates to nutritional imbalances due to HLB infection.
In order to examine how Candidatus Liberibacter asiaticus (Las) infection affects gene expression in citrus tissues, transcriptional response of leaf, stem and root tissues of Valencia sweet orange (Citrus sinensis) to Las infection was compared using Affymetrix microarray. The analysis revealed that Las reprograms several metabolic and cellular processes in citrus, and identified genes whose expression is regulated in a tissue-specific manner. Tissue specific regulation was observed for several genes and gene groups including those encoding cell wall pectins, transcriptional factors with MADS box and PHOR1 domains, G-proteins, legume-lectin family proteins, pectatelyases, SUT4, SUC6, BAP12, protein kinase THESEUS1 and vacuolar invertase, which were regulated only in leaves; major intrinsic protein family proteins, crinkly 4-like protein, SUT1, ERF5, CPRD2, CNGC1, CSLD4, and FERONIA regulated in stems; and NAT12, GLR4, DDM1, SCL14, APS kinase, resistance protein RGC2, CCR4-associated factor 1-related protein and Arabidopsis response regulator 1 regulated in roots. In another study, host response of Rangpur lime (Citrus . limonia Osbeck) which shows tolerance to the bacteria, to Las infection, was examined using suppression subtractive hybridization (SSH). Genes encoding senescence-associated protein, dehydroascorbate reductase, stress enhanced protein 1, protein kinases, miraculin-like protein 2, actin related protein 2/3 complex, checkpoint-like protein CHK1, ACC oxidase, type I proteinase inhibitor-like protein, xyloglucan endotransglycosylase, protease inhibitor, and PR10-related Cas s 1 pollen allergen were up-regulated, while genes encoding photosystem II subunit R, DNA binding protein, a putative zinc finger protein, SLL1 protein, CCR4-associated factor, metallothionein-like protein, cysteine proteinase, cinnamyl alcohol dehydrogenase-like protein were down-regulated. Many of these genes were unaffected in the susceptible C. sinensis, suggesting genotype dependent response to Las. Sequence analysis and comparison of the expression of a set of pathogenesis-related (PR) proteins is being done using quantitative real-time reverse transcription PCR. To examine the molecular response of citrus to infection by Las under field conditions, Affymetrix microarray analysis was repeated using samples collected from Valencia sweet orange in citrus grove. The analysis is currently undergoing to compare the microarray results from the greenhouse, and the citrus grove (two different times). This research has significantly advanced our understanding of how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Importantly, the manipulation of host response especially plant defense response by Las provides valuable information regarding how to manage HLB.
Using Liberibacter from the alimentary canals of psyllids as inoculum, we have tested over 560 media formulations for growth of liberibacter. Some formulations support prolonged survival of Liberibacter in culture. After 2-3 weeks in culture, masses of Liberibacter have been observed in biofilms on the psyllid alimentary canals used as a source of inoculum and streaming from the canals into the media. We will to continue testing different formulations of the media to determine if they will support planktonic growth of Liberibacter. Information obtained from the genome sequence of Liberibacter and by comparing that genome with the genome of the closely related Babaco bacterium from culture is being used to devise new media formulations. Other possible growth factors, such as diffusible signal factors produced by the psyllids, are also being evaluated.
1. Data mining for ACP continues for annotated transcriptomes (phase I) of the whole adult and gut ACP to identify candidate genes for functional analysis, with an emphasis targets for siRNA-mediated silencing. These data have been used to design RNAi solutions for bioassay. 2. Illumina sequence data for ACP ESTs (from PAVE) was analyzed based on R-stat and normalized expression levels. Pairwise comparisons were done for the Adult,Nymph and infected adult and infected nymph libraries, revealing a number of genes differentially expressed in the four pairwise comparisons, based on transcript copy number. Not all genes that are differentially expressed in the Adult and Nymph were exclusively expressed in either one. The varying range of differential expression suggests that there are well defined developmental cues. Expression of genes in all pairs and the fold change ranges from very obvious to very subtle differences. 3. Potato psylllid ESTs.The Six adult psyllid libraries (adults, dissected salivary glands and alimentary canal) were used to construct mRNA enriched from total RNA extracts (Trizol). The paired Illumina libraries were prepared for sequencing, subjected to DNA sequencing, and are currently being assembled by NCGR. DNA sequences will be organized in PAVE, our data management system that constructs a database and has tools for mining transcripts, and will allow for subsequent RNASeq quantification (Soderlund). 4. FISH probe localization was highly successful for Liberibacter solanacearum localization in dissected guts using the 16S rRNA, outer membrane protein, and abc transporter genes probes when fixation conditions were modified and the new fluorescent camera was employed. Testing with the 16S rRNA for Carsonella ruddii (primary endosymbiont, potato psyllid) as an internal control, are underway to demonstrate specificity. Optimization is underway using the 16S rRNA probe for salivary glands and the oral box. FISH results for whole psyllids indicated decolorization will be essential and that this may confound the technique’s utility for intact adult psyllids, however, parameters are being tested in an attempt to overcome this limitation. 5. Feeding studies are underway to optimize an artificial feeding assay for potato psyllid using a system developed for whitefly. Psyllids were found to remain alive on sucrose feeding solutions tested for 3-7 days.
1. mtCOI haplotyping. More samples are being received for mtCOI haplotyping from the R. Lee lab (USDA, CA) to explore baseline diversities in different locations, in an attempt to relate populations from the US and elsewhere in the Western Hemisphere to haplotypes from a primary region of endemism. All sequences in hand have been edited. 2. Time-course and transmission studies continue bioassay to assess transmission frequency in relation to Rt-PCR detection of Ca. Liberibacter solanacearum (AZ) and in Ca. L. asiaticus (FL) in individual psyllids reared on infected plant material. Results using dot blot hybridization were found to be less sensitive than RT-PCR but were generally comparable with a few exceptions; nonetheless we will continue the work using qPCR even though it is the more expensive method, results are robust. Five psyllids reared on infected tomato, and given a range of inoculation access feeds on tomato seedlings from 30 min, and 1,2,4,8,12,and 24 hr transmitted Ca. L. solanacearum 5-20%, 35, 25-30,70, 80, 90, and 95% of the time (20 plants per rep). Plants were assayed using RT-PCR and were scored for symptom development. Only the former data were used to calculate transmission frequencies because psyllid feeding causes aberrations in symptom phenotype that disallow Liberibacter-incitedsymptoms to be differentiated consistently. This above result was corroborated in two replicated studies and a third replicate is underway. Results suggest that once acquired psyllids transmit at relatively high frequency. The goal is to select individuals for light microscopy, and SEM-TEM ultrastructural observations to pin point localization following a range of different AAP and IAPs. We initiated two AAP time points, starting with 8 and 24 hours using a 24 hr IAP. IAP studies with ACP have proven too difficult owing to the apparent uneven distribution of bacteria in plants, which confounds reliable detection and transmission bioassay to further the localization effort. The goal to produce time course feeding populations for both ACP and potato psyllid is overly ambitious given the problems experienced obtaining ACPs with consistent Liberibacter titer, despite season or host. Even so, extrapolations from the more tractable potato psyllid study system, (an approach approved by the project Director) will clearly help clarify AAP and IAP time-points in relation to organs that are key to the transmission pathway. 3. As noted we are using the potato psyllid as a surrogate for the Asian citrus psyllid since they are closely related, both have digestive systems so similar that we can’t find any differences using the methods and instruments available to us (Cicero et al 2010), and, thirdly, the potato psyllid is easy to rear on tomato in our Arizona laboratory. Thanks to timely publications (Cicero et al 2010, Ammar et al 2011a, b), collaborative efforts, and FPRAC quarterly/annual reporting by the teams involved, the general motif for the transmission cycle of Ca. Liberibacter has been worked out in the potato psyllid. We are in publication mode now. Further, we are now developing the toolbox to answer more detailed questions, such as the infra-instar changes in bacterial titers, and the role of the pharate instar in the movement of bacteria from the midgut, where the anatomically operational beginning of the cycle occurs, to the stylets, where the cycle finishes. We are also moving to apply the methods, used for the above breakthrough, on the Asian citrus psyllid as a check to be sure no significant differences occur in that species.
In this fourth quarter, 208 plants of Valencia orange on Kuharske Carrizo rootstocks, inoculated with HLB infected buds on the 3rd week of April of 2011 using a standard inverted ‘T’ budding technique as describe by Ferguson (2007), were individually tested for HLB infection monthly, starting on July 11, 2011. Tree leaves per each inoculated tree were harvested to verify HLB infection by quantitative real-time PCR (qRT-PCR). Petioles and midribs of the citrus leaf were used (100 mg per sample) to extract total DNA using ‘Qiagen DNeasy Plant Mini Kit’. After DNA isolation the yield and purity of the DNA was estimated by measuring OD260 and OD260/280, respectively, with a NanoDrop Spectrophotometer ND-1000. The qRT-PCR for Candidatus.Liberibacter species was performed using 16S rDNA primer and probe sets as described by Li et al.(2006). The qRT-PCR consisted of 2 min incubation at 50 ‘C followed by 10 min incubation at 95 ‘C and 40 cycles at 95 ‘C for 15 s and 60 ‘C for 1.0 min. qRT-PCR data were analyzed using the Applied Biosystems software Version 1.4.0. The qRT-PCR results from July, August and September showed HLB infection on 0, 6, and 11 inoculated trees, respectively. Based on the low number of tree that acquired HLB infection 5 months after inoculation, starting on October, 2011 citrus trees will be re-budding with HLB infected buds. To obtain a base line of gene expression on leaves form HLB inoculated and HLB non-inoculated trees were sampled for RNA isolation on August 22, 2011. For control trees for each treatment, 10 mid-vein + petioles were pooled. For the HLB-infected trees, 18 were pooled (mid-vein + petiole only). A total of 448 frozen pooled samples were ground and immediately 1 g was weighed for RNA isolation and the rest was stored for back up as pre-ground RNA tissue. RNA was extracted from August 24 to August 30, 2011. The RNA extraction was done using the 3 day guanidine isothiocyanate (GITC) protocol and stored at -80 ‘C, gene expression will be determined by qRT-PCR. Literature cited: Ferguson J. 2007. Your Florida dooryard citrus guide. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Document HS 884. Li W., Hartung J.S. and Levy L. 2006. Quantitative real time PCR for detection and identification of Candidatus Liberobacter species associated with citrus huanglongbing. J. Microbiol. Methods 66: 104-115.
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