This project aims to provide comprehensive knowledge of the genetic and biological diversity of CTV isolates in CA and to identify and put in place mild isolates of the virus that can provide sustained protection against severe SP isolates. As the first step we are conducting molecular characterization of CTV isolates (Folimonova lab) collected from various regions in California. This approach will provide important information regarding genetic diversity of CTV populations in this region required for further selection of mild protective isolates. To this date, 53 samples received from Vidalakis lab in 2010 (as nucleic acid extracts from isolates of CCCP collection) were assayed using conventional reverse transcription PCR with CTV genotype-specific primers designed to specifically amplify sequences of the T30 or VT strains. Majority of those isolates demonstrated presence of the VT genotype, while some contained the T30 strain. Thirteen isolates appeared to contain mixed populations of both genotypes. Further analysis of those samples with primers specific to other CTV genotypes (T68, T3, T36) is in progress. The results of molecular characterization will be then evaluated along with information obtained from biological indexing of those isolates on indicator hosts in order to select mild non-stem pitting isolates as potential protective candidates against severe stem pitting isolates which share the same genotype composition. In addition to conventional reverse transcription PCR technique, we are developing a CTV genotype-specific quantitative reverse transcription PCR method that will allow us not only to determine what are the components of each isolate but also to quantify the amount of each genotype component present in isolates that contain several different isolates. Also this technique will speed up our work by allowing us to process more samples in a less amount of time. Recently we received 45 additional samples from CCTEA collection. These samples are being prepared for further characterization using the approaches described above. In order to obtain an understanding what CTV genotypes are currently present in the southern part of California where no previous eradication occurred, we are planning to conduct additional sampling of the new material from this region (Vidalakis lab). Those samples will be then used for inoculation of greenhouse trees for bioindexing characterization (Vidalakis lab) as well as sent (as nucleic acid extracts) to Folimonova lab for further molecular characterization.
The long-term goal of this project is to identify early diagnosis markers and to understand the mechanisms of plant defense responses against bacterium Candidatus Liberibacter (Ca. L.), which causes one of the most devastating citrus diseases – Citrus greening or ‘Huanglongbing’ (HLB). By examining the responses of 30 different genotypes of citrus to Florida isolates of Ca. L. asiaticus under controlled conditions in the green house, tolerant genotypes as well as sensitive genotypes of citrus have been identified (Folimonova et al., 2009). This project aims to identify important small RNAs and mRNAs that encoding signaling genes that contribute to HLB defense responses, by profiling small RNAs and mRNAs from HLB-infected tolerant and sensitive genotypes. We expect that different set of small RNAs and mRNAs will be identified from the tolerant and sensitive genotypes. HLB-induced small RNAs may target negative regulators of plant defense and HLB-suppressed small RNAs should target positive regulators of defense pathways in tolerant genotypes. mRNA profiling will help identify and validate these targets. Similarly, HLB-regulated small RNAs and mRNAs may help identify important components in pathogenicity of the greening. We chose Nules clementine as a sensitive variety and Sun Chu Cha mandarin as a tolerant one for deep sequencing analysis because both of them are mandarins and the genome sequence of a mandarin was just released which will ensure the success of our project. Currently, Dr. Folimonova’s lab is helping preparing and collecting the plant material. We will construct and sequence small RNA libraries from grafting- and Psyllid-infected Nules clementine and Sun Chu Cha mandarin use Illumina high throughput deep sequencer. Currently, we are in the processing of preparing and collecting the material to validate our small RNA results from previous studies using HLB plants that were infected by psyllid transmission. Our previous studies used only the grafting infected HLB plants from Dr. Kim Bowman’s lab at USDA lab Florida. Madam Vinous sweet orange plants (represent sensitive group) and Citrus macrophylla plants (tolerant group) that were already infected with HLB via psyllid transmission, they are at early stages of infection (about 3 months ago they we put in the psyllid room and still remain there). In addition, bacteria-regulated small RNAs often target important regulatory components in plant defense pathways. In comparison with mRNA profiling data, we will identify a series of plant regulatory genes that may contribute to HLB-tolerance and resistance responses. We propose to use high-throughput deep-sequencing approach to profile the mRNA populations of these two distinct tolerant and sensitive varieties. The newly released genome sequence of a mandarin will be essential for the data analysis. This study not only leads us to the discovery of HLB-induced small RNAs for early diagnosis marker development, but also helps us understand the pathogenicity of citrus greening, and helps us identify important components in natural defense of HLB disease.
The purpose of this California Citrus Research Board-supported project is to develop and implement methods to extend the shipping and shelf-life of fresh citrus fruits. The postharvest life of citrus fruit is often ended by fungal decay pathogens; in arid production regions of the California and Arizona, the preponderance of losses are caused by green mold and blue mold, caused by Penicillium digitatum and P. italicum, respectively. Phosphites (also termed phosphonates) are salts of phosphorous acid (H3PO3), nutritionally ineffective forms of phosphorus with an oxidation state of +5, as opposed to phosphorus compounds of oxidation state +3 that are used in fertilizers and utilized by plants. Known to control diseases caused by Oomycetes, such as brown rot of citrus, they inhibit other fungi but their activity can be modest (Guest and Grant, 1991). They merit thorough evaluation because of their relatively benign environmental aspects. Most formulations are neutral in pH and, in soil, they oxidize to phosphate. This facilitates disposal of used solutions from packinghouse tanks and drenchers, compared to solutions that contain fungicides, sodium salts, or are alkaline. Phosphites are exempt from residue tolerances for many applications. In postharvest applications to fruit inoculated the prior day, brief immersion (15-60 sec) in 1 to 3% phosphite solutions controlled both green and blue molds significantly, and were particularly effective on mandarin cultivars where another fungicide alternative treatment, sodium bicarbonate, often performs poorly. For example, a 1 min immersion at 25oC in 2% calcium or sodium phosphite reduced green mold from 63.0% among untreated ‘Murrcott’ mandarins to 12.2 and 14.5%, respectively, while 30.5% decayed after 2% sodium bicarbonate treatment. Similar treatments at 50oC with calcium or sodium phosphite further reduced green mold to 6.5 and 6.9%, respectively, while 14.3% decayed after sodium bicarbonate treatment. In most tests, heating the phosphite solution consistently improved its performance. Mixing phosphites with imazalil, thiabendazole, fludioxonil/azoxystrobin, or pyrimethanil improved the effectiveness of these fungicides. Synergy with imazalil occurred with solutions at neutral pH and not at low pH. Applications of 1 to 3% (wt/vol) solutions of calcium or potassium phosphite to run-off one week before harvest to ‘Atwood’ navel oranges reduced postharvest decay from natural or artificial infections by approximately 50%, although these very high phosphite rates, greatly above those recommended when phosphites are applied to trees for other reasons, caused some defoliation. At lower rates (less than 1%), preharvest applications of phosphite did not control postharvest Penicillium decay. Tests to control preharvest brown rot, caused by Phytopthora spp., and in progress. Brown rot is usually minor in California in most seasons, but severe outbreaks are occurring this year because of the prolonged wet conditions in groves caused by exceptionally abundant rainfall. Phosphites were effective as a postharvest treatment, but their preharvest use for postharvest decay control was not promising. Amiri and Bompeix (2011) reported similar promising results with postharvest phosphite use on pomes. Postharvest registration of potassium phosphite was granted in 2010 in the USA. Amiri, A.; Bompeix, G., 2011. Control of Penicillium expansum with potassium phosphite and heat treatment. Crop Protect. 30:222-227. Guest, D.I.; Grant, B. 1991. The complex action of phosphonates as antifungal agents. Biol. Rev. 66: 159-187.
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 journal manuscript was submitted on research showing that use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime; experiments to test this with commercial sweet oranges are underway. We continued with experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publications supported by this grant:1. Dutt, M., D.H. Lee and J.W. Grosser. 2010. Bifunctional selection-reporter systems for genetic transformation of citrus: mannose and kanamycin based systems. In Vitro Cellular & Developmental Biology-Plant 46:467-476; 2.Orbovic, V., M. Dutt and J.W. Grosser. 2010. Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars. Scientia Horticulturae 127: 262-266 Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and a commercial harvesting company (w/ Frank Rogers), a plan to collect meaningful yield and fruit quality from the St. Helena project was developed – with harvest expected later this month. Approximately 10 acres of trees planted 2.8 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The majority of trees have a significant yield and the trial is showing significant rootstock affects on precocious bearing and early fruit quality – the best selections from this trial will be ideal candidates for testing with juvenile transgenics. Also of interest is the cultural program being used at the St. Helena project that mimics OHS principals but with reduced input. The trees have been grown with a UF research slow-release fertilizer mix (in cooperation with Harrell’s Fertilizer) and daily irrigation. Two trees were confirmed with HLB the first year; but even with bad neighbors, there has been no detected additional spread of HLB during the past year. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL sweet oranges (a group of clones with Rhode Red quality that show high solids in young trees) were regenerated and successfully micrografted for further study of early flowering and transgene expression. Approximately 25 transgenic sweet orange trees from OLL selections were produced containing four different gene constructs. Progress was also made transforming OLL clones with the alternative embryogenic culture transformation system, as numerous transformed somatic embryos have been recovered. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
Objective 1 (confirm biofilm formation by X. citri subsp. citri (Xcc) in comparison with other bacteria that are well known to form biofilms). When grapefruit, Citrus macrophylla, Swingle citrumelo, and Carrizo were spray inoculated and kept under greenhouse conditions, aggregate formation was most prevalent on Swingle and Carrizo, reduced on C. macrophylla, and greatly reduced on grapefruit. Conversely, lesions were most prevalent on grapefruit, moderate on C. macrophylla, and infrequent on Swingle and Carrizo, although all four hosts developed lesions after injection-infiltration. This suggests a host interaction between biofilm formation and Xcc infection. Expression of genes, gumD, cheZ and rpf, involved directly or indirectly in biofilm formation is under evaluation. Detection of a gumD RNA fragment previously demonstrated to be a viability marker was higher in aggregated than in planktonic bacteria which is correlated with higher survival after 3 or 7 days in aggregates compared to suspension bacteria. Expression of cheZ involved in signal transduction for cell motility was initally higher at the onset of aggregate formation (3 dpi) but decreased once the biofilm was formed. This implies a role for bacterial motility at initial stage of biofilm formation. Expression of rfp involved in quorum sensing is lower in planktonic than in aggregating cells and in biofilm after 7days. This preliminary result confirms the importance of quorum sensing in the aggregation of Xcc bacteria which may serve as a target for disruption of the Xcc infection process. Objective 2. New disinfectants were evaluated for their ability to inhibit biofilm formation or disrupt bacterial aggregates. Zixvirox and Aquazix are used as surface disinfectants for food and have reported activity against biofilms. Aquazix is a mixture of hydrogen peroxide and silver as active ingredients. Zixvirox is a mixture of hydrogen peroxide and peracetic acid. Different concentrations of these products were tested against planktonic Xcc and as inhibitors of biofilm formation. To assess the effect on planktonic bacteria, culturability on LB medium plates and viability by membrane integrity (live-dead kit) assay were measured. Zixvirox at 0.03% and Aquazix at 2% were 100% effective for killing bacteria in suspension and complete disruption of cell integrity was confirmed by live dead assay. To evaluate the effect of these products on biofilm, aggregation was observed on borosilicate plates using a non stable Gfp strain and confocal laser scanning microscopy. At sublethal concentrations, Zixvirox was not completely effective for inhibition of biofilm formation because some bacterial aggregates formed. Aquafix at sublethal concentration prevented aggregation whereas bacteria were still viable in culture suspension. Although Aquazix and Zixvirox show promise as disinfectants, further assays are needed to confirm their activity as biofilm inhibitors. The effect of peroxide, CuSO4, SOPP, NaCl and NaClO at different concentrations was evaluated after 30 minute exposure of preformed biofilms on slides. Efficacy for disruption of bacterial aggregates varied with the compound. Treatments with SOPP, NaCl and NaClO disrupted the biofilm. CuSO4 or peroxide did not disrupt and may have stimulated biofilm formation based on the observed higher aggregation. Currently, assays are underway to evaluate the viability of bacteria in aggregates after bactericide treatment of the preformed biofilms. These assays are based on respiratory activity of the aggregate and fluorescence detection of non stable Gfp expressing strains.
In 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.
Preliminary work to explore optimal parameters for genetic transformation of Murraya has been carried out, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the recently optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments have been initiated using the plasmid pTLAB 21 harbored in Agrobacterium tumefaciens strain EHA 101. This vector contains the genes for kanamycin resistance as a selection agent and GFP (green fluorescent protein) as an easily observed marker. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were tested, to attempt establishment of an optimal and standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots has occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures have revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth is being monitored, as well as the stability and uniformity of GFP expression over time, and further production of new transgenic events is underway.
Progress in the project was disrupted by the unexpected departure, with no advance notice, of the previous employee responsible for the research underway. Another employee was assigned responsibility and she has made excellent efforts to retain forward momentum and to make credible progress. Seeds of Murraya paniculata were procured from the USA-ARS National Clonal Germplasm Repository for Citrus and Dates, from a commercial company (Seeds & More), and from a local source in Florida. The limited seed availability and their relatively poor quality, along with consequent low germination rates and very poor growth, have been a hurdle in the process. Such seedling materials present a major challenge for Agrobacterium based transformation efforts. Therefore, a comprehensive experiment on screening optimal conditions for in vitro seed germination was conducted. Various culture media, using Murashige and Skoog’s (MS) basal salt, with or without GA3 (3 mg/l), with varying sugar (25, and 50 g/l) and agar concentrations (4, 6 and 8 g/l), have been examined, with a goal to achieve an acceptable seed germination rate. In order to achieve the high quality (with stronger stems) of seedlings necessary for the regeneration and transformation experiments, germinated weaker seedlings were subcultured twice on MS basal medium without any plant growth regulators for another month. Stronger seedlings obtained were used as explants for regeneration and transformation experiments.
Because of successful efforts to achieve acceptable seed germination rates and to produce more vigorous seedlings in vitro, higher quality seedling explant materials have been obtained. This accomplishment has enabled work to proceed to establish a suitable and efficient protocol for in vitro regeneration of Murraya, which can then be used for the various planned transformation experiments. Research was conducted to develop a suitable and robust organogenic shoot regeneration protocol, using various parts of germinated seedlings including hypocotyls (longitudinally cut vs. no cut), epicotyls (longitudinally cut vs. no cut), roots, leaves, and cotyledons. These explants were cultured on MS basal medium supplemented with various concentrations of BA, TDZ alone, or in combination with NAA. Two suitable and reasonably effective shoot regeneration protocols have been developed and optimized for transformation experiments (a manuscript is in preparation). Simultaneously, the capacity of shoot tips and lateral buds to undergo multiple shoot formation and multiplication in vitro has been examined. Shoot tips and lateral buds were excised directly from in vitro germinated seedlings and cultured on MS medium supplemented with BA at 0, 1, 3, and 6 mg/l. Newly formed shoots were subcultured on the same medium at 3 week intervals to determine optimal conditions for maximal shoot multiplication.
Quarterly report April 2011: Reserach to explore optimal various parameters for genetic transformation of Murraya was carried out, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the recently optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments have been initiated using the plasmid pTLAB 21 harbored in Agrobacterium tumefaciens strain EHA 101. This vector contains the genes for kanamycin resistance as a selection agent and GFP (green fluorescent protein) as an easily observed marker. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were tested, and we established an optimal, standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth was monitored, as well as the stability and uniformity of GFP expression over time. Nearly all of these transformation events proved to be either chimeric or transient, so further production of new transgenic events is being pursued.
Five field studies are underway to evaluate the effects of various foliar nutrient applications on the expression of HLB in infected trees by evaluating tree nutrient status, growth, yield and visual tree appearance through photographic documentation. The first trial is a survey-type trial to monitor the health and yield of trees in Maury Boyd’s grove in Felda. We have recently completed the third Hamlin harvest on these trees and will harvest Valencia as fruit reach maturity. It is clear from this observational study that HLB infection does not kill well managed citrus trees, and that trees can be maintained with economically profitable yields of quality fruit for at least five years after known infection. Recently, a summary of the past 10 years of yield for Maury Boyd’s grove and a nearby grove practicing tree removal was completed. This summary clearly showed that MB’s yield has remained constant since HLB was discovered whereas the nearby grove’s yields are declining as the tree population declines. The second of these trials is in a heavily infected mature Hamlin grove in south Florida. Since the initiation of the project the trees in this study have received eight foliar applications of nine different treatments. Untreated trees serve as controls. The trees will be harvested on 31 January 2011 and the yield data will be compared with the previous year’s. The third trial has been discontinued because, unknown to us, the grower-cooperator began applying a foliar nutrient program to this grove, including our treatment plots. Two additional field studies were begun during 2010 in research blocks at the CREC. One of these trials involves the application of a commercially available foliar nutrient product with and without the application of SAR inducing compounds. Five treatment applications were made in 2010 and the trees (Valencia) will be harvested in spring 2011. Leaf samples have been collected and analyzed for nutrient content and a photographic record of each tree is being kept. The second of these trials involves standard and high application rates of foliar nutrients in combination with standard and elevated ACP control. Trees in this 10 acre block are Hamlin and Grapefruit. Samples have been collected from the Hamlin trees for quality analysis and total yields will be estimated when harvested. Both blocks continue to be scouted for HLB occurrence, but trees are not being removed. A hydroponics system has been constructed in an HLB approved greenhouse at the CREC. Trees are growing well in the system and have begun to show foliar symptoms of nutrient deficiencies for the different treatments. Trees are being monitored for the development of HLB symptoms and will be confirmed by PCR when suspects are detected. Once trees are known to be infected data collection of how the disease develops in trees under different nutrient deficiencies will be collected. This experiment will allow us to begin to separate nutrient and HLB effects on plant growth and development.
Abstract Citrus Huanglongbing (HLB) is caused by the phloem-restricted, Gram negative, and uncultured bacteria Candidatus (Ca.) Liberibacter (L.) africanus, Ca. L. asiaticus, and Ca. L. americanus, according with the continent in which they were first detected. Both Ca. L. asiaticus (CaLas) and Ca. L. americanus (CaLam) are transmitted by Diaphorina citrii, and are present in Brazil. Several studies about the transcriptional response of citrus plants during HLB symptoms have been reported, all of them are focused on the CaLas infection. This study proposed to evaluate the transcriptional reprogramming of sweet orange challenged with CaLam using a customized 385K microarray chip containing about 32,000 unigenes. A set of candidate genes from this study and from others was used to validate the expression profile in symptomatics and symptomless positive PCR tissue of sweet orange infected with CaLas or CaLam. We propose a global feature of the defense mechanisms of citrus in response to CaLas and CaLam. Methodology Challenge with Ca. Liberibacter spp ‘ Transcriptome experiments by microarrays were performed using plants of sweet orange (Citrus sinensis L. Osb. cv Pera) grafted onto Rangpur lime (C. limonia L. Osb.). The first challenge with Ca. Liberibacter americanus was carried out in 25 plants of four months old plants by grafting infected (PCR positive) budwoods. They were monitored bimonthly by end-point PCR for detection of the bacterium. PCR positive plants were challenged again by new grafting with infected budwoods, and pruned. Since Ca. Liberibacter americanus is heat sensitive, all plants were kept in growth chambers at 22 to 24 ‘C and 12/12 h light for six months. Full expanded leaves of three plants displaying initial symptoms of HLB and PCR positive for the bacterium, and leaves of healthy plants in the conditions were collected and stored at -80’ C. To validate specific gene expression, eight plants of sweet orange (cv Hamlin) grafted onto Rangpur lime were challenged with infected budwoods, either with Ca. Liberibacter americanus or Ca. Liberibacter asiaticus. Plants infected with Ca. L. asiaticus were kept in screen house at 28 to 30 ‘C, and plants infected with Ca. L. americanus in growth chambers at 22 to 24’C. Full expanded PCR positive leaves for Ca. L. americanus and Ca. L. asiaticus, respectively, were collected and stored at -80’ C. Genome wide transcription approach – Global transcriptional levels of diseased and healthy plants were evaluated with a Roche Nimblegen Systems customized 385K chip containing 32,000 unigenes of sweet orange. Three probes for each unigene with optimal predicted hybridization characteristics were designed to compose a probe set. Each probe set was represented onto the final array by four replicates. All probes were designed as perfect match oligonucleotides. Total RNA concentration and purity were determined from the ratio of absorbance readings at 260 and 280 nm using a Nanodrop ND8000 spectrophotometer (Nanodrop Technologies), and RNA integrity was tested in a denaturing agarose gel. Reverse transcription was performed with 1 ‘g of total RNA in a total volume of 20 ‘L with oligo(dT) primer using Revertaid H-Minus reverse transcriptase (Fermentas). The final cDNA products were diluted 50-fold prior to use in RT-qPCR. Raw data were imported to NimbleScan 2.5v software, which employs three steps of preprocessing: convolution background correction, quantile normalization, and a summarization of expression measures in a probe-level with a robust multiarray model fit (RMA) using the median polish algorithm. Unigene transcripts with p-values ‘0.05, fold change |FC| ‘ 2.0 and odds probability ‘ 0.95 were considered as differentially expressed genes (DEG). To identify relevant molecular mechanisms potentially associated with sweet orange response against Ca.L.americanus infection gene set enrichment analysis (GSEA) method, that evaluates microarray data at level of gene sets was carried out. Gene set was defined as all predicted genes that sharing the same ontology based on GO database from A.thaliana, and can be classified as biological processes, molecular functions and cellular components over-represented in a set of DEG. Specific gene expression analysis – To obtain reliable of gene expression measurements, we performed a screening of candidate reference genes of our microarray analyses, adopting the following cutoff: absolute logFC ‘0.5, average expression (AveExp ‘7.0) and standard deviation (stdev ‘0.5). Unigene transcripts were then sorted by coefficient of variation (CV) standard deviation, and LogFC. Besides that, we decided to evaluate the expression stability of 18S ribosomal and GAPDH that were usually used to normalize transcript levels. Primer efficiencies, Cq values and normalized relative quantities (NRQ) were calculated as described. The most stable reference genes were identified using the geNorm 3.5v (medgen.ugent.be/~jvdesomp/geNorm/) algorithm. Stepwise exclusion of the reference genes with the lowest stability of expression (the highest M) allows ranking the reference genes according to their expression stability. The pairwise variation also determines the minimum number of reference genes required to a reliable normalization. Based on the predicted function, retrieved from TAIR, 24 genes were selected to be validated by real time quantitative PCR (RT-qPCR). To those eigth additional genes differentially modulated in response to Ca.L.asiaticus, were included in the validation. The gene-specific primers were designed using Primer 3 (http://frodo.wi.mit.edu/primer3/) and IDT (Promega Corporation) software tools with melting temperatures of 60’C, amplicon length of 150 to 200 bp, and a GC content of 50 to 60%. Amplicon specificity was checked by 2% (w/v) agarose gel electrophoresis and by melting-curve analysis. Sequence identity was confirmed by direct sequencing of PCR products using an Applied Biosystems Model 3730 DNA sequencer. Relative quantification was carried out in a 96-well optical plate with an ABI PRISM 7500 FAST sequence detection system (Applied Biosystems) using the Fast SYBR green PCR master mix (Applied Biosystems). The standard thermal profile was used for all amplifications. All assays were performed using three technical replicates and a non-template control, as well as three biological replicates. To analyse dissociation curve profiles,program was run after the 40 cycles of PCR: 95’C for 15 sec followed by a constant increase in temperature between 60 and 95’C. Raw data of fluorescence accumulation for each individual assay were imported into R statistical package version 2.922 (R Development Core Team). Cq values were determined for each amplification by the maximum of the second derivative of the fitted sigmoid curve. The comparison of means of normalized expression values among groups were performed by a nonparametric one-way ANOVA with 1000 unrestricted permutations, followed by pair-wise comparisons with Bonferroni adjustment. Results and Discussion The complete manuscript is been submitted to an international journal for evaluation and, if accepted, to be published. Results and discussions are been focused on the following points: – Detection of the bacteria in the tissues of sweet orange. – Genome wide transcription approach. – Main affected processes or pathways in response to the infection o CaLam. – Specific gene expression by RT-qPCR. As soon as the manuscript is accepted I will send the final report of the project.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. ‘ We have greatly improved our efficiency of screening . ‘ We are modifying the vector to express more than one anti-HLB gene. ‘ We are modifying the vector to allow addition of a second vector. ‘ We are preparing to put trees into the field for testing as soon as potential freezes are over. ‘ We continue to supply infected and healthy psyllids to the research community.
A major objective of this project is to develop an understanding of how the HLB bacterium (Las) interacts with citrus genotypes to cause disease. After finding that different citrus genotypes respond differently to Las from extremely sensitive (sweet orange and grapefruit) to tolerance with minor symptoms, we have focused on the one citrus genotype that is most resistant to citrus. Las is restricted to very low levels in Poncirus trifoliata. Most plants remain PCR negative, but a few have barely detectable levels of Las. We have found that under some conditions Las appears not to be able to move through poncirus. We have plants with lower living inoculum that is highly infected with Las, but sensitive sweet orange shoots grafted on top of the poncirus plants have not become infected. We are examining the value of using Poncirus rootstocks and interstocks to reduce or prevent spread of the disease in sweet orange or grapefruit. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las for those plants can be as little as 6 weeks. We now are focusing on when and how psyllids acquire Las from newly infected plants. This information is necessary of the epidemiology models for managing HLB. We also have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or anti-psyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. We have greatly optimized conditions to allow exposed plants to become rapidly inoculated by infected psyllids. We continue be a resource of healthy and infected psyllids and plants for other laboratories.
Sensory impacts and flavor and aroma changes in HLB fruit: Hamlin fruit was harvested, juiced using a commercial extractor and pasteurized in late December, 2010. Symptomatic greening fruit was blended into healthy fruit at 0, 2.5, 5, 10, 20, 50, and 100% by fruit weight prior to juicing. These juices are being stored and will be pasteurized. Future taste panels are planned. Physiological changes in HLB fruit: We compared the visual appearance and characteristics of HLB-impacted fruit with girdled fruit to determine if restriction of carbohydrate movement caused symptoms characteristic of HLB. Girdling was performed on ‘Hamlin’ branches in groves in July and August 2010 on healthy trees. A ring (full girdled) or half ring (half girdled) of bark (‘8 mm width) around the twig located 10-cm above a fruit was removed. Leaves between the girdled region and fruit were removed. Four biological replications composed of eight trees (4 fruit/rep) each were used for girdling experiments; fruit were harvested on December 8th 2010. Relative gene expression in flavedo was analyzed using quantitative real-time PCR. Citrus glyceraldehyde-3-phosphate-dehydrogenase was used as the constitutively expressed internal calibrator. Visual analysis indicated that full-girdled (FG) fruit were similar in size to HLB symptomatic (SY) fruit, but girdled fruit were greener and not misshaped. No visual differences were seen between healthy (H) and asymptomatic (AS) fruit or ungirdled (UG) and half-girdled (HG) fruit. SY and FG fruit were lower in starch and sugar and had similar changes in juice quality than H or UG fruit, respectively. Generally, the impact on 5FG (5 months girdling) was greater than on 4FG (4 months girdling). Carotenoid content was reduced in SY when compared with H, but increased in FG flavedo when compared to UG. Expression of several genes was significantly changed in SY but not FG flavedo. Included in this list were a sulfate transporter, starch synthase, alpha-amylase, four phospholipases and two ethylene synthesis genes. Yield, peel color, fruit size and seed abortion in HLB fruit: FG fruit had significantly lower fruit diameter, fruit weight, flavedo starch and sugar content compared to HG or UG fruit. Such a pattern was similar when SY fruit were compared with AS or H fruit. Extension and education: The processor HLB posters were displayed at the following meetings: International Citrus and Beverage Conference, the Deputy Under Secretary of Agriculture visit, Florida Television visit, Florida Citrus Mutual Board Meeting, and a CRDF meeting. A total of 452 processors brochures were handed out in the fall that describe our results.
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