New outbreaks of invasive fruit flies (Diptera: Tephritidae) continue to threaten agriculture world-wide. Establishment of these pests often results in serious economic and environmental consequences associated with quarantine, control, and eradication programs. Early fruit fly detection and eradication in the United States requires deployment of large numbers of traps baited with the highly attractive male specific parapheromone lures trimedlure (TML), cue-lure (C-L), and methyl eugenol (ME) to detect such pests as Mediterranean fruit fly, Ceratitis capitata (Wiedemann), melon fly, Bactrocera cucurbitae (Coquillett), and oriental fruit fly, B. dorsalis (Hendel), respectively. The current study compared the performance of solid single lure cones and plugs in conjunction with DDVP insecticidal strips; liquid lure with naled formulations; and single, double, and triple solid lure wafers impregnated with insecticide. Treatments were placed in AWPM and Jackson traps under Hawaiian climatic conditions in habitats where B. dorsalis, C. capitata, and B. cucurbitae occur together. The overall goal of this study was to develop a more convenient, effective, and safer means to use male lures and insecticides for improved detection and male annihilation of invasive fruit flies. In survey trials near Kona, HI captures of C. capitata, B. cucurbitae, and B. dorsalis with Mallet TMR wafers were equal to those for the standard TML, ME, and C-L traps used in Florida and California. A solid Mallet TMR wafer is more convenient to handle, safer, and may be used in place of several individual lure and trap systems, potentially reducing costs of large survey and detection programs in Florida and California, and male annihilation programs in Hawaii. With confirmatory trials completed in Hawaii, further testing will be conducted in citrus orchards under California weather conditions. Through Dr. Joseph Morse of the University of California, Riverside, we will conduct weathering trials of the novel TMR dispensers in California (Riverside, Lindcove, Bakersfield, Ventura, and Costa Mesa, CA) beginning in July 2012. Climate data will be obtained from Hobo weather recorders maintained at each location. Weathered dispensers will be sent to Hawaii and Washington for bioassays and chemical analyses, respectively. Roger Vargas of US PBARC will oversee bioassays in Hawaii. Peter Cook of Farmatech and John Stark of Washington State University will collaborate on chemical analysis of wafers in North Bend, WA. Currently, approximately 30,000 sets of TML, ME, and C-L traps are maintained throughout the state. From a worker safety, convenience, and economic standpoint, Farma Tech TMR Mallet solid wafers with DDVP may be more cost effective, convenient, and safer to handle than current liquid lure and insecticide formulations (e.g. naled) used for detection programs for TML, ME and C-L responding flies in California. Cost/benefit analyses of Mallet TMR vs. standard trapping systems will be done.
The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. Money was just released in last week so no results to report.
The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. The field survey of tangerine hybrid blocks is nearly finished with approximately 1000 isolates collected. Pathogenicity testing and collecting monoconidial isolates is underway. Analysis of data from 2008-2011 is under way and a manuscript is in preparation and is waiting for the 2012 results. Plants have been prepared for a series of experiments to look at the fitness characteristics among sensitive and resistant isolates.
The first objective of this project was to hire a Florida-based faculty scientist that could be trained under Dr. Leandro Pena in Spain, for the purpose of learning the mature tissue transformation technique and transferring the technology to Florida. The scientist (Dr. Cecilia Zapata) was hired, at the end of the first year of the three year project, and traveled to Dr. Pena’s lab at the IVIA, Spain, where she was trained in all tissue culture techniques associated with citrus mature transformation, starting with preparation of the source of material at the greenhouse and ending with the acclimatization of transformants in the greenhouse. It was emphasized that the preparation of plant material needed for mature transformation is the key to successfully and consistently obtaining mature transformants, and this can only be achieved by producing budsticks in a highly controlled and clean environment. The second objective of the project was to build a greenhouse at the Citrus Research and Education Center in Florida for the purpose of creating and growing citrus for mature transformation and to establish a Mature Transformation Laboratory. A growth room was constructed instead of a greenhouse due to budget constraints. It took approximately 7 months to construct the growth room. It is currently operational after more than a year of troubleshooting. The water filtration system still needs some adjustments to be able to obtain a better water quality. The water quality is affecting the plant growth and the humidifiers. A generator needs to be purchased; without it, any prolonged electricity failure could jeopardize the whole project. The laboratory is fully operational. The third objective of the project was to obtain mature transgenic plants from the most important Florida citrus cultivars. We started using the growth room and planted the rootstocks at the beginning of April 2011. Three (3) sweet orange varieties were indexed in vitro and micrografted. The cultivars introduced were Hamlin 1-4-1, Valencia SPB 1-14-19 and Pineapple F-60-3. A calendar was established in October 2011 and firsts mature transformation experiments were performed in November 2011, all the protocols developed at the IVIA were adjusted to our specific environmental conditions and clone specificities. Mature Valencia, in our conditions, was very responsive to organogenic regeneration. We obtained positive plants, checked by PCR, and they are currently growing in the growth room. Hamlin was also transformed but was less responsive to organogenic regeneration, but we were able to obtain a plant currently growing in the growth room. Pineapple did not respond and we discarded the cultivar after the last batch in the production calendar was used. We are still waiting from results on this last experiment. We are currently introducing another clone of the same Hamlin cultivar 1-4-1 to improve its quality. A few of the initial mother plants were not as cleaned as previous determined and we introduced more plants using antimicrobial to guarantee cleanliness. It seems like yearly introductions may be necessary to maintain the quality of the material desired in the experiments. We are also cleaning the rootstocks Swingle Citrumelo and Carrizo to be transformed in future experiments.
The overall objective of this project is to develop and use a high-throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps) which causes psyllid yellows of tomato. Previous work focused on development of a system for the model plant Arabidopsis thaliana which has the best developed genetics of any plant and has been used in previous chemical genomics experiments. However, repeated attempts to infect Arabidopsis plants grown in solid culture media, liquid culture media, or hydroponics were not successful. Only plants grown in soil were infected by psyllid nymphs. The most recent experiments gave a low percentage of infected plants (about 15%), and differences between Arabidopsis lines observed previously were not repeatable. We believe that it will still be of interest to analyze the tolerance (lack of symptoms) seen in CLps infected Arabidopsis plants and are accumulating samples from CLps-positive plants for gene expression analysis. Because of the problems with Arabidopsis system, in late 2011 we began to develop a system for tomato which, as a natural host of CLps, is more easily infected. Compound leaves along with petioles from tomato plants were placed in 50 ml culture tubes with the cut end immersed in water in a microfuge tube. This design was adapted from one shown on a poster by Ammar et al. at the Citrus Health Research Forum in Denver in October. Adult psyllids are placed in the culture tubes and within 7 days most tomato leaf petioles are qPCR positive for CLps. After two weeks Ct values are typically less than 25 and about 80% of plants are infected. This system appears promising since chemicals can be introduced into the water for plant uptake. We will investigate methods to induce rapid rooting of these leaf petioles since this may increase chemical uptake. Experiments to test chemical uptake were initiated. We also began testing a transgenic tomato carrying a marker gene (GUS) driven by a pathogen responsive promoter (CaBP22). This system has facilitated detection of chemicals that activate defense pathways in Arabidopsis. Sweet orange seedlings were grown to test chemical application methods during the next quarter. This work is supported by a separate funding source, but little progress on the chemical genomics project is expected while this work is carried out.
Previously, we have shown that specific psyllid dsRNAs can be toxic to Asian citrus psyllids when the psyllids feed on citrus that have been engineered to produce these dsRNAs using a Citrus tristeza virus (CTV) expression vector. In this work, variability of dsRNA present within the tissues on which the psyllid is feeding effects toxicity to the psyllid and we have identified a threshold concentration of dsRNA needed to be toxic to adult psyllids. We have also identified a number of dsRNAs matching specific psyllid genes to which the psyllid are hypersensitive to (active at relatively low concentrations) when these dsRNAs are taken up orally through artificial diet feeding. Experiments have been initiated to express these dsRNAs in citrus and test their effects on all life stages of the psyllid when fed on these engineered plants.
The focus of this research has been to clone, express, and test the effect of suggested RNAi molecules on psyllid vectors using an artificial feeding system. We have developed a gut gene library, have isolated several sequences for critical proteins, and constructed RNAi molecules based on these sequences that will kill Asian citrus psyllids in controlled feeding experiments. We have also constructed and tested contest entrants. Several molecules have potential to control ASP. We have expressed two of the molecules in citrus using Dr. Dawson’s CTV vector, and have shown leaves from these trees are highly toxic to psyllids. There is a good correlation between RNAi expression and psyllid mortality. We will be testing other RNAi molecules using the CTV vector shortly. The sequences of the RNAi molecules and the genes they target are intellectual property. This project has given hope for a specific, environmentally friendly field control of Asian citrus psyllid without transforming trees. We are in the process of securing intellectual property rights for UF and USDA that will allow licensing, production, and marketing of this technology.
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. 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.
Huanglongbing (HLB) was first discovered in Florida in 2005. In response, Florida citrus nurseries began treating rootstock seed trees located outdoors with insecticide applications to reduce risk of psyllid transmission of ‘Candidatus Liberibacter asiaticus’ (Las), the putative causal agent. In 2008, a survey identified two ‘Carrizo’ citrange trees with symptoms of HLB. To assess the potential for seed transmission from HLB-affected seed source trees, assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008, 1557 seedlings germinated from ‘Pineapple’ sweet orange seeds from trees in Collier County were assayed by quantitative polymerase chain reaction (qPCR) using 16S rRNA gene primers. Of these seedlings, a single plant was positive for (Las+), although additional tests were negative. In 2009, no Las+ plants were detected among 332 ‘Murcott’ tangor seedlings from trees in Hendry County. From nurseries in 2008, one Las+ seedling was detected in 290 seedlings from fruit located on symptomatic branches of two ‘Carrizo’ citrange trees, but its Las+ status was not confirmed after repeated testing. In 2009, a single Las+ result was obtained for one of 100 Cleopatra mandarin seedlings, whereas no Las+ seedlings were detected for 125 seedlings from seeds from two trees of ‘Swingle’ citrumelo, 649 seedlings from four trees of ‘Kuharske’ citrange, or 100 seedlings from one tree of ‘Shekwasha’ mandarin. Despite the occasional Las+ qPCR tests, no plants developed HLB symptoms. The most probable explanation for these results is transient transmission of Las from seed obtained from HLB-affected trees with no subsequent disease establishment.
Over the past year, our research has focused on the following areas: (i) Isolation and sequencing of TAL effectors from additional citrus canker strains Sequencing of TALE genes is especially difficult due to the presence of between14 and 20 repeats of the highly sequence-related DNA binding domain. However with considerable effort, we have now determined the sequences of eight proteins from five novel strains: A44 (Argentina), Etrog (Florida), 2090 (Florida), Miami (Florida), and 93 (Brazil), with four more protein sequences nearing completion. Although these strains show variation in phenotype or host range, our engineered promoter constructs containing 14 TALE recognition sites conferred recognition to TALEs in four of the strains, with the fifth pending analysis. These results further support our aim of engineering a resistance construct that will be triggered by a broad range of canker strains. Differences do occur in some of the sequences, and we plan to investigate how these differences may influence the behavior of strains in various assays. (ii) Production and testing of stable transgenic citrus lines: As of the end of this year, we have transformed a total of 21,504, 747, and 173 explants of ‘Duncan’ grapefruit, ‘Ruby Red’ grapefruit, and sweet orange cultivars, respectively, and have 446 plants in 4 inch pots. We have tested epicotyl and cotyledon explant material and find that epicotyls are the most efficient material for use with grapefruit, whereas cotyledons appear to work best for sweet orange. The Ruby Red cultivar is the most difficult to work with, because of the difficulty in sourcing seed. Each transformant is grown through shooting, rooting and transfer to soil, and then it is analyzed by PCR for each of the construct components – promoter, gene and selectable marker. Plants are further tested by pathogen inoculation. All stable and transient transformations were made with eight distinct gene constructs and a negative control. Overall, we find the broadest and best induction using the 14 box promoter, relative to other promoter versions. We have tested three HR-inducing genes – the Bs3 gene from pepper, and the AvrGf1 and 2 genes from Xanthomonas We have not observed activity of Bs3 in citrus to date. AvrGf1 has worked well in transient assays, but we have not yet analyzed enough stable lines to identify reproducible disease resistance. We continue to test lines as they mature, and AvrGf2 lines will also be tested when they become available. (iii) A third gene option for conferring resistance The type 3 effector AvrGf2, identified from X. fuscans subsp aurantifolii strain C, is being tested as another resistance gene option because it has been observed to cause a more robust HR on grapefruit than AvrGf1. The coding sequence was fused with the Bs3- PIP14 box promoter and used in transient and stable transformation assays. In transient assays, the avrGf2 construct did confer a robust HR within 3 days as compared to 4 days using AvrGf1. Stable transformation experiments involving epicotyls of ‘Duncan’ grapefruit, ‘Ruby Red’ grapefruit, and sweet orange segments, had 42, 16 and 32 plants transferred to rooting media, respectively. More transformants are in the pipeline, and all will be subject to molecular characterization and testing.
In the third and final year of funding, Core Citrus Transformation Facility (CCTF) maintained its level of performance and produced transgenic citrus plants for many satisfied customers. Considering that the major goals of proposed project to increase the capacity of the transformation lab were met within the first year of funding, during the second and third year of project duration CCTF had a task to maintain the achieved level of operation. The number of experiments and the quantity of produced transgenic material were at the level projected in the grant proposal. By accomplishing these tasks, CCTF assisted other researchers in efforts to improve different citrus cultivars by increasing their resistance and/or tolerance to diseases. Newly placed orders for transgenic plants stayed at high level. Altogether, thirteen new orders were received for processing during the third year of funding: pY46-Carrizo; pY102-Carrizo; pY141-Carrizo; pY150-Carrizo; pCitIntra-Duncan; pAZI-Duncan; pAtBI-Duncan; pBCR2-Duncan; pDPR1-Duncan; pLP1-Hamlin; pLP1-C-mac; pLP2-Hamlin; pLP2-C-mac. During the last quarter of this funding year, work was mostly concentrated on recent orders. Fourteen Duncan plants were produced carrying a gene of interest from the p35S-TRX vector and 23 more Duncan plants were produced carrying a gene from the pSucTRX vector. Multiple Duncan plants were produced toward satisfaction of ‘WG’ group of orders: eight-pWG22-1 plants, three-pWG21-1, and four pWG25-13 plant. Also, following Carrizo plants were produced for the ‘Yale’ order: nine plants with the gene from the pY46 vector and 11 plants with the gene from the pY102 vector. Eighteen Duncan plants were produced after treatment with bacteria harboring pBCR2 vector. Three more Duncan plants were produced with the EDS5 gene and six Mexican limes with the P35 gene. Four additional Duncan plants carrying a gene from pSUC-CitNPR1 were produced. In the previous three quarters of this year, small number of plants was produced for completion of older orders including: pNAC1 (1 plant); pMKK7 (1 plant); p33 gene (3 plants); pSUC-CitNPR1 (10 plants); p7+ p10 gene (32 plants). Most of the work was done on orders received in the last quarter of the second year and those placed in the third year of funding. The latter include: 12 Duncan plants (pWG19-5 vector); 11 Duncan plants (pWG20-7 vector); 18 Duncan plants (pWG21-1 vector); 22 Duncan plants (pWG22-1 vector); seven Duncan plants (pWG24-13 vector); 17 Duncan plants (pWG25-13 vector); 15 Duncan plants (pWG27-3 vector); 36 Duncan plants (ELP3 gene); 23 Duncan plants (ELP4 gene); nine Duncan plants (EDS5 gene); 10 Hamlin plants (pLC220 vector); 26 Duncan plants (p35 gene); 11 Duncan plants (35S-TRX vector), two Duncan plants (SUC-TRX vector). Joint efforts of Citrus industry and academic institutions to find solutions against huanglongbing (HLB), canker, and other citrus diseases are getting stronger with some positive results already being published. Continued funding for CCTF which is an integral part of this community and contributes greatly towards common goal will allow for the progress to go on by keeping production of transgenic material un-interrupted and at high levels.
In the 3rd and final year of the project, significant progress was made on several fronts. Juvenile Explant Transformation Protocol R&D: Key components of our transformation system were investigated in order to improve transformation and regeneration efficiency. The best medium for citrus transformation was determined to be the MS medium. Optimum hormonal levels in tissue culture medium was determined to be 3 mgL-1 BAP supplemented with 0.5 mgL-1 NAA for trifoliate rootstocks, Mexican lime and recalcitrant citrus cultivars like Volkamer lemon and mandarin / tangerines, and 1 mgL-1 BAP for sweet oranges and grapefruits. It was determined that a 3 hour soak in an auxin rich medium containing 1 mgL-1 2,4-D, and 0.5 mgL-1 NAA with 3 mgL-1 BAP significantly improved the transformation efficiency in a number of cultivars evaluated. Optical density of the bacteria was a determining factor in the genetic transformation of citrus. Trifoliate rootstock explants could tolerate a higher OD (0.6) while optimum transformation was observed at a lower OD for sweet oranges (0.15 or 0.3). We also observed co-cultivation duration was observed to be cultivar dependent. 3 days co-cultivation duration was observed to be optimum for cultivars with a thicker epicotyl such as trifoliate rootstocks or tetraploid selections. The optimum period for co-cultivation of sweet oranges was observed to be 2 days. Addition of 1mgL-1 GA3 resulted in rapid elongation of shoots, allowing in vitro micrografting within a month of initial selection of shoots. We determined that addition of Lipoic acid ‘ an antioxidant to shoot regeneration medium following transformation dramatically enhanced the transformation efficiency. This addition has resulted in improved transformation efficiency in otherwise recalcitrant cultivars. Transgenic plants from precocious sweet orange somaclones including OLL8, B4-79, Vernia 2-1, and a precocious mandarin W. Murcott, containing the LIMA antimicrobial construct, were produced and were grafted to precocious rootstocks (Amblycarpa+ Benton and Changsha + Benton somatic hybrids) for continued early flowering-induction experiments. Seasonal effects of on regeneration potential and transformation success rate were also evaluated, and confirmed that each cultivar behaved differently based on the time of fruit harvest and seed germination. A rapid ex vitro micrografting technique suitable for propagating in vitro and young ex vitro transgenic stem pieces was developed. Combining all of these advances is expected to cut in half the time from initiating an experiment to flowering and fruiting transgenic trees, thus making juvenile transformation more competitive with mature tissue transformation. In addition we have developed an efficient protocol using cell suspension cultures that has enabled us to transform seedless or recalcitrant cultivars such as the precocious mandarin cultivar W. Murcott and the seedless cultivar Okitsu Wase satsuma. This protocol has created an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as an embryogenic cell suspension culture. This research supported 8 journal publications. Transformation with Early Flowering Genes: We have regenerated many transgenic citrus plants that include: poplar FT gene behind either the 35S or heat shock promoter; co-transformed Carrizo plants with two cassettes, one containing 35S-cft1 and the other containing AtSUC2 ‘ gus; and numerous transgenic plantlets of Hamlin and Carrizo containing P27, P28, P29, PATFT and pPTFT. All of these plants are at various stages of evaluation.
Over the first year of this project, we have established the methodologies to aseptically acquire LAS inoculum from fruits from infected trees, detect LAS via qPCR, and determine LAS cell viability via qPCR-EMA. Then, we applied these methods in 10 replicated experiments to monitor LAS viability in different culture media over time. We are currently finishing the analysis of the data from the experiments and expect to publish this data within the year. For the replicated experiments, LAS suspension obtained from seed of infected pomelo fruits has been used to inoculate replicate flasks containing four different types of media: 1/3 King’s B media (control), J50 media (50% juice from the infected fruit), G50 media (50% store-bought grapefruit juice), and G media (100% store-bought grapefruit juice). Samples were then collected every other day for 18 days. These samples were assessed via EMA-qPCR for viable cells. Results show that LAS viability is maintained longer in a juice-based media than in 1/3 King’s B media. Total DNA and viable DNA quickly approach 0 detectable genomic equivalents after a few days in the 1/3 King’s B media, while viable cells are detectable until close to the end of the experiments in the other culture media. However, LAS cell viability changes over this time in a reproducible oscillating pattern that may be an indication of cryptic growth. Two complete experiments and three partial experiments have been analyzed at this time, with the remaining analysis currently being conducted. Culture media samples were collected at both the beginning and end of each of these experiments. We are currently working to use the analytical technique ICP-OES to analyze the concentrations of various nutrients in both the LAS cells and the media to determine what nutrients, if any, change in concentration in each fraction during the course of the experiments. This could provide an indication of nutrients utilized by LAS. It will also provide a comparison between nutrient concentrations between the four different media used here. We hope to send culture samples to a colleague in Florida for similar analyses of amino acids soon. Flasks used during the experiments showed evidence of biofilm formation at the liquid-air interface. The biofilm was almost nonexistent in flasks containing 1/3 KB media, while it was most pronounced in flasks containing G50 or G media. We have recently been working to reproduce this biofilm on glass slides, but the LAS inoculum concentration in seeds is much lower this time of year so it has been difficult to obtain sufficient biofilm formation. We do see that LAS cells adhere more easily to a special charged-surface glass slide called PlusGold. Since in our previous experiments using LAS inoculum in microfluidic chambers showed the cells would not adhere much, we will try to make chambers using these glass slides instead. Once this year’s new crop of pomelo fruit is available, we will be able to test these new microfluidic chambers for LAS cell attachment.
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. 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. 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. 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. 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. 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). 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. Based on the predicted function, retrieved from TAIR, 24 genes were selected to be validated by real time quantitative PCR (RT-qPCR). 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). All assays were performed using three technical replicates and a non-template control, as well as three biological replicates. To analyse dissociation curve profile 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). The complete manuscript is been submitted to an international journal for evaluation and, if accepted, to be published. As soon as the manuscript is accepted I will send the final report of the project. If requested I can submit the preliminary version of the manuscript in confidence.
Under objective 1: Biofilm from A and Aw strains of Xcc were evaluated in time course experiments on fruit and leaf surface using scanning electronic microscopy (SEM) and confocal laser scanning microscopy (CLSM). Differences among wide and limit host range strains were shown, confirming previous results for in vitro biofilms. Initially, biofilm formation by the wide host range strains showed greater ability for aggregation than the narrow host range strains. Wide host range bacteria were aggregates comprised of a well-developed biofilm matrix whereas, narrow host range bacteria formed a labyrinth with a fiber of unknown composition. These structures were detected both by CLSM and SEM. Ongoing experiments are evaluating the differences in the matrix between the two host range strains and whether those differences are related to their motility. Under objective 2: Inefficacy of several bactericide treatments previously reported were evaluated by treating the bacteria in preformed biofilms and measuring their viability after the treatments. Viable and culturable bacteria were isolated after the treatments confirming the resistance of the aggregates to bactericidal treatments.
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