In Objectives 1 and 2, we proposed targeting specific regulators of key phage encoded virulence genes (such as the Las LexA-like repressor, LC1, a second downstream repressor, LC2, controlled in part by LC1), and a key exogenous regulator of the (lethal) phage lytic cycle encoded by Wolbachia, an important psyllid endosymbiont that is always found when Las is present. These results have so far resulted in three full length manuscripts and 14 abstracts. LC1, LC2 and the Wolbachia repressor have all been confirmed to be transcriptional repressors. All three are therefore prime targets for chemical interference. Purchase orders to have these three repressors commercially produced by synthesized were placed several months ago and all three have finally now been delivered. Both the LC1 and the Wolbachia repressor were charactereized by the commercial vendors as “difficult” to synthesize. Attempts in our lab to synthesize LC1 and the Wolbachia repressor indicated that both cause E. coli host cells to become quite “sick”, with very slow growth, likely because of the repressors’ effects on E. coli host gene regulation. These proteins were synthesized in vitro for evaluation purposes, but this is expensive for the scale needed for chemical screening purposes and slow delivery of the repressor proteins was not anticipated. Two versions of the Wolbachia repressor were delivered. One version, with a histidine (HIS) tag, failed to repress the Las target promoter, probably due to the presence of the tag. The native version, without the tag, worked as expected from the in vitro results. Chemical screening assays are now underway with all three. The Wolbachia protein has been shipped to both the De La Fuente lab in Auburn, and Duan lab at USDA-Ft. Pierce, our collaborators on a separate culturing project.
During the period of April, May, and June of 2017, most of the progress was related to application for follow-up funding to characterize HLB resistance of plants generated in the present project. With assistance from Dr. Catherine Hatcher (CRDF), Dr. McNellis has coordinated with three faculty at the University of Florida to make arrangements for HLB resistance testing for the existing plants expressing anti-NodT scFv antibody (FT-scFv). These faculty are Drs. Ozgur Batuman, Liliana Cano, and Rhuanito Ferrarezi. They have the expertise to do CLas infections and PCR-based quantification of CLas in plant tissues. We plan to submit a pre-proposal to the CRDF for funding to support HLB resistance testing of the existing trees at Fort Pierce, propagated in Dr. Tim Gottwald’s lab at the USDA facility there. This will allow us to infect and perform quantitative bacterial population analysis in the citrus trees. We plan to submit this CRDF pre-proposal by the end of August, 2017. Dr. McNellis also attended and gave a presentation at the May 22-23, 2017, Forum on Citrus Breeding and Transformation for HLB resistance hosted by the National Academies of Sciences, Engineering, and Medicine in Irvine, CA. In addition, Dr. McNellis, along with Drs. Batuman, Cano, Ferrarezi, and Vladimir Orbovic (also University of Florida) submitted a pre-proposal to the USDA Citrus Disease Research and Extension Specialty Crop grant program on May 12, 2017, and were invited to submit a full proposal as of June 28, 2017. This proposal uses the present research results from this CRDF project as the preliminary data for the proposal, and builds upon results from the present work. We plan to submit the full proposal for the August 18, 2017, deadline.
This project continues to make progress. We have grafted the new copies of the LuxI plants and they have grown well. We have collected leaf samples from these plants to test them for the presence of acylated homoserine lactone (AHL). We also have already set up a gas chromatography mass spectrometry (GC-MS) method for the detection of AHL in the LuxI plants and we were able to detect these AHL in spiked leaf samples. Our next step is to test the presence of AHL in LuxI plants. In addition, the LuxI plants are being challenged with Diaphorina citri to determine the effect of expressing of (AHL) on the pathogenicity of Candidatus Liberibacter asiaticus (CLas). Our preliminary observation showed that HLB symptoms are localized in specific areas in the LuxI plants, which means that the presence of AHL prevent the spread of CLas pathogen. In our next step, we will use polymersae chain reaction (PCR) to quantify the level of CLas in CLas-infected LuxI and control plants. This work will be accomplished by December 2017. We look forward to the results of this work hoping that it will provided valuable information to the citrus research community.
The merging and comparing of the collected data is still in progress. The most recent data addition has been from the Indian River location. In August, more new data from the South Florida area will be incorporated. Using neural networks to analyze the data is underway and a few compelling results have been obtained and need further validations. Objective 1: Leaf nutrient thresholds At this point, we have just begun to analyze the combined data using the neural network software Easy-NN, including the Indian River Data set with the Ridge data set, which is complete through May 2017. We are looking at the sample dates as snapshots in time and combined for any possible connection or correlation with HLB severity. Continuing from our previous leads using mean leaf perimeter and leaf area as outputs in generating neural networks, we have found that along with calcium and magnesium, iron and copper are looking like important variables to watch with severity of HLB infection. When we run the neural network with soil parameters from the Ridge and Indian River area, we find that the organic matter content still plays a key role as well as soil pH and soil Mg concentration. Another vital variable to explore further is the L* parameter (lightness in color of the soil). As L* increases, the mean leaf area decreases. However, on its own, this variable is not well suited for predicting HLB incidence or severity; it does help to show the importance of organic matter in tree health. Looking further into the soil parameters as inputs and using leaf thickness (grams of leaf dry mass / meter square leaf area) as an output, we find the most important soil variables to be the soil magnesium content, as well as the lightness of the soil (L*), potassium content, and soil organic matter content. These are only preliminary results and more investigation is necessary as well as increasing the data set over the next few quarters. In the next quarterly report, we hope to be able to include data from the South Florida Region. A preliminary set of samples was just delivered for image analysis and soil samples should be forthcoming. Objective 2: Determine soil conditions that favor root hair and VAM proliferation i. We have discussed further soil analysis that we would like to work on, including data about permanent wilting points as well as possibly quantifying the silicon content of the soil. Soils from the South Florida area will be included into the data set and will be measured for all of variables the other two regions have been measured for, including organic matter content, and color analysis. ii. We are investigating a new system for root hair development using Valencia and Murcott seedlings in a nutrient solution. Test runs of seedlings exposed to nutrient fog is underway.
During the reporting period, we continuously examined the effects of the two proposed genes on transient and stable transformation efficiencies of mature and juvenile citrus plant tissues. The experiments produced mixed results. We are repeating these experiments to obtain more definite conclusions. Two of the many chemicals we have tested consistently display 2-3 fold increases in transient and stable transformation efficiency. Meanwhile, our preliminary results suggest that we can also manipulate and enhance Agrobacterium-mediated transient expression and stable incorporation efficiencies of T-DNA genes. Increase in transient expression of the T-DNA gene expression in citrus tissues could help us to develop a technique to use CRISPR to produce non-transgenic mutants of citrus, which can facilitate the use of CRISPR to produce non-transgenic HLB resistant cultivars of citrus. A manuscript using Agrobacterium-mediated transient expression of CRISPR genes to produce non-transgenic mutants of a model plant species will be submitted for a consideration of publication soon.
During the reporting period, we continuously examined the effects of the proposed genes on transient and stable transformation efficiencies of mature and juvenile citrus plant tissues. We now conclude that two of the four genes examined cannot significantly enhance the transformation efficiencies of citrus tissues consistently. The other two genes are still under testing. On other hand, we tested effects of several chemicals on citrus transformation efficiencies, three of the five chemicals tested show 2-3 fold increases in transient and stable transformation efficiency. We are currently testing the effects of their combinations and developing a protocol of incorporating these chemicals in the culture media at various stages. Increases in stable transformation efficiencies of citrus will facilitate the use of transgenic technologies to create HLB resistance traits. Increase in transient expression of the T-DNA genes can help production of non-transgenic CRISPR-mediated mutants using Agrobacterium.
Our hypothesis is that application of antibacterial-producing bacteria directly to citrus root could suppress Las population in the roots and control Las. Application of antibacterials in this manner will avoid the strict restrictions of application of antibiotics on crops and ease public concerns since those bacteria are naturally present in the soil and are associated with plant roots. In order to achieve the goal, the following objectives will be conducted: Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria which are closely related to Las. We tested 27 antibacterial compound producing bacteria including Bacillus cereus, B. licheniformis, Paenibacillus polymyxa, Pseudomonas spp., Streptomyces aureofaciens, Streptomyces fradiae, Streptomyces fradiae, Streptomyces garyphalus, Streptomyces griseus, Streptomyces kanamyceticus, Streptomyces niveus, Streptomyces pristinaespiralis, Streptomyces virginiae, Streptomyces ribosidificus, Streptomyces venezuelae, Streptomyces vinaceuse, and Streptomyces capreolus. We have isolated 327 bacteria from Florida citrus groves. The antagonistic activity against Agrobacterium, Sinorhizobium meliloti, L. crescens and Xanthomonas citri pv. citri was determined. 21 strains including bacteria belonging to Paenibacillus, Burkholderia, Bacillus, and Streptomyces showed good antagonistic activity. Those isolated bacteria showing high antimicrobial activities have been sequenced to help us understand the mechanism and for identification purpose. Currently, the genome sequencing was finished and genome analysis was completed. Because Las infection also affects host resistance to Phytophthora, one common citrus pathogen in Florida, we tested the antimicrobial activity of the bacterial isolates against Phytophthora nicotinae and P. palmivora, multiple bacterial isolates showed antimicrobial activities against Phytophthora spp. Four bacterial strains: two Burkholderia, one Pseudomonas geniculata, and one Rhodococcus strains have been tested for their activity in controlling citrus HLB and canker and all showed induced plant defenses and control effect against infection by Xanthomonas citri. The HLB result is shown below. To further study the antimicrobial producing bacteria, tow Burkholderia strains have been labeled with GFP tag. Seven other strains are being labeled with GFP or RFP tag. We also investigated the antibiotic genes in the 21 antimicrobial producing bacteria that we isolated previously. These strains were inoculated to citrus roots and the colonization was determined by inoculation and recover method in lab condition using small citrus seedlings. Around 10E8 cfu were inoculated to each seedling. Approximately 10E4 cfu were recovered from roots 20 days after inoculation (dpi). In a separate experiment, two Burkholderia strains were tested and up to 10E5 cfu/g soil was recovered at five days post inoculation. Four antimicrobial producing bacterial strains belonging to Paenibacillus, Bacillus sp., and Pseudomonas geniculata were tested in field trial via a soil drench method applied every two months for one year. The treated trees were divided into the following categories based on the disease index of 0-5: 1) No symptoms or few symptoms (0-2); 2) Trees with severe HLB symptoms (3-5). One gallon of bacterial culture was applied per tree at the three concentrations: 10E6, 10E7, and 10E8 CFU/ml. Water treatment was used as negative control. The result demonstrated the applied bacteria survived better in rhizosphere soil than applied via irrigation, but the overall survival in the soil is still limited and the bacteria did not establish high population on the root surface. Application of beneficial bacteria slowed down the disease index increase and Las titers for the asymptomatic or trees showing few symptoms compared to the control, but it did not prevent the disease index and Las titers from increasing. For the trees showing severe symptoms, the applied bacteria did not show any effect on disease index and Las titers. One manuscript has been submitted to Frontiers in Microbiology. One more manuscript is being prepared.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. Grafted plants are currently growing, and will be tested for responsiveness to the elf18 ligand for EFR and for canker resistance. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and therefore the putative transformants are being screened by RT-PCR. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Work on these constructs has been discontinued due to negative effects of the constructs in citrus. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we are targeting mutating the native citrus Bs5 alleles while simultaneously replacing the gene with the effective resistance allele. Two editing constructs have been created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. Both constructs have been verified to function by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. These constructs have been prioritized for transformation into Carrizo citrange, and transformations are underway at UC Davis, with several rooted plants obtained so far. Molecular characterization of the putative transformants will be carried out at UC Berkeley. Transformants with mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
1) Trees have been in the ground for 3.5 years in a trial of 50 selections and cultivars on US-802 following no-choice ACP inoculation and several months in an ACP house. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a semi-annual basis. HLB is now widespread and the trees looking healthiest include a full sib of our best mandarin selection, and several of our best grapefruit-like hybrids. The one true grapefruit is the least healthy selection in the trial. There are eleven selections with a canopy volume 50% greater than Valencia and 28 with canopy volume >2X that of Flame. The best performers include hybrids containing Poncirus, and conventional hybrids which are predominately mandarin or pummelo. It may take 2-3 more years to clearly distinguish tolerant material. These trees are cropping this year and fruit will be used in a complementary project exploring synthesis of orange-like juice from HLB-tolerant types. 2) In June 2015 a field planting was established of: seedling trees of 133 Fortune x Fairchild hybrids from an earlier mapping study, seedlings of 27 Ponkan-like accessions, budded trees of 10 advanced ARS selections that are predominately mandarin, and budded trees of Fortune, Fairchild and Valencia. Data collection is underway. A NIFA grant is in preparation to map genes associated with tolerance. 3) Replicated trials in multiple locations are established of our best sweet-orange-like cultivars and mandarin-types. Volatiles from sweet-orange-like hybrids are so similar to sweet orange that likely can be legally named sweet orange. 4) RNA-seq compared transcriptome responses in HLB moderately tolerant Sun Chu Sha mandarin and susceptible Duncan grapefruit, to Xcc-flg22 and CLas-flg22 (most active epitope from the pathogen flagella; project initiated with Gloria Moore at University of Florida). Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with CLas, suggesting their involvement in HLB tolerance. In addition, several genes were similarly regulated by CLas-flg22 and CLas treatments. Genes identified are valuable for studying HLB tolerance mechanisms and potential for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy. Using these genes as markers, expression analysis from a group of mandarin hybrid in their responses to CLas-flg22 is underway. Highly and lowly responsive plants will be marked for long term observation of field tolerance. 5) Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, are being grown, and will be planted soon for field testing of HLB resistance. 6) Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance is completed, has revealed potentially valuable tolerance and indicates that early HLB symptoms and earlier CLas titer are unrelated to growth and cropping. In August 2010, the plants were established at Pico s farm in Ft. Pierce FL. Despite the high incidence of mottle in SugarBelle / SourOrange, it had the greatest overall increase in diameter. ‘SugarBelle’ and ‘Tango’ (which were not on the same stock as ‘Hamlin’ and so results should be viewed as comparing cultivar/rootstock combinations) were the healthiest in overall appearance in 10/15 and had the most fruit (88 per tree). 7) Our putative chimeras have not proven to be successful. We identified a chimera (Satsuma and Poncirus) from the Citrus genebank, arranged its importation, and we finally got permission to accept this material and maintain it in a quarantine death house. Cuttings of the chimera and each separate component (Owari and Poncirus) have been rooted and will be challenged by hot ACP feeding in the next quarters.
1) Assessed use of isolated leaf inoculation, and small plant destructive sampling: Isolated leaf inoculations do not readily distinguish between resistant and susceptible citrus selections, but may prove useful in identifying nearly immune material. Small plant destructive inoculation assays now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. This assay seems to be an efficient way to test transgenics that are expected to kill CLas. Recently we have had delays due to failures in ACP-inoculation and have reinitiated several challenges. 2) Data collection continues on transgenics. Transgenic plants expressing a modified thionin are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. . Rooted cutting of 167 Carrizo plants were obtained. A subset of 67 plants representing 13 independent events and wild types (4-5 replicates each) were inoculated by ACP infestation. All of the plants except 2 were confirmed CLas positive after a 2-week ACP exposure, and the titer between wild type and transgenic groups are similar at two weeks. The plants are maintained in the greenhouse for tests at 3, 9 and 12 months after inoculation. Transgenics expressing AMP D2A21 suppressed canker but not HLB with manuscript submitted for publication. Transgenics expressing LuxI from Agrobacterium, and an array of ScFv transgenics (more in 5 below) have also been propagated for testing. 3) Two new chimeral peptides (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A group of 100 Carrizo plants were obtained as rooted cuttings and will be used for HLB testing. 4) A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets citrus chloroplasts. Transcription data were obtained by RNA-Seq showing significant alteration in the transgenics. Publication submitted. 5) Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have putative transgenic Carrizo reflecting 69 events from 7 ScFv with verified transgenics ready for testing. These have been replicated by rooting and will be exposed to no-choice CLas+ ACP followed by whole plant destructive assays. 6) To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 showed significant canker resistance to spray inoculation. Paper is published. In-silico analyses are being conducted to develop citrus FLS2 optimized for sensing CLas flagellin. 7) Arabidopsis DMR6 (downy mildew resistance 6)-like genes were downregulated in more tolerant Jackson compared to susceptible Marsh grapefruit. DMR6 acts as a suppressor of plant immunity and it is upregulated during pathogen infection. In a gene expression survey of DMR6 orthologs in Hamlin , Clementine , Carrizo , rough lemon, sour orange and citron, expression levels were significantly higher in all CLas-infected trees compared with healthy trees in each citrus genotype. We developed 2 RNA silencing (hairpinRNA) constructs aimed to silencing citrus DMR6 and DLO1 respectively. Citrus DMR6 is silenced in hairpin transgenic plants and with an average silencing efficiency of 41.4%. DMR6 silenced Carrizo plants (28 independent so far) exhibit moderate to strong activation of plant defense response genes. Determination of silencing efficiency of DLO1 in transgenic plants (20 plants so gar) are ongoing. Comparison of reactive oxygen species in transgenic and nontransgenic plants treated with CLas-flg22 are underway, to determine if there is an enhancement of the broad-spectrum PAMP-triggered immunity . With targeted gene expression data, we will propagate selected plants based on the above-mentioned tests for HLB inoculations purpose. 8) Optimizing use of a SCAmpP (small circular amphipathatic peptide) platform, was conducted in collaboration with Dr. Belknap and Dr. Thomson of the Western Regional Research Center of USDA/ARS. SCAmpPs were recently identified and have tissue specific expression, including having the most abundant transcript in citrus phloem. Furthermore, members of the SCAmpP family have highly conserved gene architecture but vary markedly in the ultimate gene product. Variants of a tissue-specific SCAmpP were tested using GUS as a reporter gene: removal of the conserved intron reduced tissue specificity and deletion of non-transcribed 5 region reduced expression. Excellent phloem-specific expression is achieved in citrus when a target gene is substituted for the gene encoding the SCAmpP peptide. We are using this promoter aggressively in transgenic work 9) Third generation chimeral peptides were designed based on citrus thionins and citrus lipid binding proteins and plants have been transformed. Carrizo transformation of two constructs was completed and regenerated many seedlings. About 40 of each group are being tested for transgene insertion and level of expression. Two constructs with above gene driven by double 35S promoter have 400 explants of Ray Ruby for each. 10) Two constructs with chimeral peptides containing citrus thionin and citrus proteinase were developed with both encoding genes are under by 35S promoter and SCAmpPs promoters. Transformation of those constructs are ongoing.
The objective of this research will 1) characterize Pr-D (FP3) and its role and disease suppression; 2) investigate the dynamics of the prophages/phages in Las bacteria by revealing the variations in gene expression and recombination; and 3) identify critical elements, such as heat and chemical stress that facilitates lytic activities of the prophages. In addition, we will demonstrate whether or not the cross protection using mild strains of Las bacteria will work for the HLB pathosystem along with quantitative detection protocols for prophage-based strain differentiation. To identify the new prophage/phages in Las genome from Florida isolates we isolate DNA from mealybugs, all mealybugs tested contained a high level of iFP3 and very low levels of FP1 and FP2, contrary to psyllids in which FP1 and FP2 were the most abundant and iFP3 was absent. The iFP3 is believed to be the recombinant of FP1 and FP2. Using an Extra-Long PCR, we were able to obtain additional 6 Kb amplicon, and after sequencing confirmation. We believe that FP3 is much small than FP1 and FP2. Further characterization of FP3 on their roles in attenuating Las transmission and HLB symptoms are underway. In order to define the mechanisms that phage are employing to overcome abiotic stress, we designed and optimized specific primer sets for quantitative reverse transcription PCR (qRT-PCR) for genes within the phage region that were likely regulated by heat treatment and other stress. Because this analyses are based on mRNA transcript level and not on genomic DNA, the upregulation of phage genes reflects the relative level of transcript of active living cells. In order to ensure adequate generation of cDNA from Las, we found it necessary to use individual primers specific for the targeted region instead of generalized primers such as random hexamers. The cDNA that was generated in this fashion was then used as the template for qRT-PCR. The present analysis included three biological samples for each condition and threetechnical replicates for each sample for statistical purposes. Particular genes that were found to be upregulated included: CLIBASIA_5590 encoding an unknown protein, CLIBASIA_5610 encoding a putative phage terminase (large subunit), CLIBASIA_5665 encoding an unknown protein, CLIBASIA_5390, which has the conserved sodium: dicarboxilate symporter family domain, CLIBASIA_5525 encoding a guanylate kinase that catalyzes the reaction ATP + GMP <->ADP + GDP. Phage genes found to be downregulated included: CLIBASIA_00005 encoding an unknown protein, CLIBASIA_00010, which has an NTPase domain of typical DNA-packaging enzyme, CLIBASIA_00030 encoding a putative DNA polymerase of bacteriophage origin, CLIBASIA_5565, which has the conserved domain TolA protein and is thought to be required for the translocation of the phage DNA. This data correlated well with what was seen via our previous RNA-Seq analysis and helps reveal the transcriptional response of the phage to abiotic stress factors. Given that previous studies on thermotherapy showed an overall reduction in Las titer in citrus affected by HLB post heat treatment, harnessing the ability to control these particular genes may allow us to lower the bacterium s ability to handle stress. Based on the variations of Las prophages/phages, we recognized certain molecular mechanisms behind the symptom variations and their association with “mild strains” of Las bacteria and host tolerance/resistance. The titration dynamics between 16S DNA-based and phage gene-based results revealed the association of host tolerance with the dynamics. Construction of a transcriptional reporter system is also currently in progress for the final verification of the genes identified as being involved in stress response to heat in plants subjected to thermotherapy. This system will also allow future experimentation to rapidly identify other catalysts that can produce the same reduction in bacterial numbers as thermo-therapy. To investigate the effects of stress on the genes involved in the phage lytic cycle, we identified several phage genes that were over-expressed in citrus plant after heat treatment. These results indicated that thermotherapy has a direct effect on Las bacteria by actively regulating specific phage genes. To further evaluate genes related to stages of the phage lytic cycle, we further compared Las genes expression profile from two distinct insect vectors, psyllids, and mealybugs. Mealybugs were found to contain much higher titers of Type D when compared with psyllids, indicating that the phage may be more active in the mealybugs than in the psyllids. New results revealed Type D prophage/phage is smaller than the prophage/phage A and B, and the association of Type D with suppression of Las bacterial population and HLB symptom expression is under investigation. Using an enrichment method to acquire RNAseq data, we compared the Las transcriptomes between the two insect vectors, and revealed that psyllids samples contained more than four times reads of the Las 16s rRNA than those of mealybugs samples, indicating much higher bacterial titers in psyllids than in mealybugs. However, mealybug s transcriptome profiling showed much higher expression level of prophage genes than those in psyllids, where expression level of prophage genes was completely absent or extremely low. Interestingly, more than 2/3 of the highly expressed genes in mealybugs were identified as prophage/phage genes. Interestingly, eight genes with the highest level of expression in mealybugs were identified as the highly expressed ones in Las-infected citrus after heat treatment. These results indicates that both stresses caused by thermotherapy and in mealybugs environment triggered similar signaling pathways, and result in the expression of prophage genes that may induce lytic cycle and eventually reduce Las titer in citrus plant treated with heat stress, and maintain low titer in mealybugs. We also revealed another mobile element that co-exist as a high-copy element as the prophage/phage D (FP3) in mealybugs and periwinkle. How these mobile elements suppress the HLB are under investigation.
We have begun merging and comparing the collected data from the three different sampling locations, Ridge, South Florida and Indian River. Using neural networks to analyze the data is underway and a few compelling results have been obtained and need further validations. Objective 1: Leaf nutrient thresholds At this point, only the Ridge data, which is complete through March 2017, has been analyzed using the neural network software Easy-NN. We are looking at the sample dates as snapshots in time and combined for any possible connection or correlation with HLB severity. First, we found that the parameters of mean leaf area and mean leaf perimeter were highly correlated with severity of HLB symptoms. As has been noticed in the past, trees with severe symptoms of HLB have smaller leaves, leading to lower average leaf areas. Also with the use of ImageJ software for analysis of leaf size and shape, we have found perimeter to also be correlated with the severity of tree dieback from HLB. Using mean leaf perimeter and leaf area as outputs in generating neural networks, we have found that some of our various measurements are of more important in explaining the means for leaf area and perimeter. Looking at the Ridge area data across all dates (including all data except soil data), we found the leaf nutrients Calcium and Magnesium are of high importance. When we run the neural network with Soil parameters, we find that the organic matter content in the soil plays a role in determining the mean leaf area. As the soil L* parameter increases, which is the lightness in color of the soil, the mean leaf area decreases. However on its own this variable is not well suited for predicting HLB incidence or severity, it does help to show the importance of organic matter in tree health. Other variables of importance in the data set include, Soil Calcium and Magnesium content as well as soil pH. Looking further into the soil parameters as inputs and using Leaf Thickness (grams of leaf dry mass / meter square leaf area) as an output, we find the most important soil variables to be the soil organic matter content and the soil color variable a*, the redness of the soil. These are only preliminary results and more investigation is necessary as well as increasing the data set over the next few quarters. Including the other two areas, Indian River and South Florida, we should be able to strengthen our data set, neural network and conclusions. Objective 2: Determine soil conditions that favor root hair and VAM proliferation i. We have discussed further soil analysis that we would like to work on, including data about permanent wilting coefficient as well as possibly quantifying the silicon content of the soil. Soils from the South Florida area will be included into the data set and will be measured for all of variables the other two regions have been measured for, including organic matter content, and color analysis. ii. Using rooted cuttings and the aeroponics tanks did not yield any results. We believe this is because the micro-jet spray in the tanks causes too much disturbance and damage to the root hair development. Next, we will try with true hydroponics, growing seedlings in solutions for minimal root disturbance. Seeds are in trays for germination now.
This project continues to make progress. We have grafted the new copies of the LuxI plants and they are growing well. We plan to begin the chemical analysis (to determine if the plants are producing the targeted homoserine lactones) in June 2017. Following confirmation of HSL production, the plants will be challenged with ACP. However, this work will not be accomplished until after the end date of the project; therefore, in April 2017 we submitted a request for a no-cost extension. We look forward to the results of this work and know it will provided valuable information to the citrus research community.
This project continues to make progress. We have grafted the new copies of the LuxI plants and they are growing well. We plan to begin the chemical analysis (to determine if the plants are producing the targeted homoserine lactones) in June 2017. Following confirmation of HSL production, the plants will be challenged with ACP. However, this work will not be accomplished until after the end date of the project; therefore, in April 2017 we submitted a request for a no-cost extension. We look forward to the results of this work and know it will provided valuable information to the citrus research community.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) Conducted two more rounds of cage experiments to further test if the transgenic plants inhibit psyllid reproduction. Results showed that none of the transgenes was able to significantly reduce psyllid progeny numbers in the cage experiment. (2) Newly generated replicates of the transgenic lines were inoculated in the psyllid room and nymph-infested plants were recorded and moved to the immediate greenhouse for symptom development.
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