Project narrative: We aim to understand the specific interactions between Candidatus Liberibacter asciaticus (CLas) and the insect vector Asian citrus psyllids (ACP) to block the transmission. ‘The transmission process of CLas depends on the success of specific interactions between CLas and the insect vector ACP. The bacterium passes through the intestinal barrier to reach the hemolymph where they multiply then they must invade the salivary glandes in order to be inoculated in a new plant host while insect feeding. Passing these biological barriers needs specific interactions between CLas cells and the epithelial cells in the guts and the salivary glands cells.’ major finding since the start of project: 1- Identification of the receptors in the Asian citrus psyllids (ACP). The function of these proteins was analyzed with bioinformatics. These genes were cloned, proteins were expressed, antibody against these proteins were made. “a publication described this work is in preparation” 2- Recently, we were able to establish a new method we called it Reverse Far-Western to identify the membrane protein in CLas that bind to the receptors. “Since we have identified the receptors in ACP, we predicted the antigenic domains in the identified ACP-receptors to produce the antibodies. We already obtained antibodies against some of them. Our aim for the next few months is to identify the proteins of Clas (ligands) that adhere to ACP cells. We purified the membrane proteins from infected phloem sap (CLas membrane proteins) using TritonX114, X100. investigated proteins in PAGE show very nice membrane protein profile. We will use these purified membrane proteins for our reverse Far-Western The yeast double hybrid system will be used to validate specific interaction for each couple (receptor-ligand) Overall, our research work is carried out according to milestone of the project. Currently, 1- We are purifying Ferritin from ACP haemolyph to check if it bind to the CLas membrane protein. a potential role for ferritin as a CLas carrier is found in our work. 2- We start to establish the complexome to study the interaction of both partners (receptor-ligands) in native conditions. 3- We are targeting ACP receptor by RNAi to test if we can interfer with the interaction with CLas and subsequently the transmission.
There was no funding received until the end this quarter, so little progress could be made.
This project has been difficult from the beginning and when we were coming up on the third year, all of my Co-PIs decided they did not to continue on this project for various reasons. I did want to continue because my part of the project was going well. It took some time to sort this out but by May 9, 2012 I had e-mailed Dr. Browning for guidance. He indicated that I could submit by myself. I did this on June 19. I know it was received because Ms. Nowicki had me change something in the budget. And the executed contract was sent. Then I heard nothing. On September 28, Dr. Turpen called me about why I had not submitted paperwork. I told him I had and resent the third year paperwork to him. Again I heard nothing until an NOA was sent in the middle of November for $39,000. When someone in DSR asked Ms. Nowicki about a no-cost extension, she said there would not be one, I had until December 31 to spend the money because I had been so late in getting in the paperwork. I was not extremely late in getting in the paperwork! And of course I could not spend all of the funding in six weeks (during the holiday season) without doing things I am not comfortable doing because I have no desire to fail an audit or get in trouble with either CRDF or UF. A 6 month no-cost extension was finally executed. This allowed us to continue preliminary work on cell penetrating peptides, which led to the funding of Project 752. How to write the reports was problematic because of the gaps in funding and although I asked for guidance, I did not receive any.
There was no funding received during this quarter, so progress could not be made.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This greenhouse is completed and operational. – The first cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation are currently being propagated.
Oral uptake of dsRNA targeting specific Asian citrus psyllid genes can induce psyllid mortality and reduce Liberibacter titer in infected psyllids. Significant progress has been made with research on the use of RNAi as a means of Asian citrus psyllid control. We have made use of a Citrus tristeza virus (CTV) dsRNA expression system which when inoculated into C.Marcrophylla results in leaf phloem containing dsRNAs which target essential ACP genes. When ACP feed on leaves from these “paratrangenic” (CTV transfected) citrus plants, mortality was double that observed when the ACP fed on artificial diets containing this synthetic dsRNA, greater than 80% mortality. Currently, new versions of paratransgenic citrus are being produced to determine the best sequence to use targeting this essential gene. The use of the Ion Torrent (by Life Technologies) next generation sequencing has provided rich insight into the transcription profile of “paratransgenic” fed “sick” ACP using comparative RNA Seq analysis. The data support the specific down-regulation of the dsRNA target gene as the cause of mortality as seen by the significant perturbation of genes in the molecular pathway in which this gene functions. RNA Seq data continues to be collected from ACP fed on a variety of gene specific dsRNA containing diets. Experimentation has also begun using artificial diets containing dsRNAs synthesized by a novel “mass-production” technique that would make it practical for the use of RNAi technology as a field application. The use of topical application in conjunction with “paratransgenic” plant varieties presents a strategy for effective delivery and a multiple gene targeting employment of the RNAi pest control technology.
We hypothesized that groves with high bicarbonate stress are suffering from HLB because they support lower fibrous root density compared to groves with lower bicarbonates (less than 100 ppm) in irrigation water and/or soil pH (less than 6.5). To confirm this relationship, we surveyed 37 grove locations in Highlands and Desoto counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density is significantly related to well water pH greater than 6.5 and to soil pH greater than 6.2. Yield records from these blocks reveal that groves under high bicarbonate stress production have declined 20 percent over the last three seasons (2009-2012) in contrast to Ridge groves with low bicarbonate stress which have increased 6 percent in production even though HLB incidence has accelerated. The yield losses are correlated with less fibrous root density, which reduces root system capacity for water and nutrient uptake. Evidence from research on other crops indicates that bicarbonate impairs the root’s ability to take up important nutritional cations including Ca, Mg and K, as well as micronutrients, especially Mn and Fe. In Florida, Bryan Belcher of Davis Citrus Management has acidified irrigation water with sulfuric or N-furic acid (a mixture of urea and sulfuric acid) by injection at the well in the same way as fertigation. N-furic has the advantages of being safer to handle and providing some additional N due to the urea component, but the disadvantage is higher cost of treatment compared to sulfuric acid. Since irrigation is not necessary when it rains, acidification treatment only occurs during the dry season when the bicarbonates are loading into the wetted area under the tree. When the rain begins, these bicarbonates are flushed from the rhizosphere. Our labs and grower cooperators are also evaluating acidification of soil by amendment with elemental sulfur applied in prilled or finely ground form. Sulfur (S) releases acid when it interacts with Thiobacillus bacteria in soil to form acid (H+) ions. This process of acidification with S is slower than treatment of the water but provides for longer lasting reduction in soil pH. Sulfur can be applied in prilled form with a fertilizer spreader or with a herbicide boom as a slurry. Sulfur can also be added to dry and fertigation formulations to lower the pH by as much as one unit after repeated ground applications.
We hypothesized that groves with high bicarbonate stress are suffering from HLB because they support lower fibrous root density compared to groves with lower bicarbonates (less than 100 ppm) in irrigation water and/or soil pH (less than 6.5). To confirm this relationship, we surveyed 37 grove locations in Highlands and Desoto counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density is significantly related to well water pH greater than 6.5 and to soil pH greater than 6.2. Yield records from these blocks reveal that groves under high bicarbonate stress production have declined 20 percent over the last three seasons (2009-2012) in contrast to Ridge groves with low bicarbonate stress which have increased 6 percent in production even though HLB incidence has accelerated. The yield losses are correlated with less fibrous root density, which reduces root system capacity for water and nutrient uptake. Evidence from research on other crops indicates that bicarbonate impairs the root’s ability to take up important nutritional cations including Ca, Mg and K, as well as micronutrients, especially Mn and Fe. In Florida, Bryan Belcher of Davis Citrus Management has acidified irrigation water with sulfuric or N-furic acid (a mixture of urea and sulfuric acid) by injection at the well in the same way as fertigation. N-furic has the advantages of being safer to handle and providing some additional N due to the urea component, but the disadvantage is higher cost of treatment compared to sulfuric acid. Since irrigation is not necessary when it rains, acidification treatment only occurs during the dry season when the bicarbonates are loading into the wetted area under the tree. When the rain begins, these bicarbonates are flushed from the rhizosphere. Our labs and grower cooperators are also evaluating acidification of soil by amendment with elemental sulfur applied in prilled or finely ground form. Sulfur (S) releases acid when it interacts with Thiobacillus bacteria in soil to form acid (H+) ions. This process of acidification with S is slower than treatment of the water but provides for longer lasting reduction in soil pH. Sulfur can be applied in prilled form with a fertilizer spreader or with a herbicide boom as a slurry. Sulfur can also be added to dry and fertigation formulations to lower the pH by as much as one unit after repeated ground applications.
Stress intolerance of HLB trees is a direct consequence of a more than 30 percent loss of fibrous root density compared to non-diseased trees. This root loss may be compounded by interactions with root pathogens and pests such as Phytophthora spp. Progagules of Phytophthora spp. are elevated in the rhizosphere soil of HLB trees. When Las interacts with Phytophthora, fibrous root loss can be greater than that caused by HLB alone, depending on the grove location and time of year. Although not directly studied, HLB associated root loss has been attributed to carbohydrate starvation. Initially, canopy symptoms are not apparent in trees with significant root loss, so starch concentration in roots was determined. Surprisingly, early root loss occurred without a drop in starch. This disappearance of roots could be due to the lack of new growth to replace old roots during the normal cycle of death and regeneration of fibrous roots, or may be associated with premature root death unrelated to phloem plugging, or a combination of the two processes. Also, it is highly likely that Las damage alters the concentration of soluble sugars in roots by increasing leakage from roots that attracts and accelerates infection by root pathogens such as P. nicotianae.
Seasonal root sampling continues in two field sites with a third site identified. Sampling has already revealed seasonal variation in root infections and apparent shifts in the root flush cycle caused by Liberibacter. We have swapped out root cages twice and collected data on root flushes in response to HLB during a normal root flush and nonflushing time. Data analysis on initial root flush data in comparison to overall root density is underway and data collection will continue through the year. Some difficulty is being encountered with finding sufficient presumed healthy trees in the field, so more emphasis will be placed on greenhouse experiments as the project continues. We will continue to look for new sites with moderate infection rates and trees old enough for routine root sampling. Sampling at a rootstock trial site is underway with a full year of data on the effects of HLB on these new experimental rootstocks. This has already begun to demonstrate how these new rootstock lines respond to Liberibacter infection. We are in the final preparation stage of testing the most promising rootstock lines in more controlled greenhouse studies and are awaiting improved weather conditions for high efficiency inoculation Liberibacter.
FireWall (22.3% streptomycin sulfate; Agrosource, Inc.) was granted by an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey was to apply our published protocol for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc. Two sites in two commercial grapefruit groves and a trial site treated the last 4 out of 5 years with FireWall (including 4 sprays in 2013) were sampled for streptomycin resistance on September 24 and 25, 2013. From the last mature flush that earlier in the season was directly contacted by the FireWall in spray treatments, leaf samples with canker lesions and one sample without lesions (check) were collected at five locations (four corners and the center) in each of the grove blocks. From each sampling location, 4 fully expanded leaves with canker lesions were placed in a flask. As a streptomycin-positive Xcc check, a single leaf with canker lesions produced by injection-infiltrated with a streptomycin resistant Xcc strain was placed in the flask with four asymptomatic leaves. Total colonies recovered from KCH selective medium ranged from 6 to 107 . 104 cfu/ml of washate . The number of bacteria that grew on KCH-S from the samples ranged from 0 to 80 cfu/ml and from the inoculated checks ranged from 3 to 36 cfu/ml. The identity of each yellow colony on KCH-S as Xcc was tested with Agdia test strips. No resistant Xcc colonies were detected from the grove samples with canker lesions. Positive (resistant) Xcc were only detected from the checks that had been spiked with grapefruit leaves inoculated with a streptomycin resistant strain. Recovery of streptomycin resistant colonies validated the protocol for isolation and detection of resistant Xcc from field leaves with other bacteria present. Conclusion: No Xcc resistant to streptomycin were recovered from four commercial groves locations nor were resistant Xcc recovered from the trees in a trial at Ft. Pierce that received four sprays of FireWall or Firewall plus two sprays of Kocide in 2013. The trees in Ft. Pierce had a history of 33 sprays (11 per season) of FireWall or FireWall+ Kocide from 2009-2011. Hence, Xcc in this location have a 4 year history of high frequency exposure to streptomycin without development of resistance thus far.
Objective 1. To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface. Strains of Xanthomonas citri subsp. citri (Xcc) and other Xanthomonads sense signals from the host which facilitate the location of leaf entry points that are specific for each bacterium-host association. In addition, there are differences in swimming as well as surface motility among the wide and narrow host range strains of Xcc. Differential biofilm formation was observed in vitro and in planta among the Xanthomonas strains assayed. Minimal medium XVM2 significantly contributed to biofilm formation for every strain analyzed. Biofilm production of wide host range Xcc did not differ between minimal XVM2 and the nutrient rich LB medium. In contrast, narrow host range Xcc strains formed much more biofilm in XVM2 medium. In addition, results in planta showed that the Xanthomonas stains assayed produced much less biofilm on non-host leaves or fruits than the respective host plant. Furthermore, SEM of biofilms onleaf and fruit surfaces revealed different structure biofilm aggregates betwenn narrow and wide host range Xcc strains. Large differences in swimming motility between wide and narrow host range strains were also found. It appears that narrow host range strains of CBC partially compensate for the lack of biofilm formation with the greater aggregation in the appropriate niche and higher swimming motility. Objective 2. To investigate bifofilm formation and composition and its relationship with bacteria structures related to motility in different strains of Xcc and comparison to non-canker causing xanthomonads. Two assays were performed to determine extracellular DNA presence in biofilm matrix. DNase (SIGMA) was added to bacterial culture in XVM2 media at 0, 24, 48 and 72 hours incubation. In such assays, effect of the DNAse was clear again at the initial stages of biofilm formation (0h), confirming the role of the DNA at the first stage of the biofilm for Xcc and X. alfalfae subsp. citrumelonis. Effect of the DNase in Aw and A* strains differed according to time of incubation. No clear effect of the DNAse was found in Xanthomonas campestris pv. campestris. To visualize DNA in biofilms, bacterial aggregates on glass surface were stained with Propidium iodide and SYTO9. Fibers visualized by violet crystal were stained, confirming the fibers contain DNA. Simultaneously matrix proteins were stained with biofilm SPYPRO (Invitrogen). Most of the fibers have been identified by these two methodologies as containing a mix of DNA and proteins. Transcription of genes fimA (XAC3241), fimA (Xac3240) (Pilus type IV), fliC (flagelin), fleN (flagella regulator), pilA (fimbria), motA (flagella motor), rpfF and rpfB (quorum sensing signal) were analysed on dried and inundated biofilm as well as the planktonic bacterial stage after 72 h incubation in both LB and XVM2 culture medium. Three independent assays were started and from one of them RNA was already extracted and amplified by RT-PCR. RNA was obtained from aggregates produced at the bottom of culture flask after or before the dry process as well as aggregates of the air-liquid interface. Analysis of the results is under progress but some preliminary infers could be concluded. The assay revealed that in wide and narrow host range Xcc strains, fimA (XAC3241) expression was higher in early stage of the biofilm formation as compared to after biofilm establishment. This gene was particularly upregulated in XVM2 medium compared with the biofilm or planktonic stages of the bacteria. When planktonic and biofilm bacteria were compared, differential expression of flagella genes (fliC, fleN, motA) was found between the wide and narrow host range strains when incubated in LB medium but not in XVM2 medium. transcription of the genes’.were analysed on dried and inundated biofilm as well as planktonic bacteria stage
We initiated an Illumina NextGen DNA sequencing project comparing ‘Sun Chu Sha’ mandarin (HLB tolerant) and ‘Duncan’ grapefruit (HLB sensitive) infiltrated with Candidatus Liberibacter asiaticus flagellin 22 (CLas-flg22) peptide and water control. Between 42 and 63 millions of pare-end reads were generated from our six cDNA libiaries per genotype (three replicates for CLas-flg22 treatment and control, respectively). Using Citrus clementina genome as the reference, an average of 45% of reads for each sample were uniquely mapped reads. It was shown that over 700 genes were differentially expressed in ‘Sun Chu Sha’ mandanrin due to the treatment of CLas-flg22. In contrast, only 1 gene is differentially expressed in ‘Duncan’ grapefruit. Interestingly, in our other project comparing the effect of flg22 of citrus canker causal bacteria Xanthomonas citrus subsp. citri (Xcc-flg22) showed that a much more extensive transcript reprogramming was triggered in both ‘Sun Chu Sha’ (over 2600 genes) and ‘Duncan’ (over 1300 genes), suggesting a weaker defense eliciting ability of CLas-flg22 than Xcc-flg22’s. Functional analysis of CLas-flg22 affected genes in the two citrus genotypes is underway.
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. During this quarter we accomplished: ‘ Baseline sensitivity of Alternaria alternata population to boscalid manuscript is on-going ‘ Structure of SDH-subunits A,B,C, and D has been determined in baseline population ‘ Manuscript “QoI-resistance stability in relation to pathogenic and saprophytic fitness components of Alternaria alternata from citrus” was submitted ‘ Preliminary planning for adapting rezasurin assay for use with DMI fungicides underway
In collaboration with Mike Irey, we completed evaluation of a population of transgenic lines for their resistance to Clas. Transgenic trees were kept in a greenhouse containing CLas+ Asian Citrus psyllid (ACP) adults and trees were evaluated every 6 months for HLB for 2.5 years. In addition, psyllids were tested periodically for the presence of CLas. Although we had transgenic trees from four transgenes (LIMA, AttacinE, CEAD and NPR1) showing resistance to HLB, the results from NPR1 were the most promising. 27% of the trees containing the 35S-NPR1 construct and 57% of trees with the phloem specific AtSUC2-NPR1 construct survived and were PCR- after 30 months of inoculation. The HLB-free trees were moved to the SG field site under MTA and DPI petition (many in poor condition due to severe psyllid damage). Additional clones of the promising transgenic lines are currently being propagated for additional field testing in the Dunwoody Grove of Southern Gardens. In addition, Several newer NPR1 lines are already in the field test at the Picos Farms (USDA) and results there are promising as well. Our current goal is to combine transgenes that function by completely different mechanisms as to have a back-up to prevent the pathogen from overcoming single gene resistance in the field. We have produced 40 new transgenic plants of Hamlin cominging NPR1 with the successful AMP genes CEME or CEMA. We have successfully developed a heat inducible Cre/loxP site specific based recombination system for efficient excision of antibiotic resistance genes in citrus. In our construct, the nptII gene under the control of the NOS promoter and the Cre recombinase gene driven by either a soybean heat shock protein (hsp17.5E) promoter or a Arabidopsis thaliana small heat-shock protein (HSP20) gene promoter were flanked by two loxP recognition sites in direct orientation. An anthocyanin biosynthesis gene from Vitis vinifera (VvMYBA1) was placed outside the loxP sequence. Transformation efficiencies were similar using either soybean or the Arabidopsis promoter. Anthocyanin activity analysis on transformed Carrizo citrange (Citrus sinensis x Poncirus trifoliata) demonstrated that approximately 30-40% of transformation efficiency could be obtained following Agrobacterium mediated transformation and heat shock treatment. Molecular analyses have demonstrated that 100% selectable marker gene deletion occurred in all regenerated plants expressing anthocyanin. We have completed building a RES type structure in our greenhouse#7. Transgenic trees have reached the top of the structure and have been bent downwards. We did not observe any flowering this past year; however, downward growing branches on many of the trees have completely lost their thorns, indicating a rapid reduction in juvenility. Since this greenhouse is heated during the winter, flowering induction may be inhibited.
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