The goal is to understand how citrus interacts with Candidatus Liberibacter asiaticus (Las) infection. To achieve the goal of this research, we are conducting the following objectives: Objective 1. Identification of the receptors for Las PAMPs in susceptible and tolerant citrus varieties21 outer membrane proteins have been cloned and the putative targets in citrus are being identified using Yeast 2 hybrid system. Potential PAMPs from Las (either homologous to known PAMPs or pilin genes) LasFlaA (flagellin), LasEF-Tu, LasCSP (cold shock protein), LasSSBP (single strand binding protein) and pilin assembly genes (named LasPil85, LasPil95, LasPil105 and LasPil115) were cloned under 35S promoter and the Arabidopsis phloem specific promoter SUC2 and introduced into Agrobacterium. We are testing their receptors in Tobacco and citrus. We have identified multiple receptors for the aforementioned PAMPs and are in the process of confirmation.In addition, multiple PAMPs are being tested for their effects in inducing plant defense against Las in the greenhouse. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe have the selected PAMP receptors and are overexpressing them in citrus. The constructs have been made. Objective 3. Investigate the roles of effectors in HLB disease developmentFor the 10 selected Sec-dependent effectors (SDEs), we have conducted yeast two hybridization (Y2H) and identified their targets in Valencia sweet orange. We are in the process of confirming the targets using other approaches such as bimolecular fluorescence complementation (BiFC) and Co-Immunoprecipitation (co-IP) assays. We are conducting Y2H and SPR assays to identify their targets in Poncirus and in the tolerant variety sugar belle. We are overexpressing the SDEs in tobacco and citrus to test their effect on HLB disease development.
We continue to work with Michael Rogers on glyphosate as a treatment for citrus greening disease.
Over a period of five months in the greenhouse, we determined the optimal concentration of glyphosate that citrus Valencia saplings could tolerate as well as the frequency with which it could be applied. We learned that citrus can tolerate an 8 mM spray of glyphosate every three months. The plents would lose a few leaves at first but after three months, flush would appear and the plants appeared to recover.
The sprays were applied at monthly intervals. We learned that monthly and semi-monthly sprays were too frequent. We also learned that 25 mM sprays were lethal. Thus, those were discontinued. The source of glyphosate was RoundUp as pure glyphosate was too expensive. RoundUp would be the source that growers would use in any case.
A field experiment was then started six weeks after the greenhouse experiment. The plots were set up at Lake Alfred by Michael Rogers. Prior to the first spray, the trees were sampled to determine CLas titer. Disease severity of the trees also determined.
As we now know from the greenhouse experiment that a three-month interval allows for tree survival and continued growth, the field plot is scheduled to be sprayed again in early August. The trees will be sampled and assessed for disease severity before and after the three-month spray.
Qualitatively, the results from the field are encouraging and mimic the results from the greenhouse. Those plants sprayed with 8 mM glyphosate are recovering and have new flush. The untreated control plants have no new flush. The plants sprayed with 25 mM glyphosate were nearly killed. We are not treating those trees again. We will let them recover.
We are eager to continue this experiment over the next few months and hope to learn the effect of glyphosate on CLas titer over the next three months.
Meanwhile, the transformation of citrus to generate RoundUp ready plants in a cis-genic manner is in progress at Lake Alfred. We believe that using cis-genic plants will be the long-term solution.
In collaboration with CREC Driector Michael Rogers , we are proceedng with laboratory, greenhouse, and field experiments to determine whether glyphosate can control cirtus greening disease. In the laboratory, we determined the levels of glyphosate that inhibit L. crecesns. Glyphosate inhibits aromatic amino acid synthesis in any organism that produces these compounds. This includes plants and many bacteria, including L. crescens. Liberibacter crescens is also inhbited by glyphosate when the cells are cultured in the presence of aromitc amino acids suggesting that there may be another site of action for glyphosate in addition to the ESPS protein. Based on those experiments, we choose 8 mM and 25 mM concentrations of glyphosate to test for citrus toxicity in the greenhouse. In the greenhouse, 25 mM glyphosate was quite toxic to Valencia citrus. The plants survived 8 mM glyphosate but it delayed growth of flush by two months. We next tested the intervals at which glyphosate can be applied in the greenhouse. Two sprays of 8 mM glyphosate one month apart caused considerable leaf drop two weeks after the second spray. Two sprays of 8 mM glyphosate two months apart hurt the growth of flush but the flush is returning three weeks after the second spray. These interval sprays will continue monthly until the first and last sprays are six months apart. We expect that sprays three months apart will not significant effect citrus growth and yield. The greenhouse experiments above are six weeks ahead of the field trial. Thus, the greenhouse trial will inform us as to the best interval for spraying the field. The field experimental design was done by Michael Rogers. He also did the first spraying of the field trees nearly two months ago. As in the greenhouse, 25 mM glyphosate killed the trees while 8 mM did not. At 8 mM growth was slowed as in the greenhouse experiment. Based on the greenhouse trial, we are going to wait until three month after the first spray to spray again. Pre- and post-spray sampling is being done to assess CLas titer and disease severity. Meanwhile, Zhonglin Mou on our team is working with the citrus transformation facility to make Valencia cis-genic engineered plants that are resistant to glyphosate. This required the construction of an ESPS synthase gene that contains the one base change needed to confer glyphosate resistance. This gene is being inserted into mature and immature citrus. The selection of plants with the mutated gene is simple as it is just a glyphosate screen. These plants should be available early next year if not sooner for both greenhouse and field testing.
In collaboration with CREC Driector Michael Rogers , we are proceedng with laboratory, greenhouse, and field experiments to determine whether glyphosate can control cirtus greening disease. In the laboratory, we determined the levels of glyphosate that inhibit L. crecesns. Glyphosate inhibits aromatic amino acid synthesis in any organism that produces these compounds. This includes plants and many bacteria, including L. crescens. Liberibacter crescens is also inhbited by glyphosate when the cells are cultured in the presence of aromitc amino acids suggesting that there may be another site of action for glyphosate in addition to the ESPS protein. Based on those experiments, we choose 8 mM and 25 mM concentrations of glyphosate to test for citrus toxicity in the greenhouse. In the greenhouse, 25 mM glyphosate was quite toxic to Valencia citrus. The plants survived 8 mM glyphosate but it delayed growth of flush by two months. We next tested the intervals at which glyphosate can be applied in the greenhouse. Two sprays of 8 mM glyphosate one month apart caused considerable leaf drop two weeks after the second spray. Two sprays of 8 mM glyphosate two months apart hurt the growth of flush but the flush is returning three weeks after the second spray. These interval sprays will continue monthly until the first and last sprays are six months apart. We expect that sprays three months apart will not significant effect citrus growth and yield. The greenhouse experiments above are six weeks ahead of the field trial. Thus, the greenhouse trial will inform us as to the best interval for spraying the field. The field experimental design was done by Michael Rogers. He also did the first spraying of the field trees nearly two months ago. As in the greenhouse, 25 mM glyphosate killed the trees while 8 mM did not. At 8 mM growth was slowed as in the greenhouse experiment. Based on the greenhouse trial, we are going to wait until three month after the first spray to spray again. Pre- and post-spray sampling is being done to assess CLas titer and disease severity. Meanwhile, Zhonglin Mou on our team is working with the citrus transformation facility to make Valencia cis-genic engineered plants that are resistant to glyphosate. This required the construction of an ESPS synthase gene that contains the one base change needed to confer glyphosate resistance. This gene is being inserted into mature and immature citrus. The selection of plants with the mutated gene is simple as it is just a glyphosate screen. These plants should be available early next year if not sooner for both greenhouse and field testing.
In collaboration with CREC Driector Michael Rogers , we are proceedng with laboratory, greenhouse, and field experiments to determine whether glyphosate can control cirtus greening disease. In the laboratory, we determined the levels of glyphosate that inhibit L. crecesns. Glyphosate inhibits aromatic amino acid synthesis in any organism that produces these compounds. This includes plants and many bacteria, including L. crescens. Liberibacter crescens is also inhbited by glyphosate when the cells are cultured in the presence of aromitc amino acids suggesting that there may be another site of action for glyphosate in addition to the ESPS protein. Based on those experiments, we choose 8 mM and 25 mM concentrations of glyphosate to test for citrus toxicity in the greenhouse. In the greenhouse, 25 mM glyphosate was quite toxic to Valencia citrus. The plants survived 8 mM glyphosate but it delayed growth of flush by two months. We next tested the intervals at which glyphosate can be applied in the greenhouse. Two sprays of 8 mM glyphosate one month apart caused considerable leaf drop two weeks after the second spray. Two sprays of 8 mM glyphosate two months apart hurt the growth of flush but the flush is returning three weeks after the second spray. These interval sprays will continue monthly until the first and last sprays are six months apart. We expect that sprays three months apart will not significant effect citrus growth and yield. The greenhouse experiments above are six weeks ahead of the field trial. Thus, the greenhouse trial will inform us as to the best interval for spraying the field. The field experimental design was done by Michael Rogers. He also did the first spraying of the field trees nearly two months ago. As in the greenhouse, 25 mM glyphosate killed the trees while 8 mM did not. At 8 mM growth was slowed as in the greenhouse experiment. Based on the greenhouse trial, we are going to wait until three month after the first spray to spray again. Pre- and post-spray sampling is being done to assess CLas titer and disease severity. Meanwhile, Zhonglin Mou on our team is working with the citrus transformation facility to make Valencia cis-genic engineered plants that are resistant to glyphosate. This required the construction of an ESPS synthase gene that contains the one base change needed to confer glyphosate resistance. This gene is being inserted into mature and immature citrus. The selection of plants with the mutated gene is simple as it is just a glyphosate screen. These plants should be available early next year if not sooner for both greenhouse and field testing.
The goal is to understand how citrus interacts with Candidatus Liberibacter asiaticus (Las) infection. To achieve the goal of this research, we are conducting the following objectives: Objective 1. Identification of the receptors for Las PAMPs in susceptible and tolerant citrus varieties21 outer membrane proteins have been cloned and the putative targets in citrus are being identified using Yeast 2 hybrid system. Potential PAMPs from Las (either homologous to known PAMPs or pilin genes) LasFlaA (flagellin), LasEF-Tu, LasCSP (cold shock protein), LasSSBP (single strand binding proein) and pilin assembly genes (named LasPil85, LasPil95, LasPil105 and LasPil115) were cloned under 35S promoter and the Arabidopsis phloem specific promoter SUC2 and introduced into Agrobacterium. We will be testing their receptors in Tobacco and citrus. We have identified multiple receptors for the aforementioned PAMPs and are in the process of confirmation.Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe have the selected PAMP receptors and are overexpressing them in citrus. Objective 3. Investigate the roles of effectors in HLB disease developmentFor the 10 selected SDEs, we have conducting Y2H and identified their targets in Valencia sweet orange. We are in the process of confirming the targets using other approaches such BiFC and co-IP assays. We are conducting Y2H and SPR assays to identify their targets in Poncirus.
This project is a continuation of funding that has been provided to Southern Gardens Citrus (SGC) to provide growers and researchers with a facility to do testing to detect Candidatus Liberibacter asiaticus, the causal agent of citrus greening in Florida. This report covers the third quarter of year two funding. For the period of January 1, 2019 to March 31, 2019, a total of 6,951 samples were processed and tested by qPCR. Of these 95% were plant samples and 5% were psyllid samples. Virtually all of the plant samples were from grower, private entity, or grower research trials. To date, for the two-year project, a total of 42,466 samples have been processed and tested. Based on the current trends, it is expected that the total number of samples that will be processed during the grant period will be approximately 50,000 (budgeted amount was 60,000). If the lab sample load does not reach the budgeted amount, the final bill will be adjusted as necessary to reflect the total number of samples actually run. One trend that is changing is that more customers are requesting copy number information instead of just a positive/negative determination. It is expected that approximately 50% of the year two samples were be provided to customers with copy number determination. In addition, requests are coming in to provide testing using different primer sets and to return the DNA extracts back to the customers for additional in-house or custom testing. When possible, the SGC lab has tried to accomodate these requests.
The objectives of this study are to identify optimal pH range for root function and minimize root turnover on HLB-affected rootstocks and how uneven pH levels in the root zone (e.g. irrigated vs. row middle portions of root system) affect the overall health of the tree. This is being done in a split root system in the greenhouse where pH of different parts of the root system can be controlled an maintained. We are in the final stages of rhizotron construction to build enough for the experiments. Rhizotron construction was slightly delayed because of the late Valencia harvest this year for other projects combined with an unexpected loss of a staff member that will soon be replaced. The Masters student has assisted a member of Tripti Vashisth’s lab with the 2nd repetition of the experiment that created the foundation of this project to become familiar with techniques that will be important for maintaining pH and collecting data. We expect to initiate treatments before the end of May.
The goal is to understand how citrus interacts with Candidatus Liberibacter asiaticus (Las) infection. To achieve the goal of this research, we are conducting the following objectives: Objective 1. Identification of the receptors for Las PAMPs in susceptible and tolerant citrus varieties21 outer membrane proteins have been cloned and the putative targets in citrus are being identified using Yeast 2 hybrid system. Potential PAMPs from Las (either homologous to known PAMPs or pilin genes) LasFlaA (flagellin), LasEF-Tu, LasCSP (cold shock protein), LasSSBP (single strand binding proein) and pilin assembly genes (named LasPil85, LasPil95, LasPil105 and LasPil115) were cloned under 35S promoter and the Arabidopsis phloem specific promoter SUC2 and introduced into Agrobacterium. We will be testing their receptors in Tobacco and citrus. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe are cloning and overexpressing the selected PAMP receptors.Objective 3. Investigate the roles of effectors in HLB disease developmentFor the 10 selected SDEs, we have conducting Y2H and identified their targets in Valencia sweet orange. We are in the process of confirming the targets using other approaches such BiFC and co-IP assays. We will conduct Y2H and SPR assays to identify their targets in Poncirus.
1. Continuuing to improve the defined medium (Cruz-Munoz et al. 2018) for the culture of Liberibacter crescens, the cloest cultured relative of the citrus greening pathogen. An analysis of the amino acid requirements of L. crescens shows that only a few are required for growth. Deletion of the other amino acids from the medium results in growth but it is reduced. During the growth of L. crescens, we found that the pH of the medium increases by over 1 pH unit. Buffering this pH change reduces growth. The cause of the pH increase is under investigation. Nabil Killiny has published that the pH of citrus phloem increased from 5.7 to 6.2 after infection. As a result, we believe that studying this phenomenon in L. crescens may give us translatable results to understanding the cause of disease symptoms. 2. Monitoring of citrus groves for non-target antibiotic resistance prior to and after application of streptomycin and oxytetracycline. We have a method for the rapid detection of streptomycin in the field. We now need to test it in the field. 3. Developing second-generation antimicrobial treatments for citrus greening disease. A new antimicrobial Presto-Blue assay was developed for L. crescens on M15 defined medium. It is being tested on compounds we believe may be important for HLB control. If we have success with these, we will inform CRDF. At the moment, one compound looks to be promising. We are moving forward with a test to determine the spontaneous resistance rate in L. crescens for this compound. 4. Phosphate utilization as a strategy for HLB-disease management A greenhouse experiment is still in progress to determine whether foliar phosphate fertilization can recude citrate levels in phloem. Citrate is a preferred nutrient for Liberibacter. Phosphate fertilization is expected to reduce those levels in phloem sharply, thereby starving the pathogen. The phosphate foliar treatments are provided three times per week in the citrus macrophylla seedlings. When the plants flush, we will move them to a psyllid room in Lake Alfred. Cruz-Munoz, M., Petrone, J. R., Cohn, A. R., Munoz-Beristain, A., Killiny, N., Drew, J. C., & Triplett, E. W. (2018). Development of chemically defined media reveals citrate as preferred carbon source for Liberibacter growth. Frontiers in Microbiology, 9, 668. Killiny, N. (2017). Metabolite signature of the phloem sap of fourteen citrus varieties with different degrees of tolerance to Candidatus Liberibacter asiaticus. Physiol. Mol. Plant Pathol. 97, 20-29. doi: 10.1016/j.pmpp.2016.11.004
1. Developing a culture medium for Liberibacter asiaticus through comparative multiomics analysis with its closest cultured relative, L. crescens: Determined the optimal pH for the growth of L. crescens in M15 defined medium is 5.92, whereas in BM-7 is 6.5. Liberibacter crescens grows well in a pH range of 5.8 to 6.2. This is close to the pH of citrus phloem which is between 5.0 and 5.74, (Killiny. 2017). The level of Ca. L. asiaticus in the citrus phloem might be associated with the pH. During the culture of L. crescens, the pH of the medium rises dramatically. We are concerned that this rise may be limiting growth. As a result, we are conducting experiments to learn the source of the pH rise so that it can be mitigated. Chemically defined medium paper accepted for publication in Frontiers in Microbiology (Cruz-Munoz et al. 2018). Using several media based on M15 for culturing for Ca. L. asiaticus. In addition, various insect cell media are being tried. A cell line of the Asian citrus psyllid has been developed to determine whether Ca. L. asiaticus can be co-cultured with the insect cells. 2. Monitoring of citrus groves for non-target antibiotic resistance prior to and after application of streptomycin and oxytetracycline. A high throughput approach for the rapid assessment of streptomycin resistance has been developed and is now being tested with soil samples for citrus groves. Samples have been collected from four sites for this purpose. Streptomycin resistant bacteria have been isolated from these groves to test the efficacy of this method. To date, about 12% of soil bacteria appear to be resistant to streptomycin. More work is needed to test the level of streptomycin resistant levels in the pathogen in groves where streptomycin is being used compared to sites where it is not being use. 3. Developing second-generation antimicrobial treatments for citrus greening disease. A new antimicrobial Presto-Blue assay was developed for L. crescens on M15 defined medium. This approach was shared with representatives from Bayer who are developing their own high throughput assay against L. crescens. The new defined medium will greatly reduced the cost these assays and they should be more reproducible than BM-7 medium. We have learned that the undefined ingredients of BM-7 medium are quite variable among the manufacturers. 4. Phosphate utilization as a strategy for HLB-disease management A greenhouse experiment is in progress to determine whether foliar phosphate fertilization can recude citrate levels in phloem. Citrate is a preferred nutrient for Liberibacter. Phosphate fertilization is expected to reduce those levels in phloem sharply, thereby starving the pathogen. The phosphate foliar treatments are provided three times per week in the citrus macrophylla seedlings. Cruz-Munoz, M., Petrone, J. R., Cohn, A. R., Munoz-Beristain, A., Killiny, N., Drew, J. C., & Triplett, E. W. (2018). Development of chemically defined media reveals citrate as preferred carbon source for Liberibacter growth. Frontiers in Microbiology, 9, 668. Killiny, N. (2017). Metabolite signature of the phloem sap of fourteen citrus varieties with different degrees of tolerance to Candidatus Liberibacter asiaticus. Physiol. Mol. Plant Pathol. 97, 20-29. doi: 10.1016/j.pmpp.2016.11.004
The goal of this project is to determine whether pathogen or dsRNA exposure primes the ACP immune system to resist future infection by pathogens, including Las, and whether this effect is multigenerational. We have previously characterized the specificity and efficacy of the immune priming response in ACP (Obj. 1), characterixzed the effect of prior immune challenge on transmission (Obj. 3) and determined the transgenerational effect of pathogen-induced immune priming on Las acquisition. The current report describes our ongoing efforts to quantify the effect of RNAi-induced priming on Las acquisition (Obj 4). Adults of ACP collected from a laboratory colony free of CLas infection were starved for 3 hr, and then were subjected to dsRNA ATPase and sucrose. For each treatment, 5 cages including 10 insects in each were used. Insects were fed on diet solution consisting of 10, 100, and 1000 ng. l -1 dsRNA ATPase, 20% sucrose, and 0.5 green food coloring dye. As a control, adult of ACP was fed on sucrose 20%+ 0.5 green food dye. A cage for artificial feeding was prepared by stretching parafilm membranes on the bottom of plastic petri dish arenas. The parafilm surface was sterilized with ethanol and dried for 5 min under a sterile hood and layered with 400 L of diet solution including dsRNA, 20% sucrose, and green food coloring dye. The liquid was then covered with a second layer of stretched parafilm. During the feeding, the cages were placed in a growth chamber at 28oC. Insect were collected after 24 hrs and 5 days feeding and stored in -80 C for RNA extraction. Insects primed by exposure to artificial diet solutions with dsRNA for 24 hours were transferred to separate branches of a potted citrus plant (var. “Swingle”). Seven days after priming, insects were removed from plants, starved for 3 h, and either injected (experiment 1) with a lethal dose of S. marcescens or control treatment. After feeding or injection, the ACP that survived were allowed to reproduce on healthy of CLas-infected hosts. DNA was extracted from D. citriusing Qiagen DNeasy Blood and tissue kits (Qiagen, Hilden, Germany) per manufacture recommendations. Quality and concentration of DNA was assessed after extraction on a Nano Drop 2000 (Thermo Fisher Scientific, Waltham, MA), then standardized to 10ng/ l. CLas titers were assessed by the detection of the 16S rDNA gene by qPCR methods described by Coy et al. (2014). Plants were tested to ensure infection with CLas by qPCR following methods described by Li et al. (Li et al., 2006). No significant differences in reproductive success or acquisition by offspring were detected to date, which suggests that immune priming does not occur in response to RNAi, and that this response is unlikely to affect CLas transmission.
The goal of this project is to determine whether pathogen or dsRNA exposure primes the ACP immune system to resist future infection by pathogens, including Las, and whether this effect is multigenerational. We have previously characterized the specificity and efficacy of the immune priming response in ACP (Obj. 1), characterixzed the effect of prior immune challenge on transmission (Obj. 3) and determined the transgenerational effect of pathogen-induced immune priming on Las acquisition. The current report describes our ongoing efforts to quantify the effect of RNAi-induced priming on Las acquisition (Obj 4). Adults of ACP collected from a laboratory colony free of CLas infection were starved for 3 hr, and then were subjected to dsRNA ATPase and sucrose. For each treatment, 5 cages including 10 insects in each were used. Insects were fed on diet solution consisting of 10, 100, and 1000 ng. l -1 dsRNA ATPase, 20% sucrose, and 0.5 green food coloring dye. As a control, adult of ACP was fed on sucrose 20%+ 0.5 green food dye. A cage for artificial feeding was prepared by stretching parafilm membranes on the bottom of plastic petri dish arenas. The parafilm surface was sterilized with ethanol and dried for 5 min under a sterile hood and layered with 400 L of diet solution including dsRNA, 20% sucrose, and green food coloring dye. The liquid was then covered with a second layer of stretched parafilm. During the feeding, the cages were placed in a growth chamber at 28oC. Insect were collected after 24 hrs and 5 days feeding and stored in -80 C for RNA extraction. Insects primed by exposure to artificial diet solutions with dsRNA for 24 hours were transferred to separate branches of a potted citrus plant (var. “Swingle”). Seven days after priming, insects were removed from plants, starved for 3 h, and either injected (experiment 1) with a lethal dose of S. marcescens or control treatment. After feeding or injection, the ACP that survived were allowed to reproduce on healthy of CLas-infected hosts. DNA was extracted from D. citriusing Qiagen DNeasy Blood and tissue kits (Qiagen, Hilden, Germany) per manufacture recommendations. Quality and concentration of DNA was assessed after extraction on a Nano Drop 2000 (Thermo Fisher Scientific, Waltham, MA), then standardized to 10ng/ l. CLas titers were assessed by the detection of the 16S rDNA gene by qPCR methods described by Coy et al. (2014). Plants were tested to ensure infection with CLas by qPCR following methods described by Li et al. (Li et al., 2006). No significant differences in reproductive success or acquisition by offspring were detected to date, which suggests that immune priming does not occur in response to RNAi, and that this response is unlikely to affect CLas transmission.
To determine the optimal concentration of dsRNA priming, ACP were exposed to a series of dsRNA concentrations (10, 100, 1000 ng. l-1) in artificial diet for 24 hrs, and 5 days. ACP were subsequently transferred to artificial diet containing S. marcescens for 4 days, then transferred to C. macrophylla for 14 days. Survival of ACP was recorded every 24 hrs for 14 days. ACP survival in response to S. marcescens was lowest after feeding on 100 ng. l-1 of dsRNA T7_pGEMT for 24hrs, as compared with control (no dsRNA priming) treatments. The survival rates of ACP exposed to S. marcescens or sucrose following control (no dsRNA priming) were not significantly different. Eighty percent of ACP survived after feeing on 100 ng. l-1 dsRNA ATPase for 5 d prior to being transferred to sucrose, and was significantly higher than among ACP that fed only on sucrose prior to S. marcescens exposure. This suggests that initial (24 h) exposure to dsRNA may increase susceptibility of ACP to pathogens. Among the insects that did not survive pathogen challenge, S. marcescens was detected in 79% of ACP following priming with 100 ng. l-1 dsRNA T7_pGEMT100 fior 24 h, as compared with 9-15% of ACP not primed with dsRNA. Insects primed for 5 days on dsRNA followed by exposure to S. marcescens, 75% of insects fed with 100 ng. l-1 dsRNA ATPase were infected with the pathogen. No bacterial infection was observed in insects fed on 1000 and 10 ng. l-1 of T7_pGEMTdsRNA , or control (no dsRNA priming) treated insects. The high percentage of bacterial infection in the dsRNA-treated insects indicates that dsRNA may contribute to bacterial loads in ACP, although this effect appears to be dose-dependent. Target gene expression decreased among ACP that were primed with 1000 ng. l-1 T7_pGEMT or 100, 1000 ng. l-1 of dsRNA ATPase prior to S. marcescens exposure but this reduction was not statistically different from untreated (no dsRNA priming) ACP. It is expected that the time between feeding dsRNA and quantifying mRNA at the end of the feeding bioassay is too long long to detected dsRNA-associated changes in expression; therefore, experiments are underway to evaluated changes in expression following 24 h and 5 d exposure to dsRNA. In addition, subsequent analyses will be conducted to determine whether priming facilitates Liberibacter acquisition.
Media continued to be modified to improve the growth of L. crescens and use those results to develop better media for CLas culturing. During this period the Killiny lab provided an enormous amount of metabolomic data from citrus phloem to enhance our media formulations. Results from this work has suggested components that should be added or added at a higher concentration to the medium for the culturing of CLas including carbohydrates (a-ketoglutarate, galactose, glucose, fructose, maltose, sucrose, xylose), amino acids (alanine, arginine, asparagine, aspartate, cysteine, 2-aminobutyrate, glutamate, glycine, isoleucine, phenylalanine, proline, serine, threonine, and valine), sugar alcohols (iso-inositol, sorbitol, xylitol), organic acids (citrate, fumarate, malate, and succinate), micronutrients (B, Cu, I, Mn, Mo, Zn), vitamins (ascorbate, cobolamine, diaminopemilate, pyridoxal phosphate, riboflavin, thiamine). In past media formulations, some of these molecules were in particularly low levels, particularly thiamine which was 100-fold below the level found in media. For our defined media for L. crescens, we have always used a commercial source of Grace’s insect medium as part of the formulation. In the past, we mistakenly assumed that the levels of compounds reported by the manufacturers are the actual levels present in the Grace’s medium. The components of three commercial sources of Grace’s medium were examined by metabolomics and none of them were very close to the standard composition of Grace’s medium as listed on their labels. One of them, made by a manufacturer in Mubai, India, was very different and provided remarkable growth of L. crescens with no other added components. The growth obtained with this medium was very similar to that obtained by culture in BM-7, the standard, undefined medium used for this organism. The two other sources of Grace’s medium from Gibco and Sigma did not support L. crescens growth and a version of this maedium made by our lab with ingredients from Sigma also did not support growth. We were unable to culture CLas on the Indian-made Grace’s medium, referred to as Hi-GI. Metabolomics analysis showed that Hi-GI medium contains 10-fold higher levels of various vitamins compared to what would be expected in Grace’s medium. Sugars and organic acids (including glucose, fructose, sucrose, turanose, maltose, fumarate, alpha-ketoglutarate, malate, maleate and succinate) were also higher. To date, we haven’t been able to make a defined medium based on Hi-GI that provides the same high level of growth as does Hi-GI. This implies the chemically defined medium still requires improvements. Liberibacter crescens requires compound or combinations of compounds for optimal growth and these have not yet been discovered. Meawhile, we have a large stock of Hi-GI on hand and are modifying it in order to culture CLas.
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