All significant results remain the same as in the previous quarter.
The long term field trial continues with weekly psyllid counts and quarterly CLas infection testing. Treatments continue to have similar effects on ACP counts. Plants in both of the kaolin treatments continue to show higher growth rates than the other two treatments. The red treatment has the highest growth rate, trunk cross-sectional area, and canopy volume. Kaolin treated trees that are infected grow more than untreated-infected trees, but less than treated uninfected trees. The field trial will continue until the project ends, when we expect to have the first economic yield.
We are now performing follow-up repetitions of the MS student’s thesis work. We anticipate publication submission of this work in the Fall.
Objective 1: Investigate the efficacy of bactericides treatments for preventing new infections for young citrus trees protection.
Hypothesis: Bactericidal treatment will protect young trees from CLas colonization.
Initial leaf samples were collected prior to treatments to evaluate CLas titers in the uninfected trees.
We applied bactericide treatments from May through September. CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of CLas-infection rate in citrus leaves from May and June is described. Currently, citrus leaves tissue samples from July through September are being processed to analyze the CLas-infection rate.
*Trees were considered CLas-infected (positives) when CT values were below 35.
1. Antibiotics (monthly rotation): Prior to bactericide application (May), 15% of trees (20 trees/treatment) were CLas positive (Ct<35). After the bactericide application (June), 35% of trees were CLas positive (Ct<35).
2. Antibiotics (quarterly rotation): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After bactericide application (June), 40% trees were CLas positive (Ct<35).
3. Negative Control (insecticide + Tree defender exclusion netting): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After the bactericide application (June), 45% trees were CLas positive (Ct<35).
4. Positive Control (insecticide only): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After the bactericide application (June), 5% trees were CLas positive (Ct<35).
Counting of ACP adults using taps was conducted bi-weekly from May through September, presence of other life stages such as eggs and nymphs were scouted visually. Preliminary results showed a low ACP population in citrus locations due to the active vector management performed by farm manager. As consequence, no ACP adults were collected to analyze the CLas-infection rate using quantitative real-time PCR analysis. The overall number of eggs and nymphs were low or undetectable in citrus trees from May to September. Also, to determine the effect of citrus vegetative growth (flush-like structures) in CLas-infection rate, 1 ft.3 was used to count the number of flush-like structures per tree. Results showed that the presence of flush-like structures incremented from May to July and decreased in September.
Objective 2. Determine the effect of bactericides application frequency on Las infection of citrus.
Hypothesis: Bactericidal treatment will reduce CLas infection in mature trees.
We applied bactericide treatments from May through September. CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of CLas-infection rate in citrus leaves from May and June is described. Currently, citrus leaves tissue samples from July through September are being processed to analyze the CLas-infection rate.
*Trees were considered CLas-infected (positives) when CT values were below 35.
1. Antibiotics (monthly rotation): Prior to bactericide application (May), 100% of trees (20 trees/treatment) were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35). Although positive, bacterial titers declined in trees receiving antimicrobial treatments.
2. Antibiotics (quarterly rotation): Prior to bactericide application (May), 100% of trees were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35).
3. Positive Control (insecticide only): Prior to bactericide application (May), 100% of trees were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35).
Counting of ACP adults using taps was conducted bi-weekly from May through September, presence of other life stages such as eggs and nymphs were scouted visually. Preliminary results showed high ACP populations in treatments from May to August, excepting for June. The number of eggs and nymphs were not collected during May and first collection of June. However, populations increased from late June to August and reached high population levels. Currently, ACP adults that were collected bi-weekly are being processed to analyze the CLas-infection rate using quantitative real-time PCR analysis. Also, to determine the effect of citrus vegetative growth (flush-like structures) in CLas-infection rate, 1 ft3 was used to count the number of flush-like structures per tree. Flush was not collected during May and June. However, results showed that the presence of flush was high in July and August.
August 31, 2019 – In this quarter, we have continued to work on objectives outlined in our chronogram.
Objective 1. We have completed assessment of trees planted in our pilot study (planted 22 months ago) for CLas infection and HLB symptoms. All the non-covered trees are PCR-positive for CLas whereas all trees covered with IPC have tested negative. We are continuing with quantification of leaf drop and comparing leaf drop in both treatments; 6-month cumulative data show no significant differences in leaf drop in IPC-covered trees compared with non-covered trees. Interestingly, when counted seasonally, in spring leaf drop was significantly higher in non-covered trees as compared to IPC trees, whereas in summer, it was slightly higher inside IPCs. This fact points out a seasonal component that we will investigate as the project progresses.
In August, we have replaced the old 4-ft IPCs with new 8-ft covers, donated by The Tree Defender, Inc, because the trees had filled the volume of the cover completely. This also has opened the possibility of studying the dynamics of branch unfolding, which we are doing visually (photography documentation) and by measuring canopy growth and leaf area index. We have also assessed other pest and disease incidences inside the IPCs. We have found less incidences of canker inside IPCs and approximately equal incidences of greasy spot. However, greasy spot severity is higher inside the IPCs. We have found more incidence of other pests such as mites, armyworms, and leafrollers inside the IPCs, and a total absence of predators (beneficials). This suggests that relying only on IPC for insect control is not sufficient, and insect management must still be conducted. No psyillid have been found inside the IPCs.
Objective 2. To study the edge effect in different IPC layouts, we are now preparing to plant 700 trees of SugarBelle, Tango and Early Pride mandarins and using 3 different arrangements (targeted, alternated and patterned, as described in the proposal) of IPC. We have performed initial measurements of the tree parameters (trunk diameter, and leaf sampling, for CLas, cholorophyll and sugar analysis).
Objectives 3 and 4. We are continuing to measure fruit set and development inside the IPCs and comparing this with our CUPS planting. We are taking fruitlet and fruit samples regularly for biochemical analysis.
Outreach, Professional Presentations and Extension Activities for this quarter :
-Grower Presentation: “Growing Young Citrus Trees Under Individual Protective Covers (IPCs): What We Know After 18 Months” Citrus Expo 2019, August 15, Fort Myers, Fl.
-Industry Magazine Article: “Individual Protective Covers for Psyllid Exclusion and HLB Disease Prevention in Young Trees”. Article submitted to Citrus Industry Magazine in July to be published in October issue.
-Our Project was also noted in the September’s issue of Citrus Industry Mag’s UF/IFAS. The Citrus State Opinion Column by Jack Payne highlighted this work as an example of collaboration between growers, extension agents, and scientists in Florida. The column was entitled “Collaboration breeds solutions”.
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.
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