CLas Bacteria


CLas Inhibition with Antisense Oligonucleotides for Management of Citrus Greening Disease

Report Date: 09/15/2022   Project: 20-021   Year: 2022

CLas Inhibition with Antisense Oligonucleotides for Management of Citrus Greening Disease

Report Date: 09/15/2022
Project: 20-021   Year: 2022
Percentage Completion: 0.4
Category: CLas Bacteria
Author: Kirsten Pelz-Stelinski
Sponsor: Citrus Research and Development Foundation

. Please state project objectives and what work was done this quarter to address them: Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Our working hypothesis is that CLas-specific FANAs can be delivered using microinjection developed for RNAi-based technologies to reduce CLas in infected citrus trees. Field trials with laboratory vetted FANAs were conducted in research groves at the UF Citrus Research and Education Center. Treatments were applied to 10-year-old, CLas-infected ‘Hamlin’ trees of a standard size and CLas titer.  AUM LifeTech designed and synthesized FANA ASOs to be complementary to two CLas essential genes. As a negative control, a FANA ASO was designed as a scramble sequence with no complementarity with any citrus gene. Antibiotic application (Fireline – Oxytetracycline) and insecticide-only treatments were applied to trees as control treatments. Each treatment was applied to 15 trees in 1-acre plots, replicated three times in a randomized complete block design. Treatments were applied to both sides of the tree canopy using microinjection of dosages determined in our previous greenhouse assays. The first replicate of this experiment was conducted from May through August and consist of five treatments: untreated control (insecticide-only), oxytetracycline control (1.56 g of Fireline per tree), Scramble Control-FANA, CLas L-FANA, and CLas B H-FANA. All FANAs dosages were 625 ppm per tree. Prior to treatment, four leaves were removed from each tree, two from each side of the apex of the tree and two from each side of the base of the canopy, for initial titer (T0) using quantitative real-time polymerase chain reaction (qPCR) assays. To monitor the effect of the FANA ASOs on the CLas titer of each tree, four leaves samples were removed from the same branches as the T0 samples after 2, 7, 30, 60 and 90 days. The post-treatment CLas titer (TF) were calculated by qRT-PCR analysis, at each time. Leaf samples were run in duplicates, and the relative quantities of CLas in threes were calculated based on the comparative cycle threshold 2-..Ct method. Results: In this report, information regarding the first application of treatments is presented. At T0, CLas titters in trees were similar across treatments. Two and seven days after treatment were applied, CLas titers in trees fluctuated; and higher CLas titers were observed in FANA treated trees compared to antibiotic and insecticide treated trees. No differences were found in CLas titers among treatments 30 days after treatments were applied. However, CLas titers were lower in trees treated with L-FANA and titers were comparable with those trees treated with antibiotics 60 days after treatments were applied. Overall, trunk-injections of L-FANA and antibiotic treatments significantly reduced CLas titers in trees as compared with insecticide only or control FANA treatments. A second replicate of this experiment will be performed in September of 2022 to confirm our observations.  Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Our working hypothesis is that CLas will be specifically inhibited in psyllids by using CLas-specific FANAs, resulting in a reduction in Las acquisition and transmission by ACP in a field setting. Acquisition assay. Field assays with psyllids were conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP in June 2022. Psyllid nymphs, who develop on immature leaf tissue, acquire CLas more efficiently than adults; therefore, acquisition of CLas from FANA-treated infected citrus trees were compared with acquisition from untreated infected trees, using the treatments described in Obj.1. Seven days after treatments were applied, ten ACP (five males and five females) from uninfected laboratory cultures were caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment was replicated three times on individual trees. Following oviposition (seven days after), ACP adults (P1) were collected and preserved for CLas detection. Egg clutches were left on trees enclosed in mesh sleeves. After nymphs reached the adult stage (15 days after), psyllids (F1) and leaves from test plants were collected. The effect of FANA treatments on acquisition of CLas was assessed by comparing the CLas titer in P1 and F1 ACPs caged on treated and untreated citrus trees. Results: The results from a bioassay assessing acquisition of CLas following FANA treatments showed that ACP adults (P1) and offspring (F1) were able to acquire CLas across treatments 7  and 15 days following treatments, respectively. The lowest CLas titers were observed in P1 and F1 ACP feeding on antibiotic-treated trees; followed by L -FANA and H-FANA treatments. The highest CLas titers were observed on ACP adults and offspring feeding on Scramble-FANA and insecticides only treated trees. Overall, L -FANA, H-FANA, and antibiotic treatments reduced CLas acquisition by ACP as compared with scramble-FANA and insecticide treatments. A second replicate of this experiment will be performed in September 2022.   2. Please state what work is anticipated for next quarter: Objective 1:The second treatment application is planned for this period. Samples from the second round of treatment applications through day 30 will be processed during quarter #3. Tree health and fruit quality/yield will be assessed in quarter # 4.  Objective 2: Acquisition assays will be replicated and partial results with P1 and F1 adults will be reported in quarter #4. Inoculation assays will also be completed in quarter #4, with samples collected for CLas detection. If rootstocks are used for inoculation, plants will be held for development of CLas infection and symptom development.  3. Please state budget status (underspend or overspend, and why): Our budget is on track for the project. FANA treatments, which are the largest portion of the budget, have been purchased for the experiments. Remaining budget will be spent for sample analysis, ACP assays and colony maintenance, research plots, and personnel. Microinjectors needing replacement will be purchased next quarter.   



Continued Support of the Southern Gardens Diagnostic Laboratory

Report Date: 06/27/2022   Project: 21-002C   Year: 2022

Continued Support of the Southern Gardens Diagnostic Laboratory

Report Date: 06/27/2022
Project: 21-002C   Year: 2022
Percentage Completion: 0.8593
Category: CLas Bacteria
Author: Michael Irey
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them: This report is for the third quarter of year 1 of Project 21-002C – Continued Support for the Southern Gardens Diagnostic Laboratory.  This project provides HLB testing for resarchers, growers and homeowners. A total of 9,118 samples were run during this 3 month-period.  All samples were plant samples, mostly from large scale research trials.  To date 24,704 samples have been run.  It is expected that we will be close to the budgeted amount of 28,750 samples for the end of year 1.  During this quarter, the lab experienced some technical difficulties which were resolved by working with Dr. Ozgur Batuman’s lab by running comparison samples.  The problem was traced to using EDTA in the elution buffer.  Although the elution methodology has been the same for years, for some unknown reason it became an issue. Suppliers of the affected samples were notified and the problem was corrected.  It is suspected that the supplier of the master mix changed their formulation which resulted in the inhibition by EDTA (which has been reported in the literature as a potential inhibitor of PCR reactions).  The lab operations are back on track at this time. 2. Please state what work is anticipated for next quarter: It is expected that the number of samples will slow down for the last quarter of year 1 as this is the time of year when symptoms are not evident and the titer of the bacterium in the plant declines.  3. Please state budget status (underspend or overspend, and why): Based on current projections, it is expected that the lab will be on target for the budgeted amount of samples for year 1 (budget=28,750)    



Continued Support of the Southern Gardens Diagnostic Laboratory

Report Date: 06/27/2022   Project: 21-002C   Year: 2022

Continued Support of the Southern Gardens Diagnostic Laboratory

Report Date: 06/27/2022
Project: 21-002C   Year: 2022
Percentage Completion: 1.02
Category: CLas Bacteria
Author: Michael Irey
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them: This report is for the fourth quarter of year 1 of Project 21-002C – Continued Support for the Southern Gardens Diagnostic Laboratory.  This project provides HLB testing for resarchers, growers and homeowners. A total of 4,512 samples were run during this 3 month-period.  All samples were plant samples, mostly from large scale research trials.  To date 29,216 samples have been run, which is slightly over the budgeted amount of 28,750 samples for year 1.   2. Please state what work is anticipated for next quarter: It is expected that sample volume will be slightly higher in quarters 1 and 2 of year 2 due to CRAFT projects which are just starting to come on line.  However this remains to be seen.   3. Please state budget status (underspend or overspend, and why): For year 1, the project is essentially on budget.  A total of 29,216 samples were run versus a budget of 28,750.  As has been done in previous years, the number of samples run at the end of year 2 will be reconciled against the budgeted amount and the last payment will be adjusted up or down as needed.    



CLas Inhibition with Antisense Oligonucleotides for Management of Citrus Greening Disease

Report Date: 06/15/2022   Project: 18-018   Year: 2022

CLas Inhibition with Antisense Oligonucleotides for Management of Citrus Greening Disease

Report Date: 06/15/2022
Project: 18-018   Year: 2022
Percentage Completion: 20
Category: CLas Bacteria
Author: Kirsten Pelz-Stelinski
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them: The overall objective in this proposal is to explore whether FANA oligonucleotides, which are synthetic RNA-based synthetic molecules that mimic DNA and inhibit bacterial gene expression can be used to mitigate CLas in infected trees.The first objective of this project is to s screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Treatments were applied to 10-year-old, CLas-infected ‘Valencia’ trees of a standard size and CLas titer. FANA ASOs complementary to two CLas essential genes F(ANA-L and FANA-H), a scramble sequence control (FANA-SC), oxytetracycline (Fireline) and insecticide-only treatments are were applied to 15 trees in 1-acre plots, replicated five times in a randomized complete block design. Treatments are applied using microinjection of dosages determined in our previous greenhouse assays.Prior to treatment 1 (T1), four leaves were removed from each tree, two from each side of the apex of the tree and two from each side of th20%e base of the canopy, to determine initial CLas titer (T0).  PCR of these leaves and leaves collected post treatment was conducted during Q2 of this project to monitor the effect of the FANA ASOs on the CLas titer of each tree. Pathogen titers in trees before treatment was determined using a quantitative real-time PCR (qPCR) assay. Trees were uniformly infected with mean Ct (qPCR cycle threshold) values ranging from 24 to 25. Oxytetracyline (Fireline) was initially the most effective in preventing increases in CLas titers, followed by FANA L. CLas titers increased 2 d following all other treatment applications. The percent increase in CLas Ct value was greatest in the FANA-H and insecticide-only treatments. Sample processing is still underway to assess the effect of treatments on tree infections7, 30, and 60 d after treatment applications. We expect that the treatments will take time to move throughout the trees and reduce CLas infection.          The second objective is to evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Single females from uninfected laboratory cultures were  caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment was replicated ten times on individual trees. Due insufficient survival of ACP in March 2022, this experiment was carried out in May-June2022 and is still underway. Adult psyllids have been collected from trees and are being process to assess acquisition. F1 adults will be collected in late June to assess acquisition during nymph development and to use in inoculation assays. 2. Please state what work is anticipated for next quarter: Objective 1:Samples from first round of treatment applications through day 60 will be processed during quarter 3. A second round of treatments will be applied at the end of quarter 3. Objective 2: Results of acquisition assay #1  with P1 and F1 adults will be next quarter. Inoculation assays will also be completed in Q3, with samples collected for CLas detection. If rootstocks are used for inoculation, plants will be held for development of CLas infection and symptom development. 3. Please state budget status (underspend or overspend, and why): Our budget is on track for the project. FANA treatments, which are the largest portion of the budget, have been purchased for the experiments. Micorinjectors and research plot charges have also been spent. Remaining budget will be spent to process samples, ACP assays, and on personnel. 



How do subterranean pests and diseases affect root health of trees with and without HLB?

Report Date: 05/26/2022   Project: 19-016   Year: 2022

How do subterranean pests and diseases affect root health of trees with and without HLB?

Report Date: 05/26/2022
Project: 19-016   Year: 2022
Percentage Completion: 0.75
Category: CLas Bacteria
Author: Larry Duncan
Sponsor: Citrus Research and Development Foundation

The previous report noted that trees on Swingle citrumelo and Carrizo rootstock were located and ordered for delivery in January when they would be used to repeat the HLB-Tylenchulus semipenetrans trial. Also that Kuharske plants were added to seedlings for a rootstock trial to evaluate tolerance to Radopholus similis in traditional rootstocks and newer releases, with inoculation to occur also in January.  However, our greenhouse was disassembled without notice during the first week of January. The walls were open to the outside and a job that was to be finished in  few days was not completed for more than three months. We moved all the citrus material to our soil processing laboratory to prevent infestation by Asian citrus psyllid. Eventually, we were forced to move our nematode cultures as well, due to high ambient temperature when the cooling system was removed. As a consequence, inoculation of these experiments was not possible.         



How do subterranean pests and diseases affect root health of trees with and without HLB?

Report Date: 05/26/2022   Project: 19-016   Year: 2022

How do subterranean pests and diseases affect root health of trees with and without HLB?

Report Date: 05/26/2022
Project: 19-016   Year: 2022
Percentage Completion: .7500
Category: CLas Bacteria
Author: Larry Duncan
Sponsor: Citrus Research and Development Foundation

The commercial and potential rootstock seedlings to be screened for tolerance to burrowing nematode were moved from the laboratory to the rebuilt greenhouse in early April and inoculated with Radopholus similis in late May.  A new screenhouse was constructed and the Swingle citrumelo and Carrizo rootstock Valencia trees were moved from the laboratory, pruned to initiate new root and shoot growth.  They will be inoculated with citrus nematode and graft-inoculated with CLas in the first week of June.  A trial comparing the effects of burrowing nematode and CLas on rootstocks susceptible and resistant to burrowing nematode was terminated.  Neither pathogen significantly affected the shoot weight of either rootstock; however burrowing nematode increased the root weights (P=0.001) and the effects differed for the two rootstocks. On the susceptible Carrizo citrange the nematode increased root weight by 12% (17.2g vs 19.2g) and on the resistant Kuharske Carrizo by 24% (54.1g vs 66.8g).  Unlike the adverse effect of CLas on citrus nematode populations (July 2021 report), nearly twice as many burrowing nematodes per gram of root occurred on Carrizo seedlings infected by CLas than on seedlings only infected with burrowing nematode (65.9 vs 34.3), but the effect was not significant.  There were no burrowing nematodes recovered from the resistant rootstock Kuharske. The repeat experiment is ongoing.   The project ended on 30 April. Covid-related delays related in the NCE request and the unannounced 3-month closure of our greenhouse have delayed the completion of several experiments that include a repeat of the trials evaluating interactions between CLas and burrowing nematode described above and citrus nematode in which  the nematodes increased the virulence of Las in citrus plants, whereas Las-infected plants were less suited to nematode population growth than non-infected plants. These and the trial screening rootstock resistance to the burrowing nematode are ongoing and will be completed and reported in a supplemental report in 2022.      



CLas Inhibition with Antisense Oligonucleotides for Management of Citrus Greening Disease

Report Date: 03/15/2022   Project: 18-018   Year: 2022

CLas Inhibition with Antisense Oligonucleotides for Management of Citrus Greening Disease

Report Date: 03/15/2022
Project: 18-018   Year: 2022
Percentage Completion: 5
Category: CLas Bacteria
Author: Kirsten Pelz-Stelinski
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:The overall objective in this proposal is to explore whether FANA oligonucleotides, which are synthetic RNA-based synthetic molecules that mimic DNA and inhibit bacterial gene expression can be used to mitigate CLas in infected trees.The first objective of this project is to s screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Our working hypothesis is that CLas-specific FANAs can be delivered using microinjection developed for RNAi-based technologies to reduce CLas in infected citrus trees. We have initiated field trials with laboratory-vetted FANAs in a research grove at the UF Citrus Research and Education Center in March 2022. Treatments are applied to 10-year-old, CLas-infected ‘Valencia’ trees of a standard size and CLas titer. FANA ASOs complementary to two CLas essential genes and a scramble sequence control are synthesized by AUM LifeTech, initiated in December 2021. Antibiotic application (Fireline) and insecticide-only treatments are applied to trees as control treatments. Each treatment is applied to 15 trees in 1-acre plots, replicated five times in a randomized complete block design. Treatments are applied using microinjection of dosages determined in our previous greenhouse assays.Prior to treatment 1 (T1), four leaves were removed from each tree, two from each side of the apex of the tree and two from each side of the base of the canopy, for initial titer (T0) using quantitative real-time polymerase chain reaction (qPCR) assays.  PSamples are removed from the same branches as the T0 samples after 2, 7, 30, 60 and 90 days. The second objective is to evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Field assays with psyllids are conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP. Single females from uninfected laboratory cultures are  caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment is replicated ten times on individual trees. This experiment was initiated in March 2021, as outlined in our proposal timeline. The effect of FANA treatments on acquisition of CLas will be assessed by comparing the CLas titer in ACP caged on citrus trees before and after treatments and across time. 2. Please state what work is anticipated for next quarter: Objective 1: PCR of these leaves and leaves collected post treatment will be conducted during Q2 of this project to monitor the effect of the FANA ASOs on the CLas titer of each tree. Objective 2: Initial results after 1 treatment will be reported next quarter. 3. Please state budget status (underspend or overspend, and why): Our budget is on track for the project. FANA treatments, which are the largest portion of the budget, have been purchased for the experiments. Personnel spending has been low to date, as December 21- January 22 were primarily spent designing and synthesizng FANA molecules for the experiment.



Continued Support of the Southern Gardens Diagnostic Laboratory

Report Date: 03/08/2022   Project: 21-002C   Year: 2021

Continued Support of the Southern Gardens Diagnostic Laboratory

Report Date: 03/08/2022
Project: 21-002C   Year: 2021
Percentage Completion: .5422
Category: CLas Bacteria
Author: Michael Irey
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:

This report is for the second quarter of year 1 of project 21-002C – Continued Support of the Southern Gardens Diagnostic Laboratory. This project provides HLB testing for researchers, growers and homeowners. A total of 9,119 samples were run during the 3 month period. All samples were plant samples, mostly from large scale research trials. The majority of the samples (78%) were from the first two months of the quarter. Partly due to vacation schedules associated with the Christmas/New Year holidays and partly due to Covid-related personnel issues, less samples were run during December. However, sample volume continue to be high and it is expected that a similar level of samples will be run next quarter. To date, through the first two quarters of the project, 15,587 samples have been run.

2. Please state what work is anticipated for next quarter:

Based on the samples run to date (first two quarters, and samples run through the end of February 2022) and samples that are anticipated (including samples from CRAFT trials), it is expected that the number of samples for year one of the project will be slightly over the budgeted amount. Although it is hard to predict, we are predicting that the sample volume for the first year will be approximately 30,000-32,000 samples compared to the budgeted amount of 28,750/year.

3. Please state budget status (underspend or overspend, and why):

Based on predicted sample load for the remaining two quarters, it is expected that the total sample volume will be above the budgeted amount for year 1. As has been done in the past, it is anticipated that the final budget at the end of year 2 will be adjusted based on the final sample tally. If less samples are processed than the budgeted amount, the budget will be adjusted downward. Conversely, if more samples are processed, we will ask that the cost of the overage (expendables only) be covered. However, what is new for this project is that the CRAFT project will reimburse CRDF for samples that are submitted through the CRAFT program. To the extent that the overrun of samples is a result of CRAFT samples, CRDF may be reimbursed enough to cover the overall sample overage if there is any.



Control citrus Huanglongbing by exploiting the interactions between Candidatus Liberibacter asiaticus and citrus

Report Date: 11/24/2021   Project: 18-026   Year: 2021

Control citrus Huanglongbing by exploiting the interactions between Candidatus Liberibacter asiaticus and citrus

Report Date: 11/24/2021
Project: 18-026   Year: 2021
Percentage Completion: 1
Category: CLas Bacteria
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

The goal is to understand how citrus interacts with Candidatus Liberibacter asiaticus (Las) infection and develop improved and long term HLB management strategies. Objective 1. Identification of the receptors for Las PAMPs in susceptible and tolerant citrus varietiesPotential 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 were cloned under 35S promoter and the Arabidopsis phloem specific promoter SUC2 and introduced into Agrobacterium. We have tested their receptors in Tobacco and citrus. We have identified multiple receptors for the aforementioned PAMPs. We also hypothesized that Las outer membrane proteins might directly induce plant immune response in the phloem sieve elements because Las lives in the phloem. 21 outer membrane proteins have been cloned and the putative targets in citrus were identified using Yeast 2 hybrid (Y2H) system. Two outer membrane proteins showed positive interactions with citrus proteins based on Y2H assays which were confirmed using GST pull-down assaysIn addition, multiple PAMPs have been tested for their effects in inducing plant defense against Las in the greenhouse and at least four different PAMPs showed significant effect in manipulating plant immunity. Here, we conducted RNA-seq analyses on HLB-susceptible Valencia sweet orange and HLB-tolerant Sugar Belle mandarin in winter, spring, summer and fall. Significant variations in differentially expressed genes (DEGs) related to HLB were observed among the four seasons. For both varieties, spring had the highest number of DEGs. CLas infection stimulates the expression of immune-related genes such as NBS-LRR, RLK, RLCK, CDPK, MAPK pathway, ROS, and PR genes in both varieties. The immune responses of both varieties to CLas result in oxidative stress with induced expression of RBOH genes and suppressed expression of many genes encoding antioxidant enzymes, such as superoxide dismutase, ascorbate peroxidase, catalase, thioredoxins and glutaredoxins. HLB-positive Sugar Belle trees contained higher concentrations of maltose and sucrose, which are known to scavenge ROS. In addition, Sugar Belle showed higher expression of genes involved in phloem regeneration that might contribute to its tolerance to HLB. This study shed light on the pathogenicity mechanism of the HLB pathosystem and the tolerance mechanism against HLB, providing valuable insights into HLB management.We have the control effects of different PAMPs against HLB. Three PAMPs showed strong activity in inducing plant defenses. We analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin-encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin-encoding genes. Only flgJAtu but not flgJLas restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull-down assays demonstrated that FlgJLas interacts with FlgB but not with FliE. Ectopic expression of flaALas in A. tumefaciens mutants restored the swimming motility of .flaA mutant and .flaAD mutant, but not that of the null mutant .flaABCD. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.The type IVc tight adherence (Tad) pilus locus is a putative PAMP encoded by Las. The Tad loci are conserved among members of Rhizobiaceae, including ‘Ca. L. asiaticus’ and Agrobacterium spp. Ectopic expression of the ‘Ca. L. asiaticus’ cpaF gene, an ATPase essential for the biogenesis and secretion of the Tad pilus, restored the adherence phenotype in cpaF mutant of A. tumefaciens, indicating CpaF of ‘Ca. L. asiaticus’ was functional and critical for bacterial adherence mediated by Tad pilus. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that ‘Ca. L. asiaticus’ Tad pilus-encoding genes and ‘Ca. L. asiaticus’ pilin gene flp3 were upregulated in psyllids compared with in planta. A bacterial one-hybrid assay showed that ‘Ca. L. asiaticus’ VisN and VisR, members of the LuxR transcriptional factor family, were bound to the flp3 promoter. VisNR regulate flp3. Negative regulation of the flp3 promoter by both VisN and VisR was demonstrated using a shuttle strategy, with analysis of the phenotypes and immunoblotting together with quantification of the expression of the flp3 promoter fused to the ß-galactosidase reporter gene. Comparative expression analysis confirmed that ‘Ca. L. asiaticus’ visNR was less expressed in the psyllid than in the plant host. Further, motility and biofilm phenotypes of the visNR mutant of A. tumefaciens were fully complemented by expressing ‘Ca. L. asiaticus’ visNR together. The physical interaction between VisN and VisR was confirmed by pull-down and stability assays. The interaction of the flp3 promoter with VisR was verified by electrophoretic mobility shift assay. Taken together, the results revealed the contribution of the Tad pilus apparatus in the colonization of the insect vector by ‘Ca. L. asiaticus’ and shed light on the involvement of VisNR in regulation of the Tad locus. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe have transgenically expressed putative receptors or targets (identified in Poncirus) of Las PAMPs in Valencia sweet orange or Duncan grapefruit. They are driven by 35S promoter and phloem specific promoter AtSuc2.     We have made 6 constructs to express PAMP receptors individually or in combinations. Three overexpression lines have been generated.     Objective 3. Investigate the roles of effectors in HLB disease developmentWe have completed screening of 30 putative Las effectors and 4 of them repressed plant defense. We have completed Y2H for the four defense-suppressing effectors and identified their targets in Valencia sweet orange. Meanwhile, we have conducted CTV-mediated gene silencing of 15 putative HLB susceptibility genes in collaboration with Dawson lab. Sweet orange plants carrying the CTV constructs were inoculated with Las via grafting.  Interestingly, gene silencing of one of the putative HLB susceptible genes led to significant HLB tolerance. The plants showed mild HLB symptoms, similar growth as non-inoculated plants whereas the growth of control plants was significantly reduced and showed severe HLB symptoms. In addition, we also overexpressed the HLB S gene in Valencia sweet orange to further understand the mechanism and will inoculate them with Las once they are one year old. Here we show that a Las-secreted protein, SDE15 (CLIBASIA_04025), suppresses plant immunity and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit (Citrus × paradisi) suppresses the hypersensitive response induced by Xanthomonas citri ssp. citri (Xcc) and reduces the expression of immunity-related genes. SDE15 also suppresses the hypersensitive response triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana, suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homolog of Arabidopsis (Arabidopsis thaliana) ACCELERATED CELL DEATH 2 (ACD2). SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB.    



Continued Support of the Southern Gardens Diagnostic Laboratory

Report Date: 11/04/2021   Project: 21-002C   Year: 2021

Continued Support of the Southern Gardens Diagnostic Laboratory

Report Date: 11/04/2021
Project: 21-002C   Year: 2021
Percentage Completion: 0.225
Category: CLas Bacteria
Author: Michael Irey
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:

This report is for the first quarter of year 1 of project 21-002C – Continued Support of the Southern Gardens Diagnostic Laboratory. The project provides HLB testing for researchers, growers and homeowners. A total of 6,468 samples were run during the first quarter of the project. All samples were plant samples (either leaves or roots) and the great majority of the samples were from research projects. As noted in previous reports, there is an increasing trend towards testing that includes copy number determination which increases the turn around time as the assay is more involved than assays that just require a determination of Ct. The lab also incurred an equipment-based setback that delayed some throughput for the first quarter. This has since been resolved.

2. Please state what work is anticipated for next quarter:

The budgeted sample load for each year of the project is 28,750 samples per year. Although the number of samples run during the first quarter is below the level that would meet that level of samples, the number of samples received to date indicates that we will more than likely reach or exceed the budgeted amount. The total number of samples that have been received (not all processed yet) through the first 4 months of the project is 10,410. At this rate, the projected number of samples per year would be approximately 31,230.

3. Please state budget status (underspend or overspend, and why):

As in the previously funded project, the final budget will be adjusted based on the final sample tally. If less samples are processed than the budgeted amount, the budget will be adjusted downward. Conversely, if more samples are processed, we will ask that the cost of the overage (expendables only) be covered.



Which commercial adjuvants achieve systemic delivery of antimicrobials?

Report Date: 09/10/2021   Project: 19-023   Year: 2021

Which commercial adjuvants achieve systemic delivery of antimicrobials?

Report Date: 09/10/2021
Project: 19-023   Year: 2021
Percentage Completion: 1.1
Category: CLas Bacteria
Author: Christopher Vincent
Sponsor: Citrus Research and Development Foundation

We have completed this project, having added to it an additional experiment to confirm results and assessment of ‘Ca. L. asiaticus’ bacterial titers in all studies.   In summary we find: · Both originally proposed studies were completed, as well as an additional confirmatory study with both oxytetracycline and streptomycin.· No foliar application treatment achieved a significant systemic delivery or oxytetracycline or streptomycin, nor did any reduce ‘Ca. L. asiaticus’ titer.  · Injection of oxytetracycline exceeded the minimum effective concentration (MEC) and was greater than all adjuvants, and reduced ‘Ca. L. asiaticus’ titer.· Injection of streptomycin at the equivalent per-tree rate to labeled foliar application rates did not exceed MEC, nor did it affect ‘Ca. L. asiaticus’ titer.· Results indicate that:   o Adjuvants to not enhance delivery of oxytetracycline or streptomycin sufficiently to impact ‘Ca. L. asiaticus’ titer at labeled rates.   o Once delivered inside the plant, insufficient streptomycin moves systemically to impact ‘Ca. L. asiaticus’ titer at the equivalent per tree dose to the labeled per-acre equivalent.  PublicationKilliny, N., Hijaz, F., Gonzalez-Blanco, P., Jones, S. E., Pierre, M. O., & Vincent, C. I. (2020). Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus. Antibiotics, 9(10), 677. One additional manuscript presenting the findings of studies 2 and 3 is in preparation. A full report is submitted in a separate pdf.  



How do subterranean pests and diseases affect root health of trees with and without HLB?

Report Date: 09/10/2021   Project: 19-016   Year: 2021

How do subterranean pests and diseases affect root health of trees with and without HLB?

Report Date: 09/10/2021
Project: 19-016   Year: 2021
Percentage Completion: 0.7
Category: CLas Bacteria
Author: Larry Duncan
Sponsor: Citrus Research and Development Foundation

Recently we focused primarily on the effects of burrowing nematode on selected UFR rootstocks. Previously we planted six UFR rootstocks in sandy soil, and inoculated the soil with burrowing nematode (BN). After four months, the plants were removed from the soil, cleaned thoroughly, vacuum packed and placed in the freezer. For three of the UFR rootstocks (UFR-4, -6, and -17), we compared healthy roots to those exposed to BN. We processed the roots and extracted them with hexane for volatile metabolites, and with methanol/chloroform/water for non-volatile metabolites such as sugars, organic acids and amino acid. In all, 48 samples were processed and run on the GC-MS, 24 for VOCs (3 RS x 2 treatments x 4 replicates) and 24 for non-VOCs, the chromatograms were integrated and compounds were identified. For the citrus root volatile extracts, we found that roots with BN from UFR 4 and UFR 6 contained low levels of monoterpenes in control roots (without BN damage), but monoterpenes were increased up to 6-fold in roots with BN. However, in UFR-17, all monoterpenes except d-limonene were reduced in BN+ root extracts, suggesting a different response to nematodes than in the other two rootstocks. Thirteen sesquiterpenes and 16 sesquiterpene alcohols were tentatively identified in the root extracts, with a large number of them increased consistently in all three rootstocks with BN. These may perhaps be defense compounds. One group identified were isomers of geyrene and pregeijerene, which are known to attract entomopathic nematodes in response to feeding by root weevils. We also found that the roots contained large amounts of coumarins (at least 9 different coumarins) which were reduced by half or more in roots with BN. In the root extracts for non-volatile compound determination, we detected 14 amino acids, 7 organic acids, 8 sugars, 6 coumarins and 3 sterols. We found that the amino acids were greatly reduced in roots with BN, especially L-proline and L-asparagine. Sugars, especially the monosaccharides such as fructose and glucose increased at least two-fold in UFR-4 and UFR-17, and about 1.6-fold in UFR-6. Disaccharides, mainly sucrose, also increased dramatically in BN-infested roots. The coumarins in the hexane fractions were much higher in roots with BN unlike the methanolic fractions. In UFR-4 and UFR-17, total organic acids such as malic acid increased, but were reduced in URF-6, which was the most abundant organic acid found in the roots. Citric acid decreased in all three rootstocks exposed to BN. Three sterols were tentatively identified as sitosterol, stigmasterol and campestrol, and the levels of these compounds was low.These chemical response data need to be correlated with the degree of root damage to determine which rootstock is more tolerant to burrowing nematode, and further investigation of the the phytochemicals responsible for the response to the pathogen attack is warranted. New rootstock evaluations  – We received the seeds from the USDA in Ft. Pierce for evaluating their susceptibility to nematodes. These included US-802, 812, 897, 942, 1283, 1284, 1516. After germination, the rootstock seedling were moved outside to encourage growth, repotted into larger pots, and are now about 6 inches tall and 7 months old. We hope they will be ready for evaluations soon. We plan to challenge these rootstocks with burrowing nematodes to determine their susceptibility/tolerance.       



Control citrus Huanglongbing by exploiting the interactions between Candidatus Liberibacter asiaticus and citrus

Report Date: 07/30/2021   Project: 18-026   Year: 2021

Control citrus Huanglongbing by exploiting the interactions between Candidatus Liberibacter asiaticus and citrus

Report Date: 07/30/2021
Project: 18-026   Year: 2021
Percentage Completion: 0.85
Category: CLas Bacteria
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

The goal is to understand how citrus interacts with Candidatus Liberibacter asiaticus (Las) infection and develop improved and long term HLB management strategies. Objective 1. Identification of the receptors for Las PAMPs in susceptible and tolerant citrus varietiesPotential 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 were cloned under 35S promoter and the Arabidopsis phloem specific promoter SUC2 and introduced into Agrobacterium. We have tested their receptors in Tobacco and citrus. Specifically, we are identifying the receptors in HLB susceptible variety Valencia sweet orange and HLB resistant variety Poncirus and HLB tolerant variety Sugar Belle. We have identified multiple receptors for the aforementioned PAMPs and are in the process of confirmation using pull-down assay or co-immunoprecipitation assays. We also hypothesized that Las outer membrane proteins might directly induce plant immune response in the phloem sieve elements because Las lives in the phloem. 21 outer membrane proteins have been cloned and the putative targets in citrus are being identified using Yeast 2 hybrid (Y2H) system and surface plasmon resonance (SPR) assay. Two outer membrane proteins showed positive interactions with citrus proteins based on Y2H assays. We are further confirming the interactions using GST pull-down assaysIn addition, multiple Las PAMPs have been tested for their effects in inducing plant defense against Las in the greenhouse and at least four different Las PAMPs showed significant effect in inducing plant immunity. We are testing whether those Las PAMPs can inhibit Las titers after foliar spray in the greenhouse. We have conducted RNA-seq analyses of Poncirus and sweet orange and we currently analyzing the data. We are testing the control effects of different PAMPs against HLB. Three PAMPs showed strong activity in inducing plant defenses. We have completed the trials in the greenhouse. We are spraying different PAMP products in field trials by spraying on newly planted young sweet orange trees. Significant control effect has been observed for certain PAMP products against HLB. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe are transgenically expressing putative receptors or targets (identified in Poncirus) of Las PAMPs in Valencia sweet orange or Duncan grapefruit. They are driven by 35S promoter and phloem specific promoter AtSuc2. We will conduct Las inoculation via grafting or psyllid transmission once the transgenic plants are about one year old.     For those identified receptors or targets, we are sequencing the promoter regions in Valencia, Sugar Belle, and Poncirus to compare their differences. If the native promoter of Poncirus is strong enough, we will use Poncirus promoter to drive the expression of PAMP receptors or other target genes to avoid concerns about 35S promoter or AtSUC2 promoter. We are also driving the expression of one defense inducing gene using a pathogen-inducing promoter. Several plants expressing the constructs were generated. Testing of those plants showed that they resonded to canker. We will test whether they are resistant to HLB. Right now, we are propagating to more plants for testing. We have made 6 constructs to express PAMP receptors individually or in combinations. We are expressing them in sweet orange. Three overexpression lines have been generated.    Objective 3. Investigate the roles of effectors in HLB disease developmentWe have completed screening of 30 putative Las effectors and 4 of them repressed plant defense. We are screening another 20 putative Las effectors and 3 more effectors that suppress plant defense. We have completed Y2H for the four defense-suppressing effectors and identified their targets in Valencia sweet orange. Confirmation of the targets is ongoing using coimmunoprecipitation and BiFC assays. Meanwhile, we have conducted CTV-mediated gene silencing of 15 putative HLB susceptibility genes in collaboration with Dawson lab. Sweet orange plants carrying the CTV constructs were inoculated with Las via grafting.  Interestingly, gene silencing of one of the putative HLB susceptible genes led to significant HLB tolerance. The plants showed mild HLB symptoms, similar growth as non-inoculated plants whereas the growth of control plants was significantly reduced and showed severe HLB symptoms. We are characterizing the putative mechanism of the HLB S gene. We are conducting genome editing of the identified HLB S gene of Valencia sweet orange and Duncan grapefruit to generate HLB resistant or tolerant citrus. In addition, we also overexpressed the HLB S gene in Valencia sweet orange to further understand the mechanism and will inoculate them with Las once they are one year old. We will continue to test other targets of putative effector genes. In addition, we hypothesized the effectors might induce plant defense in Poncirus and Sugar Belle. We are conducting Y2H to identify putative targets of effectors in Poncirus and Sugar Belle. We have conducted RNA-seq analyses of Sugar Belle. Data analyses have identified some interesting informations regarding chemicals to control HLB. We are summarizing the data for publication. One manuscript entitled Citrus CsACD2 is a target of Candidatus Liberibacter asiaticus in Huanglongbing disease has been published by Plant Physiology.  We have identified the binding sites of CsACD2 with SDE15. We have identified another important effector.We have conducted y2h for identification of the targets of CLas effector genes.   In total, six promising HLB susceptibility genes were identified. One line with silencling one of the target gene remains healthy despite being infected wiht CLas for over one year.     



Control citrus Huanglongbing by exploiting the interactions between Candidatus Liberibacter asiaticus and citrus

Report Date: 05/06/2021   Project: 18-026   Year: 2021

Control citrus Huanglongbing by exploiting the interactions between Candidatus Liberibacter asiaticus and citrus

Report Date: 05/06/2021
Project: 18-026   Year: 2021
Percentage Completion: 0.79
Category: CLas Bacteria
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

The goal is to understand how citrus interacts with Candidatus Liberibacter asiaticus (Las) infection and develop improved and long term HLB management strategies. Objective 1. Identification of the receptors for Las PAMPs in susceptible and tolerant citrus varietiesPotential 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 were cloned under 35S promoter and the Arabidopsis phloem specific promoter SUC2 and introduced into Agrobacterium. We have tested their receptors in Tobacco and citrus. Specifically, we are identifying the receptors in HLB susceptible variety Valencia sweet orange and HLB resistant variety Poncirus and HLB tolerant variety Sugar Belle. We have identified multiple receptors for the aforementioned PAMPs and are in the process of confirmation using pull-down assay or co-immunoprecipitation assays. We also hypothesized that Las outer membrane proteins might directly induce plant immune response in the phloem sieve elements because Las lives in the phloem. 21 outer membrane proteins have been cloned and the putative targets in citrus are being identified using Yeast 2 hybrid (Y2H) system and surface plasmon resonance (SPR) assay. Two outer membrane proteins showed positive interactions with citrus proteins based on Y2H assays. We are further confirming the interactions using GST pull-down assaysIn addition, multiple Las PAMPs have been tested for their effects in inducing plant defense against Las in the greenhouse and at least four different Las PAMPs showed significant effect in inducing plant immunity. We are testing whether those Las PAMPs can inhibit Las titers after foliar spray in the greenhouse. We have conducted RNA-seq analyses of Poncirus and sweet orange and we currently analyzing the data. We are testing the control effects of different PAMPs against HLB. Three PAMPs showed strong activity in inducing plant defenses. We have completed the trials in the greenhouse. We are spraying different PAMP products in field trials by spraying on newly planted young sweet orange trees. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe are transgenically expressing putative receptors or targets (identified in Poncirus) of Las PAMPs in Valencia sweet orange or Duncan grapefruit. They are driven by 35S promoter and phloem specific promoter AtSuc2. We will conduct Las inoculation via grafting or psyllid transmission once the transgenic plants are about one year old.     For those identified receptors or targets, we are sequencing the promoter regions in Valencia, Sugar Belle, and Poncirus to compare their differences. If the native promoter of Poncirus is strong enough, we will use Poncirus promoter to drive the expression of PAMP receptors or other target genes to avoid concerns about 35S promoter or AtSUC2 promoter. We are also driving the expression of one defense inducing gene using a pathogen-inducing promoter. Several plants expressing the constructs were generated. Testing of those plants showed that they resonded to canker. We will test whether they are resistant to HLB. Right now, we are propagating to more plants for testing. We have made 6 constructs to express PAMP receptors individually or in combinations. We are expressing them in sweet orange.    Objective 3. Investigate the roles of effectors in HLB disease developmentWe have completed screening of 30 putative Las effectors and 4 of them repressed plant defense. We are screening another 20 putative Las effectors and 3 more effectors that suppress plant defense. We have completed Y2H for the four defense-suppressing effectors and identified their targets in Valencia sweet orange. Confirmation of the targets is ongoing using coimmunoprecipitation and BiFC assays. Meanwhile, we have conducted CTV-mediated gene silencing of 15 putative HLB susceptibility genes in collaboration with Dawson lab. Sweet orange plants carrying the CTV constructs were inoculated with Las via grafting.  Interestingly, gene silencing of one of the putative HLB susceptible genes led to significant HLB tolerance. The plants showed mild HLB symptoms, similar growth as non-inoculated plants whereas the growth of control plants was significantly reduced and showed severe HLB symptoms. We are characterizing the putative mechanism of the HLB S gene. We are conducting genome editing of the identified HLB S gene of Valencia sweet orange and Duncan grapefruit to generate HLB resistant or tolerant citrus. In addition, we also overexpressed the HLB S gene in Valencia sweet orange to further understand the mechanism and will inoculate them with Las once they are one year old. We will continue to test other targets of putative effector genes. In addition, we hypothesized the effectors might induce plant defense in Poncirus and Sugar Belle. We are conducting Y2H to identify putative targets of effectors in Poncirus and Sugar Belle. We have conducted RNA-seq analyses of Sugar Belle. Data analyses have identified some interesting informations regarding chemicals to control HLB. We are testing them in the greenhouse right now. One manuscript entitled Citrus CsACD2 is a target of Candidatus Liberibacter asiaticus in Huanglongbing disease has been accepted by Plant Physiology.  We are investigating the binding sites of CsACD2 with SDE15. We have tested the effect of effectors in suppressing plant immune responses caused by PAMPs. We have identified another important effector. In total, six promising HLB susceptibility genes were identified.    



How do subterranean pests and diseases affect root health of trees with and without HLB?

Report Date: 03/21/2021   Project: 19-016   Year: 2021

How do subterranean pests and diseases affect root health of trees with and without HLB?

Report Date: 03/21/2021
Project: 19-016   Year: 2021
Percentage Completion: 0.65
Category: CLas Bacteria
Author: Larry Duncan
Sponsor: Citrus Research and Development Foundation

Citrus Nematode Exp 1  – The experiment was terminated in early January.  Plants were removed from pots and weighed (tops and rinsed/blotted roots).  Aliquots of fibrous roots were collected for metabolomic analysis and CLas determination (1g), nematode infection rate (2g), and moisture content determination (5g). Females and offspring were separated from roots by comminuting in a dilute sodium hypochlorite solution; juvenile and male nematodes were extracted from soil on Baermann funnels. The females/g root (458) on the susceptible rootstock Carrizo citrange exceeded the damage threshold by more than twofold and those on the resistant rootstock Swingle citrumelo were about half the number considered damaging (103).  Roots also exhibited symptoms of infection by Phytophthora nicotiana and the presence of the oomycete in soil was confirmed in all treatments. The contaminant originated from the orchard in which the nematode inoculum was obtained. The effects of rootstock, treatment and their interaction were all highly significant and therefore the results from each rootstock were considered separately.  Treatment with citrus nematode or CLas reduced the root weight of Carrizo citrange by 57% and 55%, respectively and by 60% when combined. An expected additive interaction between these pathogens would  reduce roots by 80%, therefore, the interaction of these organisms on root weight was antagonistic.  Citrus nematode and CLas reduced the root weight of Swingle citrumelo by 20% and 26%, respectively and by 4% when combined. An expected additive interaction between these pathogens would  reduce roots by 41%, therefore, the interaction of these organisms on root weight was antagonistic.  In the nematode susceptible rootstock there was a highly significant inverse relationship between the root weight and the nematode offspring recovered per gram of roots; however, HLB had no significant effect on nematode infection. The nematode resistant rootstock had significantly fewer females in roots (P=0.01) and offspring per rootweight (P=0.001) when infected by CLas. qPCR measurement of CLas infection in roots and tops of plants is ongoing.