We have no new results to report except that COVID-related delays in materials procurement have been overcome and quantification of oxy-tetracylcine and streptomycin for the final two experiments are now in process, and will be completed in the coming weeks. We have now completed the field portions of the studies proposed, as well as of a follow-up study to confirm the results of both of the first studies. The field study of delivery of oxytetracycline (OTC) has now been published in the journal Antibiotics (Killiny et al., 2020). Overall the results conclude that:1. OTC moved systemically, though a significant portion was trapped in the leafs into which it originally came in contact.2. Most foliarly applied OTC was lost. This was determined by comparison with injection. Less tha3. No adjuvant delivered significant quantities of OTC. 4. Only injection reduced polulation of HLB-causing Candidatus Liberibacter asiaticus (CLas). COVID-19-related delays in supplies have delayed quantification of streptomycin, though we now have assurances that the supplies will be delivered before the start of 2021. In the meantime, OTC quantification from the follow-up study, and CLas quantification are progressing for the follow-up study and the streptomycin-specific study. We anticipate publication of results within the 4 remaining months of the project. 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.
Purpose: Investigate the effects of underground pests on the severity of HLB in citrus trees co-infected with citrus nematode (Tylenchulus semipenetrans) or burrowing nematode (Radopholus similis) compared to HLB alone.Progress Summary: We confirmed that the roots of all of the trees treated with the two types of nematodes were successfully colonized and samples have been taken to examine the damage. Damage to the cortex of fibrous roots is the most common visual symptom (by staining and microscopy). Citrus Nematode Exp 1 – The experiment was established and primary infections with HLB and citrus nematode were confirmed. We had a low number of HLB trees test positive so we regrafted the HLB treatment trees in September. Samples for a second PCR is scheduled for January2021. The trees that received citrus nematode are yellowish with thin foliage, and trees on Swingle rootstock are more vigorous. The population density of the citrus nematode has increased from a moderate level of >680 in summer to >2100 juvenile and male nematodes per 100 cm3 soil in October, which now exceeds the damage threshold. As a consequence the 200 plants (10 treatments, 20 reps) were randomized in a blocked design for growth until the trial terminates in Spring 2021. Burrowing Nematode Exp 2 – 60 Val trees (15 trees x 4 TR) were established. Leaf samples were taken to confirm HLB infection (9 months). Burrowing nematode infection will be measured from soil core/root fragment samples in January 2021.Burning Nematode Exp 3 – UFR rootstocks planted in sandy soil, inoculated with burning nematode – these plants look very bad compared to non-inoculated controls. The roots have been recovered, washed and stained, and examined under the microscope. These roots show evidence of cortex damage to the fibrous roots. Root samples from the same plants were taken for metabolite analysis (by GC-MS), currently stored at -80 ºC until analysis.Future rootstock evaluations – USDA in Ft. Pierce confirmed that they will send us seeds for germination, to evaluate their susceptibility to nematodes during the next year. These include US-802, 812, 897, 942, 1283, 1284, 1516. These will be added to our evaluation effort, as it seems they have not been screened for their tolerance/susceptibility to nematodes.
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. 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. 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. The data is under analyses. 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 continued with work on Objectives 1 and 2, determining the effect of CLas infection on nematode development/populations, and vice versa. The objectives are reiterated for convenience below:1) Determine the effect of CLas infection on subsequent development of citrus parasitic nematodes and their damage to the plants. Hypothesis 1a: CLas alters nematode development and population growth. Hypothesis 1b: Concomitant nematode and CLas infection results in synergistic damage to citrus trees.2) Determine the subsequent effect of citrus parasitic nematodes on CLas infection. Plants will be first attacked by nematodes for 3 months and then graft-inoculated with CLas. Hypothesis 2: CLas symptoms develop more rapidly in nematode damaged plants.Progress on objectivesSince the last report, we sampled leaves from the primary (Exp. 1) study trees (100 trees each Val/SW, 100 trees Val/CZO) inoculated with citrus nematode and HLB to study the interactions between the two pathogens. After testing for HLB by PCR, we found that the infection rate was low (between 5 and 20%), but it is early (6 months), and we anticipate that the rate will be higher at the next check (9 months post-inoculation). Trees that were inoculated with citrus nematodes in late 2019 and early 2020 were check for nematode infection rate in early June. Six, nematode-infected plants were chosen randomly among the treatments and two small soil cores (1.5 cm dia. x 12 cm deep) were taken and combined from each pot. Nematodes were separated from soil in the samples using a sucrose centrifugation technique and counted. The mean number (and standard error) of citrus nematodes per 100 cm3 soil was 681 (175), a level comparable to that of well-infested groves during the summer months. Soil pH of the samples was 7.1, an optimum level for citrus nematode infection. In the upcoming months we plan to begin assessing and comparing root damage among the five treatments, as well as enumerate the nematode populations.In addition to the primary experiment, we added two new experiments:Burrowing nematode (Experiment 2)We initiated a new experiment with 30 trees of Valencia on Kuharske rootstock (nematode tolerant), and 30 trees of Val/Carrizo rootstock (nematode susceptible). 15 of each rootstock were graft-inoculated with HLB+ budwood. All of these trees were inoculated with burrowing nematode (750 nematodes of all life stages per plant) in 10 ml water divided between two holes in soil adjacent to each plant.Rootstock evaluations (Experiment 3)A third experiment was initiated to evaluate rootstocks for their tolerance to burrowing nematode. We planted seedlings of 7 new rootstocks in a soil bed, and inoculated it with the burrowing nematodes. The new rootstocks include: UFR-1, UFR-2, UFR-4, UFR-5, UFR-6, UFR-15 and UFR-17, five replicates each. These rootstocks will be sampled periodically for root damage and nematode infection rate to evaluate their susceptibility to burrowing nematode.
We completed implementation of the field study of delivery of oxytetracycline (OTC). OTC in the leaves to which the foliar sprays were applied have been quantified. OTC quantification in the leaves that were protected from direct contact with the sprays is in process. Results to-date indicate that no commercial adjuvant delivered significant quantities of OTC. Of all adjuvants, only Flotek 1, an experimental adjuvant, had significantly more OTC than the treatment with water and no OTC. All others were statistically similar to no OTC (Water AB). Based on the MIC developed by Li et al. (2019), no adjuvant achieved this minimum threshold. Injection, however exceeded 4x the MIC. In all cases, the same amount of was applied to each plant. When considering this, we considered injection to represent the foliar concentration when 100% of the treatment entered the plant, and the Water AB treatment to be the quantity when nothing was delivered. Using this standardization we calculated the efficiency of delivery of each treatment. The most efficient treatment, Flotek 1, had a mean of less than 5%. We completed the field study of streptomycin and samples are being processed. We have requested an NCE because COVID-19-related delays have prevented us from acquiring materials for streptomycin quantification. We also intend to repeate OTC and streptomycin applications with select adjuvants to confirm results. We have presented results to the Citrus Expo audience, and have submitted a manuscript for publication in a peer-reviewed journal.
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. 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. 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. The data is under analyses. One manuscript entitled Citrus CsACD2 is a target of Candidatus Liberibacter asiaticus in Huanglongbing disease has been accepted by Plant Physiology.
As noted in the last report, the experiment was established to meet the early objectives to obtain the trees and establish the primary infections with HLB and citrus nematode. We anticipate being able to begin testing trees for HLB in June.The trees in this experiment were maintained with irrigation and fertilization in a screenhouse. This was a fortuitous experimental schedule, because the potential to conduct research did not exist due to the UF response to the global pandemic. Nematode and CLas infection rates will be determined in the next reporting period and, if satisfactory, plants will be arranged in a randomized design for a one-month period of growth prior to the evaluation of treatment effects.
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. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe are transgenically expressing 12 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 patogen-inducing promoter. Several plants expressing the constructs were 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. One manuscript has been submitted to Plant Physiology regarding one putative HLB susceptibility gene. Genome modification of the putative S gene is ongoing. We are also editing two more putative S genes that were identified recently.
We completed implementation of the field study of delivery of oxytetracycline (OTC). OTC in the leaves to which the foliar sprays were applied have been quantified. OTC quantification in the leaves that were protected from direct contact with the sprays is in process. Results to-date indicate that no commercial adjuvant delivered significant quantities of OTC. Of all adjuvants, only Flotek 1, an experimental adjuvant, had significantly more OTC than the treatment with water and no OTC. All others were statistically similar to no OTC (Water AB). Based on the MIC developed by Li et al. (2019), no adjuvant achieved this minimum threshold. Injection, however exceeded 4x the MIC. In all cases, the same amount of was applied to each plant. When considering this, we considered injection to represent the foliar concentration when 100% of the treatment entered the plant, and the Water AB treatment to be the quantity when nothing was delivered. Using this standardization we calculated the efficiency of delivery of each treatment. The most efficient treatment, Flotek 1, had a mean of less than 15%.
The experiment was established to meet the early objectives to obtain the trees and establish the primary infections with HLB and citrus nematode. We anticipate being able to begin testing trees for HLB in June.We obtained 200 Valencia trees (100 on Swingle rootstock/100 on Carrizo RS) from the Rasnake Citrus Nursery. Trees were transplanted to soil suitable for propagating citrus nematodes (CN): a soil mix of 3 parts autoclaved sand: 1 part organic matter. The trees were allowed to acclimate to the soil mixture for one month, after which they were divided into five equal groups of 20 trees per rootstock (control and 4 treatments) and placed in outside 400-mesh screen cages, The five treatments consist of:TR1 Control (non-treated). Valencia/Swingle (20); Valencia/Carrizo (20)TR2 HLB only 20 trees per rootstock were graft graft-inoculated with HLB+ Valencia budwood from our collection of infected trees, to establish the primary HLB infection.TR3 CN only 20 trees per rootstock received inoculation with citrus nematodes (Tylenchulus semipenitrans) to establish the primary nematode infection.TR 4 HLB1+CN2 20 trees per rootstock were first grafted with HLB+ budwood and then inoculated with CN after the initial HLB inoculation.TR 5 CN1+HLB2 20 trees per rootstock received the CN inoculation first, which was then followed with HLB graftingA second nematode inoculation occurred for the treatments receiving CN to increase the nematode titer.
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. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe are transgenically expressing 12 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. 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. We have developed new methods for testing PAMP triggered immunity in citrus. 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. One manuscript has been submitted regarding one putative HLB susceptibility gene. Genome modification of the putative S gene is ongoing.
Major accomplishments:
1. The frequency of streptomycin resistance in Liberibacter crescens was determined in the lab. One in 500 million cells are spontaneously resistant to streptomycin.
2. Gene mutation identified for streptomycin resistance – the rpsL gene.
3. Developed rapid method to assess frequency of streptomycin in the field for CLas and non-target bacteria.
4. No spontaneous resistance observed for oxytetracycline suggesting that CLas resistance to oxytetracyline will take a very long time to occur.
5. Streptomycin resistance strains of L. crescens are not resistant to oxytetracycline.
6. Resistance to currently available antimicrobials should not arise quickly in the field.
7. Developed a defined culture medium for L. crescens that alow for more rapid and accurate antimicrobial testing.
8. Discovered that HLB symptoms may be the result of ammonia production by the pathogen.
9. Discovery of preferred carbon sources of L. crescens led to discovery of citrate as the preferred carbon source.
10 Optimal citrate concentrations for the growth of L. crescens are very close to the levels of citrate in phloem and the psyllid hemolymph.
11. Citrate use by Liberibacter leads to discovery that foliar phosphate fertilization may reduce CLas infection.
12. A new antimicrobial assay for L. crescens was developed for rapid antimicrobial discovery. New assay was shared with representatives from Bayer.
13. Sulbactim, erythromycin, and thiamphenicol considered primary candidates for CLas inhibition based on their probable phloem mobility and their inhibition of L. crescens at low concentrations.
14. Glyphosate was found to inhibit L. crescens at levels below that required to kill plants.
15. Citrus sensitivity to glyphosate was tested in the field. Although citrus is less sensitive to glyphosate than most plants, it is still sensitive enough to prevent the use of glyphosate for HLB control.
16. With Prof. Mou, an altered citrus ESPS synthase gene was made and transformed into Arabidopsis and citrus. The altered gene conferred glyphosate resistance in both plants.
17. The number of glyphosate resistant citrus plants are not being propogated to get enough plant to test the effect of glyphosate on HLB infection.
18. A cisgenic line of glyphosate resistance is now being generated by the CREC citrus transformation lab.
19. We are confident that glyphosate applications to glyphosate resistance citrus, will prevent HLB.
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.
Cruz-Munoz, M., Munoz-Beristain, A., Petrone, J.R., Robinson, M.A., & Triplett, E.W. 2019. Growth parameters of Liberibacter crescens suggest ammonium and phosphate as essential molecules in the Liberibacter-plant host interface. BMC Microbiology 19:222.
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 varieties
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 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 assays
In 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.
Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing Las
We are transgenically expressing 12 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.
Objective 3. Investigate the roles of effectors in HLB disease development
We 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. 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.
In this reporting period, plant uptake and phytotoxicity studies were performed on several (previously reported) multi-metal materials. Plant uptake study was performed using Citrus reshni (Cleopatra mandarin) seedling in the greenhouse. Eleven-month old seedlings were foliar sprayed with a hand-pump sprayer with the following treatments: 300 ml of MM25C75M (800 µg/ml Cu2+, 2400 µg/ml Mg2+), MM25C75Z (800 µg/ml Cu2+, 2400 µg/ml Zn2+), MM10C45M45Z (300 µg/ml Cu2+, 1350 µg/ml Mg2+, 1350 µg/ml Zn2+) and deionized water as untreated control (UTC). The pots were covered with Parafilm to prevent treatment solutions to contact with soil and seedling roots. After 72 h incubation, plants were carefully removed from the soil and gently washed with 1% detergent and 0.1% HCl. Leaves, roots, and stem sections were separated after washing and left in an oven (Cabela’s Inc, Sydney, NE) at 50 ºC for 72 h. The dried leaves, stems and roots were pulverized with mortars and pestles. One gram of dry powder of leaves, stems, and roots was acid digested (EPA method 3050 B “Acid Digestion of Sediments, Sludge, and Soil). After digestion, the digestate was filtered with Whatman No. 42 filter paper and the final volume was 20 ml. Cu, Zn and Mg content in leaves, stems, and roots were quantified with Atomic Absorption Spectroscopy (AAS). MM25C75M (142.8 ± 28.4 µg/ml Mg2+) showed significant higher Mg2+ uptake roots compare to untreated control (71.6 ± 0.73 µg/ml Mg2+). MM25C75M (Leaves: 0.54 ± 0.16 µg/ml Cu2+, Roots:1.82 ± 0.09 µg/ml Cu2+, Stems: 0.29 ± 0.08 µg/ml Cu2+) showed significant Cu2+ uptake by leaves, roots and stems compare to untreated control (Leaves: 0.16 ± 0.01 µg/ml Cu2+, Roots: 0.84 ± 0.06 µg/ml Cu2+, Stems: 0.09 ± 0.01 µg/ml Cu2+). MM25C75Z (1.85 ± 0.04 µg/ml Cu2+) showed significant Cu2+ uptake by roots compare to untreated control (0.84 ± 0.06 µg/ml Cu2+). MM25C75Z (5.6 ± 0.13 µg/ml Zn2+) and MM10C45M45Z (7.7 ± 0.03 µg/ml Zn2+) showed significant Zn2+ uptake by roots compare to untreated control (2.2 ± 0.004 µg/ml Zn2+). According to our results, MM25C75M showed significant leaf uptake of Mg2+ and Cu2+ and movement to roots. MM25C75Z showed significant leaf uptake of Zn2+ and Cu2+ and movement to roots. Similarly, MM10C45M45Z showed significant leaf uptake of Zn2+ and movement to roots. Phytotoxicity of MM25C75M, MM25C75M, and MM10C45M45Z and selected controls was evaluated on Citrus reshni (Cleopatra mandarin) eleven-month seeding in the greenhouse. All the materials were foliar sprayed by using a hand-operated pump mist sprayer at 800 µg/ml of Cu2+. Mg(NO3)2 (2400 µg/ml Mg2+), Zn(NO3)2 (2400 µg/ml Zn2+), and Cu(NO3)2 (800 µg/ml Cu2+) were selected as controls. Visual observations were conducted at 24, 48, and 72 h post-spray applications. After 3 days of incubation, Zn(NO3)2 and Cu(NO3)2 treated plants showed severe leaf browning deformation. MM25C75M (800 µg/ml Cu2+, 2400 µg/ml Mg2+), MM25C75Z (800 µg/ml Cu2+, 2400 µg/ml Zn2+), and MM10C45M45Z (600 µg/ml Cu2+, 2700 µg/ml Mg2+, 2700 µg/ml Zn2+) exhibited no damage to the plant. This year citrus canker field trial includes both MM25C75M and MM25C75Z.
We have designed and synthesized Fe oxide based material for facilitating systemic movement of EPA approved Cu bactericide/fungicide such as Cu salt of fatty acid. Research results on this material will be shared in future reports.
Field trials for efficacy against HLB and canker were continued with sprays every 21 days through October. Harvest for the second year of the grapefruit field trial was completed in the 2nd week of November, yield was weighed and canker rated, fruit size and juice quality are underway after a delay due to equipment failure. Data analysis of this years results and planning for the final year of field trials are ongoing.
Material characterization under various conditions were continued. Studies with citrus seedling were initiated to determine the rate of vascular movement of the metalic content in the stem leaves. Spectroscopy studies were also carried out to study potential biochemical changes of the plants as a result of the treatment. Optimization of the treatment conditions will continue in the next reporting period. Data analysis of the different results is underway with implementation of multivariate analysis.
We determined aquatic toxicity as an endpoint study of where these metal based bactericides would end up within the environment and affect the organisms there. We designed this experiment in accordance with the approved IACUC on fathead minnows with time point readings every 12h run for 48 hours. We tested various concentration ranges of the treatments to determine at what point within a 48h span the population of the fish would be reduced by half. Each concentration was done in triplicates and the data shown is a result of those averages. The LC50 was recorded for each treatment which includes (concentrations in ppm for the respective metal compounds): NAC-ZnO (0.6, 0.4, 0.2), NAC-ZnO+Cu (0.2/0.05), NAC-ZnS (0.6, 0.4, 0.2), NAC-ZnS+Cu (0.2/0.04), ZnO CR-41 (0.6, 0.4, 0.2), Zinc Nitrate (0.6, 0.4, 0.2), NAC (0.5, 1), Copper Sulfate (0.05), Kocide 3000 (0.4), Mg-Sol (0.6, 0.4, 0.2), Mg-Sol+Cu (0.6/0.15, 0.4/0.1, 0.2/0.05), TMN 113 (0.6, 0.4, 0.2), TMN 113+Cu (0.6/0.15, 0.4/0.1, 0.2/0.05), TMN 113+Cu+Mg (0.6/0.15/0.15, 0.4/0.1/0.1, 0.2/0.05/0.05), Magnesium Nitrate (0.6, 0.4, 0.2), and an untreated control (0).
LC50 results: NAC-ZnO (0.6) 12h, NAC-ZnO (0.4) 24h, NAC-ZnO (0.2) 48h, NAC-ZnO+Cu (0.2/0.05) 12h, NAC-ZnS (0.6) 48h, NAC-ZnS (0.4) 48h, NAC-ZnS (0.2) 48h, NAC-ZnS+Cu (0.2/0.04) 48h, CR-41 (0.6) 12h, CR-41 (0.4) 12h, CR-41 (0.2) 12h, Zinc Nitrate (0.6) 12h, Zinc Nitrate (0.4) 12h, Zinc Nitrate (0.2) 12h, Copper Sulfate (0.05) 24h, Kocide (0.4) 12h, NAC (0.5) 48h, NAC (1) 48h, Mg-Sol (0.6) 48h, Mg-Sol (0.4) 48h, Mg-Sol (0.2) 48h, Mg-Sol+Cu (0.6/0.15) 48h, Mg-Sol+Cu (0.4/0.1) 48h, Mg-Sol+Cu (0.2/0.05) 48h, TMN113 (0.6) 48h, TMN113 (0.4) 48h, TMN113 (0.2) 48h, TMN113+Cu (0.6/0.15) 12h, TMN113+Cu (0.4/0.1) 12h, TMN113+Cu (0.2/0.05) 24h, TMN113+Cu+Mg (0.6/0.15/0.15) 12h, TMN113+Cu+Mg (0.4/0.1/0.1) 12h, TMN113+Cu+Mg (0.2/0.05/0.05) 24h, Magnesium Nitrate (0.6) 48h, Magnesium Nitrate (0.4) 48h, Magnesium Nitrate (0.2) 48h, and Untreated (0) 48h.
Notable findings: NAC-ZnS, NAC control and Magnesium only treatments (Mg-Sol & Magnesium Nitrate) did well in terms of not killing and in some cases did better at keeping the fish active and alive over the course of the experiment compared to the untreated control. NAC-ZnS+Cu and Mg-Sol+Cu also did not kill the fish despite the addition of copper which is unexpected in the case of Mg-Sol+Cu due to the large amount of Cu present. Every time Cu was added in another treatment, such as NAC-ZnO, the respective LC50 dropped. Copper sulfate and Kocide controls exhibited high toxicity. TMN113 by itself performed much better when compared to industry controls ZnO CR-41 & Kocide 3000. NAC-ZnO (0.6) and all Zinc Nitrate treatments exhibited similar toxicity to Kocide 3000 and ZnO CR-41. Copper sulfate, even at such a low concentration (0.05) killed all fish after 24h, similar to TMN113+Cu. In the case of TMN113+Cu+Mg, all concentrations killed fish within 24h.
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