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.
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.
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.
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.
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.
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.
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 identified 21 genes that might contribute to the tolerance of Poncirus to 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. 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 have identified another important effector. In total, six promising HLB susceptibility genes were identified.
1. Please state project objectives and what work was done this quarter to address them:Objective 1: Investigate efficacy of bactericide treatments for preventing new infections. Objective 2. Determine the effect of bactericide application frequency on Las infection of citrus. Objective 3: Quantify the effect of repeated inoculation of the efficacy of bactericides. During the final quarter of the project, we completed two final treatment applications for experiments addressing objectives 1-3. Samples (leaf, ACP, flush) were collected as in previous quarters. All remaining leaf samples from previous quarters and the final quarter were processed (DNA extraction, qPCR) to determine the CLas titers in trees in response to treatments. 2. Please state what work is anticipated for next quarter: No additional work is expected. The project is complete, and this is the final report. 3. Please state budget status (underspend or overspend, and why):The budget for the project was underspent. The underspent funds were is a result of savings on the treatment materials used in this project (antimicrobials, insecticides. A portion of these materials were donated; therefore, the projected budget for these materials was lower than initially anticipated. Another factor contributing to the underspend was a reduction in activities during spring 2020. Personnel were unable to come in and work in the labs (April and May 2020) or the field (April 2020). One field treatment was missed as a result.. The reduction in salary and material spending contributed to the reduced funds spent. Executive Summary AbstractThe purpose of this project was to identify the most efficacious use of antimicrobial treatments that are commercially available in order to reduce Las inoculation pressure and prevent infection in young trees. Overall, the data indicate that bactericidal treatments did not prevent CLas colonization in young citrus trees. However, the results suggest that bactericides in combination with insecticides could be useful in preventing CLas infections in the first year of new citrus tree plantings. As the trees mature, additional protection should be considered. Additionally, citrus trees receiving monthly Firewall/Fireline applications had higher numbers of ACP adults and eggs and lower flush production across treatments. The use of Tree Defender enclosures and insecticide alone showed better and more prolonged (more than one year) protection of young citrus trees against CLas infection compared with antimicrobial treatment after one year. More frequent (monthly) bactericidal applications in combination with insecticides reduced CLas titers on mature trees as compared to quarterly Firewall/Fireline applications or insecticide only. Additionally, the number of eggs in flush declined in response to antimicrobial treatment. Further studies should evaluate bactericidal application frequency on younger and productive citrus trees (commercial citrus groves rather than in research groves) as a preventative and active method against CLas infections. Additionally, the data suggest that bactericides in combination with insecticides should be used when flush is highly abundant to reduce the numbers of eggs in flush for disruption of CLas transmission.
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.
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 leaves into which it originally came in contact.
2. Most foliarly applied OTC was lost. This was determined by comparison with injection.
3. No adjuvant delivered greater quantities of OTC than water with no adjuvant.
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.
Publication
Killiny, 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.
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.
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