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.
All significant results remain the same as in the previous quarter.
The long term field trial continues with weekly psyllid counts and quarterly CLas infection testing. Treatments continue to have similar effects on ACP counts. Plants in both of the kaolin treatments continue to show higher growth rates than the other two treatments. The red treatment has the highest growth rate, trunk cross-sectional area, and canopy volume. Kaolin treated trees that are infected grow more than untreated-infected trees, but less than treated uninfected trees. The field trial will continue until the project ends, when we expect to have the first economic yield.
We are now performing follow-up repetitions of the MS student’s thesis work. We anticipate publication submission of this work in the Fall.
Objective 1: Investigate the efficacy of bactericides treatments for preventing new infections for young citrus trees protection.
Hypothesis: Bactericidal treatment will protect young trees from CLas colonization.
Initial leaf samples were collected prior to treatments to evaluate CLas titers in the uninfected trees.
We applied bactericide treatments from May through September. CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of CLas-infection rate in citrus leaves from May and June is described. Currently, citrus leaves tissue samples from July through September are being processed to analyze the CLas-infection rate.
*Trees were considered CLas-infected (positives) when CT values were below 35.
1. Antibiotics (monthly rotation): Prior to bactericide application (May), 15% of trees (20 trees/treatment) were CLas positive (Ct<35). After the bactericide application (June), 35% of trees were CLas positive (Ct<35).
2. Antibiotics (quarterly rotation): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After bactericide application (June), 40% trees were CLas positive (Ct<35).
3. Negative Control (insecticide + Tree defender exclusion netting): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After the bactericide application (June), 45% trees were CLas positive (Ct<35).
4. Positive Control (insecticide only): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After the bactericide application (June), 5% trees were CLas positive (Ct<35).
Counting of ACP adults using taps was conducted bi-weekly from May through September, presence of other life stages such as eggs and nymphs were scouted visually. Preliminary results showed a low ACP population in citrus locations due to the active vector management performed by farm manager. As consequence, no ACP adults were collected to analyze the CLas-infection rate using quantitative real-time PCR analysis. The overall number of eggs and nymphs were low or undetectable in citrus trees from May to September. Also, to determine the effect of citrus vegetative growth (flush-like structures) in CLas-infection rate, 1 ft.3 was used to count the number of flush-like structures per tree. Results showed that the presence of flush-like structures incremented from May to July and decreased in September.
Objective 2. Determine the effect of bactericides application frequency on Las infection of citrus.
Hypothesis: Bactericidal treatment will reduce CLas infection in mature trees.
We applied bactericide treatments from May through September. CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of CLas-infection rate in citrus leaves from May and June is described. Currently, citrus leaves tissue samples from July through September are being processed to analyze the CLas-infection rate.
*Trees were considered CLas-infected (positives) when CT values were below 35.
1. Antibiotics (monthly rotation): Prior to bactericide application (May), 100% of trees (20 trees/treatment) were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35). Although positive, bacterial titers declined in trees receiving antimicrobial treatments.
2. Antibiotics (quarterly rotation): Prior to bactericide application (May), 100% of trees were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35).
3. Positive Control (insecticide only): Prior to bactericide application (May), 100% of trees were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35).
Counting of ACP adults using taps was conducted bi-weekly from May through September, presence of other life stages such as eggs and nymphs were scouted visually. Preliminary results showed high ACP populations in treatments from May to August, excepting for June. The number of eggs and nymphs were not collected during May and first collection of June. However, populations increased from late June to August and reached high population levels. Currently, ACP adults that were collected bi-weekly are being processed to analyze the CLas-infection rate using quantitative real-time PCR analysis. Also, to determine the effect of citrus vegetative growth (flush-like structures) in CLas-infection rate, 1 ft3 was used to count the number of flush-like structures per tree. Flush was not collected during May and June. However, results showed that the presence of flush was high in July and August.
August 31, 2019 – In this quarter, we have continued to work on objectives outlined in our chronogram.
Objective 1. We have completed assessment of trees planted in our pilot study (planted 22 months ago) for CLas infection and HLB symptoms. All the non-covered trees are PCR-positive for CLas whereas all trees covered with IPC have tested negative. We are continuing with quantification of leaf drop and comparing leaf drop in both treatments; 6-month cumulative data show no significant differences in leaf drop in IPC-covered trees compared with non-covered trees. Interestingly, when counted seasonally, in spring leaf drop was significantly higher in non-covered trees as compared to IPC trees, whereas in summer, it was slightly higher inside IPCs. This fact points out a seasonal component that we will investigate as the project progresses.
In August, we have replaced the old 4-ft IPCs with new 8-ft covers, donated by The Tree Defender, Inc, because the trees had filled the volume of the cover completely. This also has opened the possibility of studying the dynamics of branch unfolding, which we are doing visually (photography documentation) and by measuring canopy growth and leaf area index. We have also assessed other pest and disease incidences inside the IPCs. We have found less incidences of canker inside IPCs and approximately equal incidences of greasy spot. However, greasy spot severity is higher inside the IPCs. We have found more incidence of other pests such as mites, armyworms, and leafrollers inside the IPCs, and a total absence of predators (beneficials). This suggests that relying only on IPC for insect control is not sufficient, and insect management must still be conducted. No psyillid have been found inside the IPCs.
Objective 2. To study the edge effect in different IPC layouts, we are now preparing to plant 700 trees of SugarBelle, Tango and Early Pride mandarins and using 3 different arrangements (targeted, alternated and patterned, as described in the proposal) of IPC. We have performed initial measurements of the tree parameters (trunk diameter, and leaf sampling, for CLas, cholorophyll and sugar analysis).
Objectives 3 and 4. We are continuing to measure fruit set and development inside the IPCs and comparing this with our CUPS planting. We are taking fruitlet and fruit samples regularly for biochemical analysis.
Outreach, Professional Presentations and Extension Activities for this quarter :
-Grower Presentation: “Growing Young Citrus Trees Under Individual Protective Covers (IPCs): What We Know After 18 Months” Citrus Expo 2019, August 15, Fort Myers, Fl.
-Industry Magazine Article: “Individual Protective Covers for Psyllid Exclusion and HLB Disease Prevention in Young Trees”. Article submitted to Citrus Industry Magazine in July to be published in October issue.
-Our Project was also noted in the September’s issue of Citrus Industry Mag’s UF/IFAS. The Citrus State Opinion Column by Jack Payne highlighted this work as an example of collaboration between growers, extension agents, and scientists in Florida. The column was entitled “Collaboration breeds solutions”.
The goal is to understand how citrus interacts with Candidatus Liberibacter asiaticus (Las) infection. To achieve the goal of this research, we are conducting the following objectives: Objective 1. Identification of the receptors for Las PAMPs in susceptible and tolerant citrus varieties21 outer membrane proteins have been cloned and the putative targets in citrus are being identified using Yeast 2 hybrid system. Potential PAMPs from Las (either homologous to known PAMPs or pilin genes) LasFlaA (flagellin), LasEF-Tu, LasCSP (cold shock protein), LasSSBP (single strand binding protein) and pilin assembly genes (named LasPil85, LasPil95, LasPil105 and LasPil115) were cloned under 35S promoter and the Arabidopsis phloem specific promoter SUC2 and introduced into Agrobacterium. We are testing their receptors in Tobacco and citrus. We have identified multiple receptors for the aforementioned PAMPs and are in the process of confirmation.In addition, multiple PAMPs are being tested for their effects in inducing plant defense against Las in the greenhouse. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe have the selected PAMP receptors and are overexpressing them in citrus. The constructs have been made. Objective 3. Investigate the roles of effectors in HLB disease developmentFor the 10 selected Sec-dependent effectors (SDEs), we have conducted yeast two hybridization (Y2H) and identified their targets in Valencia sweet orange. We are in the process of confirming the targets using other approaches such as bimolecular fluorescence complementation (BiFC) and Co-Immunoprecipitation (co-IP) assays. We are conducting Y2H and SPR assays to identify their targets in Poncirus and in the tolerant variety sugar belle. We are overexpressing the SDEs in tobacco and citrus to test their effect on HLB disease development.
We continue to work with Michael Rogers on glyphosate as a treatment for citrus greening disease.
Over a period of five months in the greenhouse, we determined the optimal concentration of glyphosate that citrus Valencia saplings could tolerate as well as the frequency with which it could be applied. We learned that citrus can tolerate an 8 mM spray of glyphosate every three months. The plents would lose a few leaves at first but after three months, flush would appear and the plants appeared to recover.
The sprays were applied at monthly intervals. We learned that monthly and semi-monthly sprays were too frequent. We also learned that 25 mM sprays were lethal. Thus, those were discontinued. The source of glyphosate was RoundUp as pure glyphosate was too expensive. RoundUp would be the source that growers would use in any case.
A field experiment was then started six weeks after the greenhouse experiment. The plots were set up at Lake Alfred by Michael Rogers. Prior to the first spray, the trees were sampled to determine CLas titer. Disease severity of the trees also determined.
As we now know from the greenhouse experiment that a three-month interval allows for tree survival and continued growth, the field plot is scheduled to be sprayed again in early August. The trees will be sampled and assessed for disease severity before and after the three-month spray.
Qualitatively, the results from the field are encouraging and mimic the results from the greenhouse. Those plants sprayed with 8 mM glyphosate are recovering and have new flush. The untreated control plants have no new flush. The plants sprayed with 25 mM glyphosate were nearly killed. We are not treating those trees again. We will let them recover.
We are eager to continue this experiment over the next few months and hope to learn the effect of glyphosate on CLas titer over the next three months.
Meanwhile, the transformation of citrus to generate RoundUp ready plants in a cis-genic manner is in progress at Lake Alfred. We believe that using cis-genic plants will be the long-term solution.
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