December 2017 The objectives of this proposal are 1) To determine the temperature and relative humidity optima for Guignardia citricarpa pycnidiospore infection and production on citrus twigs, leaf litter, and fruit; 2) To determine the relative potential of Guignardia citricarpa to form pycnidiospores on citrus twigs, leaf litter, and fruit; 3) To determine whether Guignardia citricarpa can survive and reproduce on citrus debris on grove equipment. In 2018 we have recorded the number of spores from field collected dead twigs from 2016 to August 2017. We have found relatively low numbers of spores from the twigs and we are unsure if this because limited numbers of spores are being produced or that we are not effectively capturing them. We have also started to evaluate the level of P. citricarpa DNA from the twig bark. We consistently find low level presence of P. citricarpa DNA but it increases over time. More data will tell us if this is a seasonal effect. The first four temperatures of the temperature and relative humidity (RH) experiment were conducted with twigs. Low numbers of pycnidia to none were formed at %RH less than 100%. This does not correspond with previous data but we have determined that the drying conditions post inoculation is likely the culprit. The fungus is not dead as pycnidia can form when the lower %RH treatments are placed at 100% RH for an additional month. We are working to resolve this problem with a less rigorous drying regimen. The temperature and relative humidity experiment on leaf tissue was more successful. Both temperature and relative humidity had a significant effect on the number of pycnidia formed on the leaf disks. As expected, pycnidia formation was slower at cooler temperatures (16 and 20C). Relative humidities less than 85% did not form pycnidia. Unfortunately, when the DNA data was examined, it was revealed the the growth had been the endophyte P. capitalensis, which may not have the same sporulation pattens as P. citricarpa. We will be repeating this experiment with autoclaved leaves. This is not as satisfying because we cannot look a the the infection process as well but at least we will be better able to study what P. citricarpa is doing rather than the endophyte. Decontamination work continued this year adding the factor of debris to the study. Debris significantly reduced the efficacy of the decontamination materials but they are still effective at the recommended rates of quaternary ammonium and bleach. Bleach was more affected by the presence of debris than quaternary ammonium. Interestingly, higher volumes of disinfectant were more effective than lower volumes.
The long term field trial continues with weekly psyllid counts. Treatments continue to have similar effects on ACP counts. The kaolin effect continues to result in differences in CLas infection. A July sampling for qPCR assesment of CLas infection indicated that there is no difference in infection between foliar insecticide and untreated control treatments with means near 75% infectction (approximately 1 year after planting). Approximately 30% of undyed kaolin plants were infected, and 15% of red-dyed kaolin plants were detected as infected. These infection results are promising, but still preliminary. Additionally plants in both of the kaolin treatments continue to show higher growth rates than the other two treatments. We harvested a small but important harvest from the kaolin treatments but not from the controls. The Master’s student funded by this project has defended his proposal has completed one experiment and nearly completed two more regarding kaolin films dyed different colors. Intitial results are that all colors reduce leaf temperature. The dyed films additionally reduce photosynthetically active radiation (PAR) below that of the white kaolin. Red produces a moderate (~30% reduction), while the other colors dramatically reduce PAR. White kaolin increased maximum photosynthesis, whle red kaolin was not significantly different from control or white kaolin. The other colors reduced photosynthesis. Both red and white result in less dramatic midday depression. We are now planning an experiment to test whether red is improving water status under water limiting conditions. A final experiment will begin in December, using similar treatments as the first potted experiment, but controling for quantity of PAR to isolate quality effects alone.
The goal of this project is to determine whether pathogen or dsRNA exposure primes the ACP immune system to resist future infection by pathogens, including Las, and whether this effect is multigenerational. We have previously characterized the specificity and efficacy of the immune priming response in ACP (Obj. 1), characterixzed the effect of prior immune challenge on transmission (Obj. 3) and determined the transgenerational effect of pathogen-induced immune priming on Las acquisition. The current report describes our ongoing efforts to quantify the effect of RNAi-induced priming on Las acquisition (Obj 4). Adults of ACP collected from a laboratory colony free of CLas infection were starved for 3 hr, and then were subjected to dsRNA ATPase and sucrose. For each treatment, 5 cages including 10 insects in each were used. Insects were fed on diet solution consisting of 10, 100, and 1000 ng. l -1 dsRNA ATPase, 20% sucrose, and 0.5 green food coloring dye. As a control, adult of ACP was fed on sucrose 20%+ 0.5 green food dye. A cage for artificial feeding was prepared by stretching parafilm membranes on the bottom of plastic petri dish arenas. The parafilm surface was sterilized with ethanol and dried for 5 min under a sterile hood and layered with 400 L of diet solution including dsRNA, 20% sucrose, and green food coloring dye. The liquid was then covered with a second layer of stretched parafilm. During the feeding, the cages were placed in a growth chamber at 28oC. Insect were collected after 24 hrs and 5 days feeding and stored in -80 C for RNA extraction. Insects primed by exposure to artificial diet solutions with dsRNA for 24 hours were transferred to separate branches of a potted citrus plant (var. “Swingle”). Seven days after priming, insects were removed from plants, starved for 3 h, and either injected (experiment 1) with a lethal dose of S. marcescens or control treatment. After feeding or injection, the ACP that survived were allowed to reproduce on healthy of CLas-infected hosts. DNA was extracted from D. citriusing Qiagen DNeasy Blood and tissue kits (Qiagen, Hilden, Germany) per manufacture recommendations. Quality and concentration of DNA was assessed after extraction on a Nano Drop 2000 (Thermo Fisher Scientific, Waltham, MA), then standardized to 10ng/ l. CLas titers were assessed by the detection of the 16S rDNA gene by qPCR methods described by Coy et al. (2014). Plants were tested to ensure infection with CLas by qPCR following methods described by Li et al. (Li et al., 2006). No significant differences in reproductive success or acquisition by offspring were detected to date, which suggests that immune priming does not occur in response to RNAi, and that this response is unlikely to affect CLas transmission.
The goal of this project is to determine whether pathogen or dsRNA exposure primes the ACP immune system to resist future infection by pathogens, including Las, and whether this effect is multigenerational. We have previously characterized the specificity and efficacy of the immune priming response in ACP (Obj. 1), characterixzed the effect of prior immune challenge on transmission (Obj. 3) and determined the transgenerational effect of pathogen-induced immune priming on Las acquisition. The current report describes our ongoing efforts to quantify the effect of RNAi-induced priming on Las acquisition (Obj 4). Adults of ACP collected from a laboratory colony free of CLas infection were starved for 3 hr, and then were subjected to dsRNA ATPase and sucrose. For each treatment, 5 cages including 10 insects in each were used. Insects were fed on diet solution consisting of 10, 100, and 1000 ng. l -1 dsRNA ATPase, 20% sucrose, and 0.5 green food coloring dye. As a control, adult of ACP was fed on sucrose 20%+ 0.5 green food dye. A cage for artificial feeding was prepared by stretching parafilm membranes on the bottom of plastic petri dish arenas. The parafilm surface was sterilized with ethanol and dried for 5 min under a sterile hood and layered with 400 L of diet solution including dsRNA, 20% sucrose, and green food coloring dye. The liquid was then covered with a second layer of stretched parafilm. During the feeding, the cages were placed in a growth chamber at 28oC. Insect were collected after 24 hrs and 5 days feeding and stored in -80 C for RNA extraction. Insects primed by exposure to artificial diet solutions with dsRNA for 24 hours were transferred to separate branches of a potted citrus plant (var. “Swingle”). Seven days after priming, insects were removed from plants, starved for 3 h, and either injected (experiment 1) with a lethal dose of S. marcescens or control treatment. After feeding or injection, the ACP that survived were allowed to reproduce on healthy of CLas-infected hosts. DNA was extracted from D. citriusing Qiagen DNeasy Blood and tissue kits (Qiagen, Hilden, Germany) per manufacture recommendations. Quality and concentration of DNA was assessed after extraction on a Nano Drop 2000 (Thermo Fisher Scientific, Waltham, MA), then standardized to 10ng/ l. CLas titers were assessed by the detection of the 16S rDNA gene by qPCR methods described by Coy et al. (2014). Plants were tested to ensure infection with CLas by qPCR following methods described by Li et al. (Li et al., 2006). No significant differences in reproductive success or acquisition by offspring were detected to date, which suggests that immune priming does not occur in response to RNAi, and that this response is unlikely to affect CLas transmission.
This project contains two objectives: 1) Control HLB by optimization of application of SA and its analogs. We are testing the control effect of SA and its analogs, e.g., ASM, Imidacloprid, DL-2-aminobutyric, 2,6-dichloro-isonicotinic acid, and 2,1,3 Benzothiadiazole via trunk injection in field trial. Oxytetracycline is used as a positive control, whereas water was used as a negative control. SA, Acibenzolar-S-methyl (ASM), benzo (1,2,3) thiadiazole-7-cabothionic acid S-methyl ester (BTH), and 2,6-dichloroisonicotinic acid (INA) have also been applied twice onto selected trees by foliar spray in November, 2015 during fall flush, arch 2016 during spring flush, and February 2017 during spring flush. In addition, three field trials for different compounds including SA are being conducted. Materials were applied once onto selected trees by foliar spray in September, 2016 during late summer-fall flush, were applied to selected trees by soil drench in September, 2016 during late summer-fall flush, in early March and June 2017. Trunk injection in August and September, 2016 during summer and late summer-fall flush. Trunk injection of SA showed significant control effect against HLB. The data for trunk injection has been collected and a manuscript has been submitted for publication. HLB disease severity,disease incidence surveys and Las titers were conducted before spray treatment in October, 2015 and at 6 months after the 1st application in April, 2016 and April 2017. SA analogs resulted in increased fruit yield in 2016, but not in 2017 probably due to hurricane damage and also slowed down the progress of Las titers compared to control. To compare the effect of suppressing SA hydroxylase, we also screened multiple SecA inhibitors which suppress the secretion of important virulence factors. Two effective SecA inhibitors have been tested in vitro. At least one SecA inhibitor has been shown to be specific against Las, but not E. coli. We are also investigating the possibility of modifying pathway of citrus to produce more SA in citrus using CRISPR. As experiment scheduled, SA, ASM, BTH and INA were applied to selected trees by foliar spray in March 2018 during spring flush. Admire, SA, ASM, BTH and INA were applied to selected trees by soil drench in March 2018 during spring flush. SA, ASM, BTH and INA were applied to selected trees by trunk injection from March to April 2018 during spring flush. HLB disease severity surveys and Las titer assays will conducted for treatments in April 2018 at 24 months after 1st application of soil drench or trunk injection and at 30 months after 1st application of foliar spray. 2) Control HLB using a combination of SA, SA analogs or SA hydroxylase inhibitors. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design are being tested for their inhibitory effect against SA hydroxyalse. To further identify SA hydroxylase inhibitors or SA analogs that are not degraded by SA hydroxylase, we have expressed SA hydroxylase in tobacco and Arabidopsis. Overexpression of SA hydroxylase decreased HR induced by Pseudomonas spp, indicating that SA hydroxylase degrades SA. We have qualified SA with HPLC and conducted SAR related genes expression analysis. We have identified multiple SA analogs and tested whether they can be degraded by SA hydroxylase. 4 SahA inhibitors were trunk-injected during fall flush. Las titers and HLB disease severity of the treated trees are being tested periodically. One manuscript entitled: ‘Candidatus Liberibacter asiaticus’ Encodes a Functional Salicylic Acid (SA) Hydroxylase That Degrades SA to Suppress Plant Defenses” has been published by MPMI.
The goal of the proposed study is to understand the mechanism of survivor trees. 1. Understanding the role of endophytic microbes from survivor trees. Three healthy and three HLB infected trees were selected for phytobiome analysis from Gapway grove based on the Las QPCR detection results. The microorganisms collected from this experiment were classified as three types: rhizosphere, rhizoplane and endosphere communities. The DNA and RNA samples were sequenced. Multiple known beneficial microorganisms, such as Bradyrhizobium, Lysobacter and Variovorax showed significantly higher relative abundance and activity in rhizoplane microbiome despite of health status. However, several beneficial taxa, including Rhodopseudomonas, Achromobacter, Methylobacterium and Chitinophaga, showed higher relative abundance and activity in healthy rhizoplane microbiome compared with rhizosphere community in healthy trees but not in HLB samples. By performing comparison between healthy and HLB samples, we found several phyla, such as Proteobacteria, Acidobacteria and Bacteroidetes were enriched in healthy root-associated microbiome. HLB altered the rhizoplane microbiome by recruiting more functional features involved in autotrophic life cycle such as carbon fixation, and abandoning the functional genes involved in microbe-host interactions identified above, collectively resulting in downward spiral in rhizoplane microbiome-host interaction. This seems to suggest the manipulation of the root microbiome is necessary. However, the challenge is how to maintain a beneficial microbiome which is under study now. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. As shown in Objective 1, Bradyrhizobium and Burkholderia are the most abundant bacteria that have shown dramatic changes between survivor trees and HLB diseased trees. We determined the contribution of Burkholderia to the citrus hosts. We isolated multiple Burkholderia strains. We selected two representative strains A53 (Burkholderia metallica) and A63 (Burkholderia territori) to inoculate citrus plants using the soil drench method. The results demonstrated that the two strains could successfully colonize the root surface and maintain a relative high population even seven months after inoculation. We then conducted a greenhouse study to evaluate the effects of the selected strains on the plant fitness. One manuscript entitled: “Characterization of antimicrobial-producing beneficial bacteria isolated from Huanglongbing escape citrus trees “has been apublished by Frontiers in Microbiology. One more manuscript on the effect of induced systemic resistance against disease by rhizospheric bacteria has been accepted for publication by Phytopathology. In addition, we grafted the roots from survivor trees to healthy and HLB diseased trees in greenhouse to check the effect of endophyte changes on the grafted trees. Since endophytes appear to be enriched from the rhizosphere, we also used the soil from the survivor trees to plant both healthy and HLB diseased trees in the greenhouse. We also grafted shoots from survivor trees to further understand the putative mechanisms. Shoots from more survival trees are being grafted. We are also characterizing the potential mechanism why some branches are Las free. Multiple plants successfully grafted with leaf branches from survivor trees were subject to a test for citrus attractiveness to ACP. No significant effect on response of ACP to the grafted trees from the control. We have grafted more trees with branches from survivor trees to test their effect on Las and ACP. Consortium of bacteria of different combinations are being used to test their effect on Las and ACP. One manuscript entitled: “Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome” has been published by Microbiome.
The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). This project contains two objectives: 1) Control HLB by optimization of application of SA and its analogs. We are testing the control effect of SA and its analogs, e.g., ASM, Imidacloprid, DL-2-aminobutyric, 2,6-dichloro-isonicotinic acid, and 2,1,3 Benzothiadiazole via trunk injection in field trial. Oxytetracycline is used as a positive control, whereas water was used as a negative control. SA, Acibenzolar-S-methyl (ASM), benzo (1,2,3) thiadiazole-7-cabothionic acid S-methyl ester (BTH), and 2,6-dichloroisonicotinic acid (INA) have also been applied twice onto selected trees by foliar spray in November, 2015 during fall flush, arch 2016 during spring flush, and February 2017 during spring flush. In addition, three field trials for different compounds including SA are being conducted. Materials were applied once onto selected trees by foliar spray in September, 2016 during late summer-fall flush, were applied to selected trees by soil drench in September, 2016 during late summer-fall flush, in early March and June 2017. Trunk injection in August and September, 2016 during summer and late summer-fall flush. Trunk injection of SA showed significant control effect against HLB. The data for trunk injection has been collected and a manuscript has been submitted for publication. HLB disease severity,disease incidence surveys and Las titers were conducted before spray treatment in October, 2015 and at 6 months after the 1st application in April, 2016 and April 2017. SA analogs resulted in increased fruit yield in 2016, but not in 2017 probably due to hurricane damage and also slowed down the progress of Las titers compared to control. To compare the effect of suppressing SA hydroxylase, we also screened multiple SecA inhibitors which suppress the secretion of important virulence factors. Two effective SecA inhibitors have been tested in vitro. At least one SecA inhibitor has been shown to be specific against Las, but not E. coli. We are also investigating the possibility of modifying pathway of citrus to produce more SA in citrus using CRISPR. One manuscript entitled: “Control of Citrus Huanglongbing (HLB) via Trunk Injection of Plant 1 Activators and Antibiotics” has been published by Phytopathology. 2) Control HLB using a combination of SA, SA analogs or SA hydroxylase inhibitors. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design are being tested for their inhibitory effect against SA hydroxyalse. To further identify SA hydroxylase inhibitors or SA analogs that are not degraded by SA hydroxylase, we have expressed SA hydroxylase in tobacco and Arabidopsis. Overexpression of SA hydroxylase decreased HR induced by Pseudomonas spp, indicating that SA hydroxylase degrades SA. We have qualified SA with HPLC and conducted SAR related genes expression analysis. We have identified multiple SA analogs and tested whether they can be degraded by SA hydroxylase. 4 SahA inhibitors were trunk-injected during fall flush. Las titers and HLB disease severity of the treated trees are being tested periodically. One manuscript entitled: ‘Candidatus Liberibacter asiaticus’ Encodes a Functional Salicylic Acid (SA) Hydroxylase That Degrades SA to Suppress Plant Defenses” has been published by MPMI.
The goal of the proposed study is to understand the mechanism of survivor trees. 1. Understanding the role of endophytic microbes from survivor trees. Three healthy and three HLB infected trees were selected for phytobiome analysis from Gapway grove based on the Las QPCR detection results. The microorganisms collected from this experiment were classified as three types: rhizosphere, rhizoplane and endosphere communities. The DNA and RNA samples were sequenced. Multiple known beneficial microorganisms, such as Bradyrhizobium, Lysobacter and Variovorax showed significantly higher relative abundance and activity in rhizoplane microbiome despite of health status. However, several beneficial taxa, including Rhodopseudomonas, Achromobacter, Methylobacterium and Chitinophaga, showed higher relative abundance and activity in healthy rhizoplane microbiome compared with rhizosphere community in healthy trees but not in HLB samples. By performing comparison between healthy and HLB samples, we found several phyla, such as Proteobacteria, Acidobacteria and Bacteroidetes were enriched in healthy root-associated microbiome. HLB altered the rhizoplane microbiome by recruiting more functional features involved in autotrophic life cycle such as carbon fixation, and abandoning the functional genes involved in microbe-host interactions identified above, collectively resulting in downward spiral in rhizoplane microbiome-host interaction. This seems to suggest the manipulation of the root microbiome is necessary. However, the challenge is how to maintain a beneficial microbiome which is under study now. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. As shown in Objective 1, Bradyrhizobium and Burkholderia are the most abundant bacteria that have shown dramatic changes between survivor trees and HLB diseased trees. Members of Burkholderia and Bradyrhizobium have been known to benefit plants. We determined the contribution of Burkholderia to the citrus hosts. We isolated multiple Burkholderia strains. We selected two representative strains A53 (Burkholderia metallica) and A63 (Burkholderia territori) to inoculate citrus plants using the soil drench method. The results demonstrated that the two strains could successfully colonize the root surface and maintain a relative high population even seven months after inoculation. We then conducted a greenhouse study to evaluate the effects of the selected strains on the plant fitness. Salicylic acid (SA)-mediated ISR is an important benefit of beneficial bacteria to the plant host. We determined the expression of three SA mediated ISR marker genes, SAM, PR1 and PR2, of the inoculated trees. Plants treated with strain A53 exhibited a significant upregulation of PR2 gene at 3 dpi compared with negative control plants. A63 induced expression of the SAM gene at 5 dpi and the PR1 gene at 7 dpi. Similarly, Actigard induced the PR1 and SAM gene expression at 5 and 7 dpi. Large scale experiment is ongoing. One manuscript has been accepted for publication by Frontiers in Microbiology. In addition, we grafted the roots from survivor trees to healthy and HLB diseased trees in greenhouse to check the effect of endophyte changes on the grafted trees. Since endophytes appear to be enriched from the rhizosphere, we also used the soil from the survivor trees to plant both healthy and HLB diseased trees in the greenhouse. We also grafted shoots from survivor trees to further understand the putative mechanisms. Shoots from more survival trees are being grafted. We are also characterizing the potential mechanism why some branches are Las free. Multiple plants successfully grafted with leaf branches from survivor trees were subject to a test for citrus attractiveness to ACP. No significant effect on response of ACP to the grafted trees from the control. Consortium of bacteria are being used to test their effect on Las and ACP. One manuscript entitled: “Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome” has been published by Microbiome.
The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). This project contains two objectives: 1) Control HLB by optimization of application of SA and its analogs. We are testing the control effect of SA and its analogs, e.g., ASM, Imidacloprid, DL-2-aminobutyric, 2,6-dichloro-isonicotinic acid, and 2,1,3 Benzothiadiazole via trunk injection in field trial. Oxytetracycline is used as a positive control, whereas water was used as a negative control. SA, Acibenzolar-S-methyl (ASM), benzo (1,2,3) thiadiazole-7-cabothionic acid S-methyl ester (BTH), and 2,6-dichloroisonicotinic acid (INA) have also been applied twice onto selected trees by foliar spray in November, 2015 during fall flush, arch 2016 during spring flush, and February 2017 during spring flush. In addition, three field trials for different compounds including SA are being conducted. Materials were applied once onto selected trees by foliar spray in September, 2016 during late summer-fall flush, were applied to selected trees by soil drench in September, 2016 during late summer-fall flush, in early March and June 2017. Trunk injection in August and September, 2016 during summer and late summer-fall flush. Trunk injection of SA showed significant control effect against HLB. The data for trunk injection has been collected and a manuscript has been submitted for publication. HLB disease severity,disease incidence surveys and Las titers were conducted before spray treatment in October, 2015 and at 6 months after the 1st application in April, 2016 and April 2017. SA analogs resulted in increased fruit yield in 2016, but not in 2017 probably due to hurricane damage and also slowed down the progress of Las titers compared to control. To compare the effect of suppressing SA hydroxylase, we also screened multiple SecA inhibitors which suppress the secretion of important virulence factors. Two effective SecA inhibitors have been tested in vitro. At least one SecA inhibitor has been shown to be specific against Las, but not E. coli. We are also investigating the possibility of modifying pathway of citrus to produce more SA in citrus using CRISPR. One manuscript entitled: “Control of Citrus Huanglongbing (HLB) via Trunk Injection of Plant 1 Activators and Antibiotics” has been published by Phytopathology. 2) Control HLB using a combination of SA, SA analogs or SA hydroxylase inhibitors. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design are being tested for their inhibitory effect against SA hydroxyalse. To further identify SA hydroxylase inhibitors or SA analogs that are not degraded by SA hydroxylase, we have expressed SA hydroxylase in tobacco and Arabidopsis. Overexpression of SA hydroxylase decreased HR induced by Pseudomonas spp, indicating that SA hydroxylase degrades SA. We have qualified SA with HPLC and conducted SAR related genes expression analysis. We have identified multiple SA analogs and tested whether they can be degraded by SA hydroxylase. 4 SahA inhibitors were trunk-injected during fall flush. Las titers and HLB disease severity of the treated trees are being tested periodically. One manuscript entitled: ‘Candidatus Liberibacter asiaticus’ Encodes a Functional Salicylic Acid (SA) Hydroxylase That Degrades SA to Suppress Plant Defenses” has been published by MPMI.
The goal of the proposed study is to understand the mechanism of survivor trees. 1. Understanding the role of endophytic microbes from survivor trees. Three healthy and three HLB infected trees were selected for phytobiome analysis from Gapway grove based on the Las QPCR detection results. The microorganisms collected from this experiment were classified as three types: rhizosphere, rhizoplane and endosphere communities. The DNA and RNA samples were sequenced. Multiple known beneficial microorganisms, such as Bradyrhizobium, Lysobacter and Variovorax showed significantly higher relative abundance and activity in rhizoplane microbiome despite of health status. However, several beneficial taxa, including Rhodopseudomonas, Achromobacter, Methylobacterium and Chitinophaga, showed higher relative abundance and activity in healthy rhizoplane microbiome compared with rhizosphere community in healthy trees but not in HLB samples. By performing comparison between healthy and HLB samples, we found several phyla, such as Proteobacteria, Acidobacteria and Bacteroidetes were enriched in healthy root-associated microbiome. HLB altered the rhizoplane microbiome by recruiting more functional features involved in autotrophic life cycle such as carbon fixation, and abandoning the functional genes involved in microbe-host interactions identified above, collectively resulting in downward spiral in rhizoplane microbiome-host interaction. This seems to suggest the manipulation of the root microbiome is necessary. However, the challenge is how to maintain a beneficial microbiome which is under study now. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. As shown in Objective 1, Bradyrhizobium and Burkholderia are the most abundant bacteria that have shown dramatic changes between survivor trees and HLB diseased trees. Members of Burkholderia and Bradyrhizobium have been known to benefit plants. We determined the contribution of Burkholderia to the citrus hosts. We isolated multiple Burkholderia strains. We selected two representative strains A53 (Burkholderia metallica) and A63 (Burkholderia territori) to inoculate citrus plants using the soil drench method. The results demonstrated that the two strains could successfully colonize the root surface and maintain a relative high population even seven months after inoculation. We then conducted a greenhouse study to evaluate the effects of the selected strains on the plant fitness. Salicylic acid (SA)-mediated ISR is an important benefit of beneficial bacteria to the plant host. We determined the expression of three SA mediated ISR marker genes, SAM, PR1 and PR2, of the inoculated trees. Plants treated with strain A53 exhibited a significant upregulation of PR2 gene at 3 dpi compared with negative control plants. A63 induced expression of the SAM gene at 5 dpi and the PR1 gene at 7 dpi. Similarly, Actigard induced the PR1 and SAM gene expression at 5 and 7 dpi. Large scale experiment is ongoing. One manuscript has been accepted for publication by Frontiers in Microbiology. In addition, we grafted the roots from survivor trees to healthy and HLB diseased trees in greenhouse to check the effect of endophyte changes on the grafted trees. Since endophytes appear to be enriched from the rhizosphere, we also used the soil from the survivor trees to plant both healthy and HLB diseased trees in the greenhouse. We also grafted shoots from survivor trees to further understand the putative mechanisms. Shoots from more survival trees are being grafted. We are also characterizing the potential mechanism why some branches are Las free. Multiple plants successfully grafted with leaf branches from survivor trees were subject to a test for citrus attractiveness to ACP. No significant effect on response of ACP to the grafted trees from the control. Consortium of bacteria are being used to test their effect on Las and ACP. One manuscript entitled: “Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome” has been published by Microbiome.
September 2017 The objectives of this proposal are 1) To determine the temperature and relative humidity optima for Guignardia citricarpa pycnidiospore infection and production on citrus twigs, leaf litter, and fruit; 2) To determine the relative potential of Guignardia citricarpa to form pycnidiospores on citrus twigs, leaf litter, and fruit; 3) To determine whether Guignardia citricarpa can survive and reproduce on citrus debris on grove equipment. The samples that were previously incubated and dried are now ready for the DNA extraction process. 480 of the 960 samples were processed by removing the bark and homogenizing the tissue using liquid nitrogen and bead beating. The DNA was recovered using the MioBio PowerSoil kit. Another 1,536 samples were processed for incubation. This includes tagging, weighing, measuring midpoint diameter, and recovery and quantification of spore suspension from rinsate. Spore suspension was taken from 672 samples before the incubation period. The spore suspension was quantified and stored at -20 C. An additional 1,152 samples were taken from the field in Immokalee, FL. Our controlled study is schedule to begin in early October and preparations for this large project have already begun. This project will analyze the impact of relative humidity and temperature on growth and dissemination of Phyllosticta citricarpa. A postdoc was hired in September and has started to confirm that the inoculation techniques and containers with the salt solutions will work adequately to move the leaf litter section forward. Work on the effect of FDACS recommended disinfectants (200 ppm bleach or 2000 ppm quaternary ammonium) on conidia germination was conducted. Effective concentrations to inhibit either 50% or 90% of conidia germination for 2 quat products, Canker Solve and C-Quat, and bleach were found to be well below 5 ppm for all products. Bleach was about ten times more effective but is not as stable as quat. The disinfectants have been preliminarily evaluated in the presence of finely ground plant debris (twigs and leaves as would be found on mowers or hedgers). Testing of sodium hypochlorite was conducted during this quarter and is almost complete. Results show that at the lowest ratio of disinfectant to debris (100 l treatment), 1500 ppm a.i. was required to reduce spore germination to zero. This is notably higher than the recommended rate of 200 ppm. Efficacy of sodium hypochlorite is significantly reduced by the presence of debris and, in fact, is not recommended by CHRP for use on dirty equipment. Results of our work demonstrate that the presence of debris significantly reduces the efficacy of disinfectant, but this can be mitigated by using a large volume, relative to the amount of debris. Therefore, the results of this study illustrate that when decontaminating equipment with disinfectant, it should be applied to the point of run-off, rather than lightly sprayed on.
To determine the optimal concentration of dsRNA priming, ACP were exposed to a series of dsRNA concentrations (10, 100, 1000 ng. l-1) in artificial diet for 24 hrs, and 5 days. ACP were subsequently transferred to artificial diet containing S. marcescens for 4 days, then transferred to C. macrophylla for 14 days. Survival of ACP was recorded every 24 hrs for 14 days. ACP survival in response to S. marcescens was lowest after feeding on 100 ng. l-1 of dsRNA T7_pGEMT for 24hrs, as compared with control (no dsRNA priming) treatments. The survival rates of ACP exposed to S. marcescens or sucrose following control (no dsRNA priming) were not significantly different. Eighty percent of ACP survived after feeing on 100 ng. l-1 dsRNA ATPase for 5 d prior to being transferred to sucrose, and was significantly higher than among ACP that fed only on sucrose prior to S. marcescens exposure. This suggests that initial (24 h) exposure to dsRNA may increase susceptibility of ACP to pathogens. Among the insects that did not survive pathogen challenge, S. marcescens was detected in 79% of ACP following priming with 100 ng. l-1 dsRNA T7_pGEMT100 fior 24 h, as compared with 9-15% of ACP not primed with dsRNA. Insects primed for 5 days on dsRNA followed by exposure to S. marcescens, 75% of insects fed with 100 ng. l-1 dsRNA ATPase were infected with the pathogen. No bacterial infection was observed in insects fed on 1000 and 10 ng. l-1 of T7_pGEMTdsRNA , or control (no dsRNA priming) treated insects. The high percentage of bacterial infection in the dsRNA-treated insects indicates that dsRNA may contribute to bacterial loads in ACP, although this effect appears to be dose-dependent. Target gene expression decreased among ACP that were primed with 1000 ng. l-1 T7_pGEMT or 100, 1000 ng. l-1 of dsRNA ATPase prior to S. marcescens exposure but this reduction was not statistically different from untreated (no dsRNA priming) ACP. It is expected that the time between feeding dsRNA and quantifying mRNA at the end of the feeding bioassay is too long long to detected dsRNA-associated changes in expression; therefore, experiments are underway to evaluated changes in expression following 24 h and 5 d exposure to dsRNA. In addition, subsequent analyses will be conducted to determine whether priming facilitates Liberibacter acquisition.
The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). This project contains two objectives: 1) Control HLB by optimization of application of SA and its analogs. We are testing the control effect of SA and its analogs, e.g., ASM, Imidacloprid, DL-2-aminobutyric, 2,6-dichloro-isonicotinic acid, and 2,1,3 Benzothiadiazole via trunk injection in field trial. Oxytetracycline is used as a positive control, whereas water was used as a negative control. SA, Acibenzolar-S-methyl (ASM), benzo (1,2,3) thiadiazole-7-cabothionic acid S-methyl ester (BTH), and 2,6-dichloroisonicotinic acid (INA) have also been applied twice onto selected trees by foliar spray in November, 2015 during fall flush, arch 2016 during spring flush, and February 2017 during spring flush. In addition, three field trials for different compounds including SA are being conducted. Materials were applied once onto selected trees by foliar spray in September, 2016 during late summer-fall flush, were applied to selected trees by soil drench in September, 2016 during late summer-fall flush, in early March and June 2017. Trunk injection in August and September, 2016 during summer and late summer-fall flush. Trunk injection of SA showed significant control effect against HLB. The data for trunk injection has been collected and a manuscript has been submitted for publication. HLB disease severity,disease incidence surveys and Las titers were conducted before spray treatment in October, 2015 and at 6 months after the 1st application in April, 2016 and April 2017. To compare the effect of suppressing SA hydroxylase, we also screened multiple SecA inhibitors which suppress the secretion of important virulence factors. Two effective SecA inhibitors have been tested in vitro. At least one SecA inhibitor has been shown to be specific against Las, but not E. coli. We are also investigating the possibility of modifying pathway of citrus to produce more SA in citrus using CRISPR. One manuscript entitled: “Control of Citrus Huanglongbing (HLB) via Trunk Injection of Plant 1 Activators and Antibiotics” has been accepted for publication by Phytopathology. 2) Control HLB using a combination of SA, SA analogs or SA hydroxylase inhibitors. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design are being tested for their inhibitory effect against SA hydroxyalse. To further identify SA hydroxylase inhibitors or SA analogs that are not degraded by SA hydroxylase, we have expressed SA hydroxylase in tobacco and Arabidopsis. Overexpression of SA hydroxylase decreased HR induced by Pseudomonas spp, indicating that SA hydroxylase degrades SA. We have qualified SA with HPLC and conducted SAR related genes expression analysis. We have identified multiple SA analogs and tested whether they can be degraded by SA hydroxylase. One manuscript entitled: ‘Candidatus Liberibacter asiaticus’ Encodes a Functional Salicylic Acid (SA) Hydroxylase That Degrades SA to Suppress Plant Defenses” has been published by MPMI.
The goal of the proposed study is to understand the mechanism of survivor trees. 1. Understanding the role of endophytic microbes from survivor trees. Three healthy and three HLB infected trees were selected for phytobiome analysis from Gapway grove based on the Las QPCR detection results. The microorganisms collected from this experiment were classified as three types: rhizosphere, rhizoplane and endosphere communities. The DNA and RNA samples were sequenced. Multiple known beneficial microorganisms, such as Bradyrhizobium, Lysobacter and Variovorax showed significantly higher relative abundance and activity in rhizoplane microbiome despite of health status. However, several beneficial taxa, including Rhodopseudomonas, Achromobacter, Methylobacterium and Chitinophaga, showed higher relative abundance and activity in healthy rhizoplane microbiome compared with rhizosphere community in healthy trees but not in HLB samples. By performing comparison between healthy and HLB samples, we found several phyla, such as Proteobacteria, Acidobacteria and Bacteroidetes were enriched in healthy root-associated microbiome. HLB altered the rhizoplane microbiome by recruiting more functional features involved in autotrophic life cycle such as carbon fixation, and abandoning the functional genes involved in microbe-host interactions identified above, collectively resulting in downward spiral in rhizoplane microbiome-host interaction. This seems to suggest the manipulation of the root microbiome is necessary. However, the challenge is how to maintain a beneficial microbiome which is under study now. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. As shown in Objective 1, Bradyrhizobium and Burkholderia are the most abundant bacteria that have shown dramatic changes between survivor trees and HLB diseased trees. Members of Burkholderia and Bradyrhizobium have been known to benefit plants. We determined the contribution of Burkholderia to the citrus hosts. We isolated multiple Burkholderia strains. We selected two representative strains A53 (Burkholderia metallica) and A63 (Burkholderia territori) to inoculate citrus plants using the soil drench method. The results demonstrated that the two strains could successfully colonize the root surface and maintain a relative high population even seven months after inoculation. We then conducted a greenhouse study to evaluate the effects of the selected strains on the plant fitness. Salicylic acid (SA)-mediated ISR is an important benefit of beneficial bacteria to the plant host. We determined the expression of three SA mediated ISR marker genes, SAM, PR1 and PR2, of the inoculated trees. Plants treated with strain A53 exhibited a significant upregulation of PR2 gene at 3 dpi compared with negative control plants. A63 induced expression of the SAM gene at 5 dpi and the PR1 gene at 7 dpi. Similarly, Actigard induced the PR1 and SAM gene expression at 5 and 7 dpi. Large scale experiment is ongoing. In addition, we grafted the roots from survivor trees to healthy and HLB diseased trees in greenhouse to check the effect of endophyte changes on the grafted trees. Since endophytes appear to be enriched from the rhizosphere, we also used the soil from the survivor trees to plant both healthy and HLB diseased trees in the greenhouse. We also grafted shoots from survivor trees to further understand the putative mechanisms. Shoots from more survival trees are being grafted. We are also characterizing the potential mechanism why some branches are Las free. We are testing the effect of application of isolates on plant defenses and attractiveness to psyllids. One manuscript entitled: “Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome” has been published by Microbiome.
The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). This project contains two objectives: 1) Control HLB by optimization of application of SA and its analogs. We are testing the control effect of SA and its analogs, e.g., ASM, Imidacloprid, DL-2-aminobutyric, 2,6-dichloro-isonicotinic acid, and 2,1,3 Benzothiadiazole via trunk injection in field trial. Oxytetracycline is used as a positive control, whereas water was used as a negative control. SA, Acibenzolar-S-methyl (ASM), benzo (1,2,3) thiadiazole-7-cabothionic acid S-methyl ester (BTH), and 2,6-dichloroisonicotinic acid (INA) have also been applied twice onto selected trees by foliar spray in November, 2015 during fall flush, arch 2016 during spring flush, and February 2017 during spring flush. In addition, three field trials for different compounds including SA are being conducted. Materials were applied once onto selected trees by foliar spray in September, 2016 during late summer-fall flush, were applied to selected trees by soil drench in September, 2016 during late summer-fall flush, in early March and June 2017. Trunk injection in August and September, 2016 during summer and late summer-fall flush. Trunk injection of SA showed significant control effect against HLB. The data for trunk injection has been collected and a manuscript has been submitted for publication. HLB disease severity,disease incidence surveys and Las titers were conducted before spray treatment in October, 2015 and at 6 months after the 1st application in April, 2016 and April 2017. To compare the effect of suppressing SA hydroxylase, we also screened multiple SecA inhibitors which suppress the secretion of important virulence factors. Two effective SecA inhibitors have been tested in vitro. At least one SecA inhibitor has been shown to be specific against Las, but not E. coli. We are also investigating the possibility of modifying pathway of citrus to produce more SA in citrus using CRISPR. One manuscript entitled: “Control of Citrus Huanglongbing (HLB) via Trunk Injection of Plant 1 Activators and Antibiotics” has been accepted for publication by Phytopathology. 2) Control HLB using a combination of SA, SA analogs or SA hydroxylase inhibitors. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design are being tested for their inhibitory effect against SA hydroxyalse. To further identify SA hydroxylase inhibitors or SA analogs that are not degraded by SA hydroxylase, we have expressed SA hydroxylase in tobacco and Arabidopsis. Overexpression of SA hydroxylase decreased HR induced by Pseudomonas spp, indicating that SA hydroxylase degrades SA. We have qualified SA with HPLC and conducted SAR related genes expression analysis. We have identified multiple SA analogs and tested whether they can be degraded by SA hydroxylase. One manuscript entitled: ‘Candidatus Liberibacter asiaticus’ Encodes a Functional Salicylic Acid (SA) Hydroxylase That Degrades SA to Suppress Plant Defenses” has been published by MPMI.
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