A major focus of this research is to screen combinatorial small molecule libraries for activity against the Asian citrus psyllid using feeding bioassays. The first library that will be screened is a peptide library synthesized by Torrey Pines Institute for Molecular Studies (TPIMS). The TPIMS researchers have finished the first round of peptide synthesis and purification. The first peptides are designed to serve as negative controls for experiments involving the Torrey Pines combinatorial libraries. In order to design a negative control, the concept of a random sequence was employed. The best approach to making a negative control when nothing is known about the system is to make randomly selected sequences. 20 different peptides were selected for synthesis. While the sequences chosen were random, 10 of the peptides were biased to contain residues that made the final peptides more likely to be soluble in water. Of the 10 water soluble biased peptides 3 were purified and provided to USDA for testing. The USDA researchers have initiated feeding trials with these peptides and are optimizing concentration ranges. Also, specific proteins within the psyllid saliva secreted during feeding have been identified using the USDA bioassay to collect salivary components. This has provided insight into how the psyllids target the citrus phloem as their food source. This information is being used to develop a screen for inhibitors of this enzyme as a means of blocking the feeding process. Psyllid colony maintenance has been ramped up to provide enough adult psyllids for the bioassay survey and experiments have been initiated to optimize the diet for feeding immature psyllid life stages. Optimizing the diets for the immature stages will allow testing of inhibitors for activity across all stages of the psyllid that feed in citrus.
Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars. This is the 1st year, 1st quarter progress report; our grant started December 1, 2018. In the last three months, we have processed all the paperwork needed to establish the grant and begin spending funds at ARS. We have identified a qualified and interested post-doctoral researcher, Dr. Tamara D. Collum, who we will be hiring with grant funding. However, all ARS hiring actions, even those using soft funds, are currently on hold at the Department of Agriculture level. Now that the department has a full-year budget, we hope this hold will be lifted shortly so we can bring Tami on board in the next couple weeks. Objective 1 (development of transgenic constructs) is close to completion and work has begun in the Stover lab on objective 2 (production of transgenic citrus lines). For objective 6 (Additional Approach: Phloem limited citrus tristeza virus vectors will be used to express the His-FLAG-tagged ribosomal protein in healthy and CLas infected citrus) inserts have been assembled and sent to Dr. Dawson’s lab for inclusion in CTV vectors and subsequent introduction into citrus.
The objective of this study is to determine how different rotation schedules of commonly used insecticides with different modes of action such as dimethoate (acetylcholinesterase inhibitor), imidacloprid and thiamethoxam (acetylcholine receptor), diflubenzuron (inhibitor of chitin biosynthesis), abamectin 3 % + thiamethoxam 13.9 % (chloride channel allosteric modulator and acetylcholine receptor), fenpropathrin (sodium channel modulator), and cyantraniliprole (ryanodine receptor modulator) may impact the level of insecticide resistance Asian citrus psyllid (ACP) populations. An associated goal is to determine if ACP populations can be managed to reduce resistance in those populations where it already exists to a particular insecticide under rotations. Finally, the rotations must be effective in managing existing ACP populations to acceptable grower standards. We have selected two locations where resistance to neonicotinoid insecticides has been demonstrated and is known to exist. At each location, three rotational schemes of insecticides for ACP management will be established in 5 acre plots in Lake Alfred and 4.2 acre plots in Wauchula. The trees are 1-2 years old Hamlin trees with a variety of rootstocks. We are collecting adult ACP currently from these locations to determine their baseline insecticide resistance levels compared with a susceptible laboratory population of ACP using a leaf dip assay. Field populations have been collected from the Wauchula site and bioassays are underway currently. We will be collecting psyllids from the Lake Alfred site shortly. We will use commercial formulations of dimethoate, fenpropathrin, imidacloprid and cyantraniliprole to determine baseline resistance levels for these populations. Five to six concentrations of each insecticide will be tested and replicated 5 times. We will begin insecticide applications to fully evaluate our rotation treatments in early April 2019. We will collect samples chosen at random from the central rows for both the Lake Alfred and Wauchula sites. The plots will be sampled weekly beginning in late March 2019. The tap sample method will be used to sample adults. Ten samples will be taken per plot. For eggs and nymphs, 10 randomly selected flush samples will be taken per plots and number of eggs and nymph per flush samples will be counted. When counts of adults, eggs or nymphs in any plot reaches a predetermined threshold, a spray will be applied with the next insecticide in the rotation. Also, we will collect adults from the rotation sites to determine the relative expression of ten CYP4 and six GST genes that are implicated in insecticide resistance in ACP compared with the laboratory susceptable population. Finally, our goal is the development of a more refined method of an effective insecticide resistance management strategy. Our newly developed methods will be have positive impact on suppression of ACP populations by stabilizing or reducing resistance and will be economically viable.
A study was designed to investigate the capacity of field collected Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Liviidae) to develop resistance to the pyrethroid insecticide, fenpropathrin and determine the biochemical and genetic mechanisms of resistance to this popularly used chemistry for ACP management. We established an insecticide resistant strain of ACP in the greenhouse. The selected adult ACP population was originally collected from commercial citrus groves from Wachula, FL on July 15, 2018. They were treated with the LC50 concentration of fenpropathrin for nine generations of continuous rearing and then for a subsequent seven generations at a higher insecticide concentration. Bioassays were conducted using the bottle bioassay to assess the resistance of adults during each successive generation. Insecticides were dissolved to make 5-7 concentrations in acetone that gave 0 to 100% mortality. Control ACP were treated with acetone. Selection was performed by exposing adults to treated glass vials at the LC50 concentration. The ACP that survived were released in rearing cages to serve as parents of the next generation. The value of LC50 was increased from 0.12 to 3.71 ng/ L after nine generations. The resistance ratio was 30.91 fold. Biochemical assays were performed with detoxifying enzymes, namely esterase (EST), glutathione S-transferase (GST) and cytochrome P450 monooxygenase (P450). These were quantified every two generations and compared with the laboratory susceptible population. The activity ratio of EST enzymes was 1.530 fold higher for the selected population compared with the laboratory population at eight generations and there were significant differences between the two populations (p < 0.001). The activity of GST enzyme was 1.486 fold higher, and for P450 the activity ratio was 1.10 fold higher for the selected population compared to the laboratory population. There were significant differences between the two populations for GST activity (p = 0.038) and for P450 activity (p = 0.045). Future experiments are planned to better understand the genetic basis of resistance by determining the expression levels of genes involved in the detoxification of pyrethorid insecticides. The results of this study provide insight into the development of insecticide resistance and designing appropriate resistance management strategies for ACP.
Psyllids feeding on treated plants may ingest antimicrobials, which may have the potential to harm psyllids due to their reliance on bacterial endosymbionts for survival. Antimicrobial treatments may negatively affect a variety of psyllid biological features, including fecundity, transmission capacity, life span, developmental time, and behavior. The current objective was to evaluate the effect of dietary exposure to antibiotics on D. citri survival in a greenhouse bioassay. Feeding solutions containing oxytetracycline (Fireline) or streptomycin (Firewall) were orally administered to D. citri. D. citri mortality was significantly higher when insects fed on artificial diet solutions containing 1mg ml-1 oxytetracycline, 5mg ml-1 oxytetracycline, or imidacloprid. Approximate 40% and 100% mortality occurred among D. citri that fed on 5 mg ml-1 of oxytetracycline after 3-d and 10-d, respectively. Greater mortality occurred in response to the high oxytetracycline concentration than the low concentration on day 3 (25%) and day 10 (63%). D. citri mortality in response to untreated diet was approximately 20% and 30% on days 3 and 10, respectively. Neither 5 mg ml-1 nor 1 mg ml-1 streptomycin exhiwas associated with significant D. citri mortality as compared with untreated diet. After 30d, 40% of D. citri that fed on untreated or streptomycin diets survived. Subsequent assays will evaluate the effect of foliar applied antimicrobial treatments on D. citri survival and CLas transmission.
During this initial 4-month period of the Project, spanning from December 1st 2018 to March, we have followed the chronology developed in our proposal. Our Project has 4 main objectives: Objective 1. Assessing tree growth and absence of psyllids and HLB disease symptoms (including CLas bacteria titer) under protective covering. Objective 2. Assessment of alternative netting approaches involved in targeted , alternated or patterned setup of IPC in groves for more cost-effective protection. Objective 3. Monitoring the transition from vegetative to reproductive stage in the covered trees as compared to the uncovered. Objective 4. Comparing IPC with CUPS-like systems. Objective 1: We assessed the trees (Valencia on Swingle) planted in our pilot study 14 months ago for HLB . Whereas 1/3 of the uncovered trees in the trial are already positive for HLB, all trees covered with IPC have tested negative. In addition we are quantifying leaf drop and comparing leaf drop in both treatments. We are now in the process of planting trees of the varieties Tango , SugarBelle and US Early Pride with our collaborators in Polk County, as described in the proposal. Objective 2: This objective will start later this spring in both SWFREC grove and in our Polk County locations. Objective 3: The trial for this objective starts this month in Polk county and has started already in our grove at SWFREC. We have seen an significant advancement in flushing timing in IPC-covered trees. We have not seen any differences in blooming rate or intensity in IPC trees as compared to uncovered trees. We are assessing and rating the type of flushes each tree individually bears. Objective 4: We are currently planting 100 trees from Tango , SugarBelle and US Early Pride in our CUPS facility, after trellis installation. As expected, a Ms student, Susmita Gaire, has joined the Project and is performing part of this work for her Masters degree. Results from this Project have been already presented at the Citrus Show in Fort Pierce this past January, and an update will be presented in Riverside at the IRCHLB meeting later this month.
March 2019 Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp. In this first quarter, we found a field site with sufficient black spot to conduct the skirting and fungicide timing trial. We scouted 100 rows for presence/abscence of black spot and chose the best 96. The plots have been laid out and calculations were done for the fungicides and organized with the grower to apply with an airblast sprayer. Two sites have been scouted for a fungicide spray trial. The details will be determined once the incidence and severity have been collected. We plan to be able to use at least 5 products and possibly more. The remaining activities have been bureaucratic in nature. The postdoctoral researcher in South Africa has been appointed from 1 March 2019, and preparations for genotyping-by-sequencing of a collection of Phyllosticta citricarpa isolates from USA has been initiated. There are also continuing contract negotiation between CRI and UF which we hope will be resolved shortly so funds can be received and the postdoc can continue to work on the project. As we work to collect isolates for objective 3, Jeff Rollins has been working with the UF administration to make sure all of the necessary permits are in place for him to be able to travel to Cuba and collect isolates to determine the mating type and species identifications. We are waiting to recieve MAT 1-1 DNA from Australia and South Africa to use as positive controls for our experiments.
The purpose is to evaluate the control effect of bactericides via trunk injection. This proposal addresses the following CRDF CPDC-18 Research Priorities: 1A, 1C, and 1D. To achieve the goal of the research, we are conducting the following objectives:Objective 1. To illustrate whether application of bactericides via trunk injection could efficiently manage citrus HLB and how bactericides via trunk injection affects Las and HLB diseased trees.Three field trials have begun to investigate how the application of bactericides via trunk injection affects citrus growth, production, HLB symptom development, and Las population in different aged trees at different levels of HLB disease severity. We evaluated the inhibitory activity of OTC against Las in greenhouse and field experiments. Citrus trees were trunk-injected with OTC, and leaves were inspected for Las populations and OTC residues using qPCR and HPLC assays respectively, at various times after OTC treatment. We have acquired data about the MBC of OTC in planta. We will repeat this experiment. Objective 2. To examine the dynamics and residues of bactericide injected into citrus and systemic movement within the vascular system of trees and characterize the degradation metabolites of bactericides in citrus. A field trial has begun to determine the concentrations of bactericides in leaf, stem, root, flower, and fruit using HPLC at the following time points: 2, 7, 14, 28 days, 2, 4, 6, 8, 10, 12 months after injection at different doses. Objective 3. To determine whether trunk injection of bactericides could decrease Las acquisition by Asian citrus psyllids (ACP).In this objective, we will determine whether trunk injection of bactericides at three different doses could decrease Las acquisition by ACP in greenhouse and in the field. We are conducting the experiment right now. Objective 4. To monitor resistance development in Las against bactericides and evaluate potential side effects of trunk injection of bactericides. Las-specific primers were designed to target the putative binding sites of OTC in 16SrRNA gene of Las. Plant genomic DNA was extracted from citrus trees received OTC injection for three years. PCR were performed with the primers and DNA samples, and the products were purified and subjected to DNA sequencing. No mutation was identified yet. We will continue to monitor the resistance development against OTC and Streptomycin.
January 2019 The objectives of this proposal are 1) To determine the temperature and relative humidity optima for Guignardiacitricarpa 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 this project we established that Phyllosticta citricarpa (syn. Guignardia citricarpa) conidia are being formed in dead twigs in the field. We were only able to collect low numbers but suspect this may have been influenced by the methods used. The conidia are less likely to be present in the dry winter months but are frequently present at other times of the year. The amount of conidia was found to be influenced by rain in the preceeding two weeks, the average temperature, days with measurable rainfall in the last 7 days and an interaction between the accumulated rainfall and average temperature. When we investigted the presence of P. citricarpa DNA in the bark of the same twigs we found that the amount was influenced most by the average temperature, days with measurable rainfall in the last 7 days, days with measurable rainfall over 28 days and an interaction with average temperature and days with measurable precipitation. We also were able to link the bark DNA quantities with areas in the grove known to have a high incidence and severity of symptomatic fruit and trees. We found that the more symptomatic trees had a greater amount of P. citricarpa DNA in their twigs than those with low severity. This establishes that there is a connection between the amount of P. citricarpa in the canopy with the severity of fruit infection. What remains to be shown is whether the twigs are the initial inoculum source for the fruit or a sign of the overall infection level. We also conducted a large factorial experiment to look at the effects of temperature and relative humidity on the infection of twigs and leaves by P. citricarpa. We found that relative humidity was very important for the development of pycnidia and conidia on twigs. Relative humidity levels below 90% greatly reduced the number of pycnidia or conidia formed. However, the amount of P. citricarpa DNA present in the twigs showed that the fungus did not die but in some cases continued to increase it’s biomass greatly. Temperature was also very important and extremes in the temperature profiles did not allow for structure formation or in some cases biomass growth. Sporulation was more affected by temperature than relative humidity. This allows us to see when production of conidia is most likely to estimate the greatest inoculum potential. We also investigated the effect of disinfectants on spores in debris. It is known that debris can cause disinfectants to loose their potency and so it was shown in this project. However, it was found that the potency could be regained if a large enough volume of the disinfecant was used. Results of the completed research are consistent with recommendations from FDACS in regards to efficacy of recommended disinfectants. The finding that efficacy diminishes when spores are associated with citrus debris offers an opportunity to update recommendations for hedging operations and other activities that may generate significant amounts of fine debris to ensure that debris is fully saturated with disinfectant solutions.
December 2018 The objectives for this proposal are 1) Conduct ground and aerial applications of fungicides to determine the efficacy and economics of fungicide treatments; 2) Determine if Luna Sensation has enough systemic activity to protect flowers from before they fully develop and open; 3) Determine if the period flowering of trees affected by huanglongbing can be narrowed to eliminate the offseason bloom that contributes to the PFD inoculum increase in groves. In 2018, two field trials were conducted to evaluate the efficacy of various fungicides as well as some fungicide programs for management of PFD. In the product screening trial (Trial 1), the top five products all contained ferbam when the number of fruit were evaluated. In the program trial (Trial 2), only one treatment was recommended so only the first product in a rotation was used. The results from Trial 2 were more confusing and few consistancies occurred among treatments. For example, Enable performed the best in one program but there was another program that started with Enable that had signficantly fewer fruit. In both trials, disease was extremely low and it is difficult to determine if the number of fruit per tree side was solely due to the treatments or other inherent differences among trees. An aerial trial was conducted in 2018 as well as and found that there were more fruit per tree in the ground application than the aerial application but this may be because the trees had a slightly larger canopy in ground application block. There were no fungicide treatment differences observed. An attempt to conduct a more controlled Luna Sensation trial in the greenhouse failed because insufficient bloom was induced to undertake the experiment. This was despite an attempt to induce flowers with cold and drought for 2 months. The second year of bloom synchronization was undertaken in 2018. Off-season bloom was suppressed with gibberellic acid (GA) in Navel and Valencia trees. In both cultivars, the major blooms were compressed by GA compared to the untreated control and fewer flowers were observed. Trees treated with napthaleneacetic acid (NAA; synthetic auxin) did not have a similar effect. Despite the significant reduction in bloom with GA, there was no signficant reduction of yield compared to the untreated control. Applications for the upcoming flowering period have been commenced and it is anticipated that fruit number data will be collected by July. Minor changes were done to the Citrus Advisory System (CAS) in 2018 to correct some minor problems. Additionally, the fungicde recommendations, in part from this project, were linked to CAS. Improvement have been made to the leaf wetness duration estimates for stations that do not have leaf wetness probes or the probes have been shown to be malfunctioning. Field validation of the CAS has continued in 2018 at 2 sites, Polk City and Fort Meade. In both sites, we compared an untreated control to the PFD-FAD, a new model CAS, and weekly fungicide applications. There were no significant differences among the treatments for the number of fruit per tree despite there being 3 weekly applications and 2 or 1 (respectively) for the PFD-FAD system. No applications were triggered by the CAS. Since there was no significant differences among the treatments, it means that no applications was the best forecast and significant cost savings could be had by using the CAS.
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. We have completed the project. Application of plant defense inducers including SA have positive effect on HLB diseased trees at the early stage of HLB infection, but have no effect on HLB diseased trees at the late stage of infection. One manuscript entitled: “Developing citrus Huanglongbing management strategies based on the severity of symptoms in HLB-endemic citrus-producing regions” has been accepted for publication by Phytopathology.
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. We have completed the project. Application of plant defense inducers including SA have positive effect on HLB diseased trees at the early stage of HLB infection, but have no effect on HLB diseased trees at the late stage of infection. One manuscript entitled: “Developing citrus Huanglongbing management strategies based on the severity of symptoms in HLB-endemic citrus-producing regions” has been accepted for publication by Phytopathology.
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 LasQPCR 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. We also determined the core microbiome based on the analysis of healthy citrus collected from 8 different countries. We have completed the project. We have identified multiple beneficial bacteria which promote citrus growth. However, the beneficial bacteria can only slow down the HLB disease progress, but can not reduce Las population in planta.
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 LasQPCR 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. We also determined the core microbiome based on the analysis of healthy citrus collected from 8 different countries. We have completed the project. We have identified multiple beneficial bacteria which promote citrus growth. However, the beneficial bacteria can only slow down the HLB disease progress, but can not reduce Las population in planta.
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 LasQPCR 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. We also determined the core microbiome based on the analysis of healthy citrus collected from 8 different countries. We have completed the project. We have identified multiple beneficial bacteria which promote citrus growth. However, the beneficial bacteria can only slow down the HLB disease progress, but can not reduce Las population in planta.
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